Research ArticleBacopa monnieri Extract (CDRI-08) Modulates the NMDAReceptor Subunits and nNOS-Apoptosis Axis in Cerebellum ofHepatic Encephalopathy Rats

Papia Mondal and Surendra Kumar Trigun

Biochemistry Section Department of Zoology Banaras Hindu University Varanasi 221005 India

Correspondence should be addressed to Surendra Kumar Trigun sktrigungmailcom

Received 27 November 2014 Revised 9 February 2015 Accepted 9 February 2015

Academic Editor Con Stough

Copyright copy 2015 P Mondal and S K Trigun This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Hepatic encephalopathy (HE) characterized by impaired cerebellar functions during chronic liver failure (CLF) involves N-methyl-D-aspartate receptor (NMDAR) overactivation in the brain cellsBacopamonnieri (BM) extract is a knownneuroprotectantThe present paper evaluates whether BM extract is able to modulate the two NMDAR subunits (NR2A and NR2B) andits downstream mediators in cerebellum of rats with chronic liver failure (CLF) induced by administration of 50mgkg bwthioacetamide (TAA) ip for 14 days and in the TAA group rats orally treated with 200mgkg bw BM extract from days 8 to14 NR2A is known to impart neuroprotection and that of NR2B induces neuronal death during NMDAR activation Neuronalnitric oxide synthase- (nNOS-) apoptosis pathway is known tomediate NMDAR led excitotoxicityThe level of NR2Awas found tobe significantly reduced with a concomitant increase of NR2B in cerebellum of the CLF rats This was consistent with significantlyenhanced nNOS expression nitric oxide level and reduced Bcl2Bax ratio Moreover treatment with BM extract reversed theNR2ANR2B ratio and also normalized the levels of nNOS-apoptotic factors in cerebellum of those rats The findings suggestmodulation of NR2A and NR2B expression by BM extract to prevent neurochemical alterations associated with HE

1 Introduction

The patients with liver cirrhosis develop a serious nervoussystem disorder known as hepatic encephalopathy (HE) [1]It is characterized by wide spectrum of neuropsychiatricsymptoms related to motor dysfunction cognitive impair-ment and disturbed sleep wake cycle [2ndash4] Most of the livercirrhotic patients have been found to show minimal to overtHE symptoms [5 6] characterized mainly by the impairedmotor functions [2 7ndash9] which is considered to be associatedwith deranged cerebellar functions [10 11] Some recentfindings from our lab also suggest that cerebellum showsgreater susceptibility to undergo neurochemical changes inthemodels of chronic type HE [12ndash14]Therefore cerebellumwas selected for the present study

Based on the studies conducted to understandpathophys-iology of HE it has been suggested that the level of glutamatean important excitatory neurotransmitter increases in thesynaptic cleft due to the increased blood and brain ammonia

level during liver dysfunction resulting into overactivationof the ionotropic N-methyl-D-aspartate receptors (NMDAR)[1] And there is a general agreement that activation ofglutamate-NMDA receptor-nNOS pathway constitutes mainneurochemical aberrations associated with HE [1 14]

Functional NMDAR a tetrameric protein complex com-prises of two subunits of a constitutive glycine binding NR1and remaining two of glutamate binding NR2 from amongstNR2A NR2B NR2C and NR2D subunits Importantlycombination of different NR2 subunits is suggested to conferunique electrophysiological properties to this neurotransmit-ter receptor [15 16] For example alterations in the ratioof NR2A versus NR2B of NMDAR have been found tobe associated with the changes in long term potentiation(LTP) and long term depression (LDP) functions duringhippocampal plasticity involved in memory consolidation[17] In addition NR2A dominating combination of NMDARis demonstrated to provide neuroprotection but NR2B rich

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 535013 8 pageshttpdxdoiorg1011552015535013

2 Evidence-Based Complementary and Alternative Medicine

NMDAR is known to drive the postsynaptic neuron towardsapoptosis during glutamate excitotoxicity [14] Thus regulat-ing NMDAR function by altering its composition withoutpharmacological blockage of the channel could be a uniqueand novel cerebral mechanism to prevent NMDAR overacti-vation led neurological disorders

It is now evident that neuronal nitric oxide synthase(nNOS) via modulating NO level in the brain cells playscritical roles in transmitting NMDAR led neurophysiologicalchanges under different pathophysiological conditions [1819] A threshold level of NO is essential to activate NO-cGMP signaling to maintain NMDAR dependent memoryconsolidation and cognition functions [18] However excessof NO is known to induce apoptosis and neuronal death [2021] Such multimodal roles of NMDAR-nNOS axis in braincells are orchestrated by a molecular link between NMDARand nNOS protein The NR2 subunits of NMDAR complexthrough their tSXV motifs connect with the postsynapticdensity protein-95 (PSD-95) which in turn via its PDZdomain interacts with the nNOS in the postsynaptic neurons[19 22] This constitutes the main mechanism of NMDARactivity led NO production and subsequent changes in theneuronal functions Particularly during HE activation ofNMDAR leads to the increased calcium influx which inturn activates nNOS and thereby overproduces NO in thepostsynaptic neurons [1 9 13] Thus increased glutamate ledNMDAR activation via activation of nNOS is consideredcritically involved in developing HE associated neuropsychi-atric problems in the patientsanimals [13 23 24]

Obviously NMDAR becomes choice of an importanttherapeutic target for the neurobiologists for HE manage-ment [25 26] Some earlier studies conducted in vivo and invitro using NMDAR antagonists indeed demonstrated desir-able results however this approach was found to produceundesirable neurological complications during the clinicaltrials [25 27] This is not surprising as NMDAR activity iscritical for maintaining normal neurophysiology includinghigher order brain functions and memory consolidationmechanisms [17] Therefore instead of blocking NMDARchannel modulation of NMDAR activity by alterations inits functional composition and downstream signaling seemsto be of special scientific merit However this evolvingconcept needs to be examined in the animal models withexcitotoxic neurological problems Since development of HEis related with NMDAR led excitotoxicity [1] and that herbalformulations are now evident to modulate brain chemistryin many ways the present work was undertaken to evaluatewhether Bacopa monnieri (BM) extract a known neuropro-tectant is able tomodulate NMDAR composition and relateddownstream events in cerebellum of the CLF induced HErats

Amongst a goodnumber of herbal drugs availableBacopamonnieri extract has been widely evaluated as a memoryenhancer adaptogenic anti-inflammatory analgesic antipy-retic sedative and antiepileptic agent [28] Also some studiessuggest its neuroprotective roles against epilepsy (neuroexci-totoxic outcome) by modulating serotonergic receptor [29]and against Parkinsonrsquos and Alzheimerrsquos disease via alteringdopaminergic signaling [30] and cholinergic [31] receptors

respectively Moreover molecular mechanisms underlyingthese BM extract effects remain largely unexplored

Importantly though information is limited efficacy ofBM extract has also been shown against glutamate toxicity viamodulating NMDAR1 gene expression and in turn affectingglutamatergic signaling [32] In our previous reports wehave observed a direct association between overexpressionof the constitutive NR1 nNOS activation and enhanced NOproduction in cerebellum of the CLF rats exhibiting HEcharacteristics [2 13 14] Importantly we could also observereciprocal expression of NR2A and NR2B in the cerebellumof those rats (data from this paper) This tempted us toinvestigate whether administration of BME is able to alter thisunusual NR2A2B composition and thus NMDAR-nNOSpathway in the cerebellum of the HE rats

2 Materials and Methods

21 Chemicals Chemicals used were of analytical grade sup-plied by E-Merck and Sisco Research Laboratory (India)Acrylamide NN1015840-methylenebisacrylamide NNN1015840N1015840-te-tramethylethylenediamine (TEMED) phenylmethyl sulpho-nyl fluoride (PMSF) bromophenol blue and Ponceau werepurchased from Sigma-Aldrich USA Primary antibodiesused were procured from the following companies rabbitmonoclonal 120573-actin from Sigma Aldrich rabbit monoclonalanti-NR2B from Invitrogen rabbit monoclonal NR2A fromEpitomics rabbit polyclonal Bcl2 andBax fromCell SignalingTechnology and rabbit polyclonal nNOS from Santa CruzBiotechnology Rabbit and mouse horseradish peroxidase(HRP) conjugated secondary antibodies were obtained fromGenei ECL western blotting detection kit was purchasedfromThermo Scientific

The ethanolic extract of Bacopa monnieri extract (BMCDRI-08) containing 6428 bacoside A and 2711 baco-side B was obtained from the Lumen Research FoundationChennai India

22 Animals Inbred adult female albino rats weighing 150ndash160 g were used in this study The rats were kept in separatecages fed with the recommended diet and maintained atstandard conditions of 12 h light and dark period at roomtemperature (25plusmn2∘C) in an animal houseThe use of animalsfor the present study was approved by the InstitutionalAnimal Care and Use Committee (IACUC) Animal EthicalCommittee (AEC) of the BanarasHinduUniversity Varanasi

23 Induction of Chronic Liver Failure (CLF)HE and Treat-ment Schedule The CLFHE model of neuroexcitotoxicityin adult albino rats was induced by the administration ofthioacetamide (TAA) as standardized previously [2] Forthis rats were randomly divided into three groups with 6rats in each Group A control (C) administered with 09saline ip once daily for 14 days Group B CLFHE groupadministered with 50mgKg bw TAA ip once daily for 14days Group C CLF + Bacopa extract (CLF + BM) Ratsin Group C were orally administered with ethanolic extractof BM extract (CDRI-08 200mgKg bw) suspended in 1

Evidence-Based Complementary and Alternative Medicine 3

gum acacia once daily starting from 8th day onwards till14th day and were administered 4 h after the TAA treatmentThe dose of BM was selected which was able to recover TAAinduced neurobehavioural deficit in the rats All the rats weresacrificed on 15th day The cerebellum was dissected out andstored at minus80∘C for further experiments

24 Preparation of Cerebellar Extracts As described earlier[13] mitochondria free cerebellar extracts were prepared inan extraction medium consisting of 400mM sucrose 1mMEDTA 02mM benzamidine 01mM phenylmethylsulfonylfluoride (PMSF) and 002 heparin The extracts werecentrifuged initially at 12000timesg for 15min and finally at19000timesg for 45min at 4∘C The supernatant obtained wascollected as the cytosolic fractionsThe protein content in thetissue extract wasmeasured by Lowrymethod [33] using BSAas standard

25 Western Blotting As described previously [13] thecytosolic fractions containing 60 120583g proteinlane were sepa-rated on 10 SDS-PAGE and electrotransferred to nitrocel-lulose membrane at 50mA and run overnight at 4∘C Proteintransfer was checked via Ponceau staining The membranewas then placed in a blocking solution of 5 skimmed milkin 1X PBS for 2 h followed by washing in PBS 3 times Themembranes were then separately processed for immunode-tection of NR2A NR2B nNOS Bcl2 and Bax using mono-clonalpolyclonal anti-NR2A (1 1000) anti-NR2B (1 1000)anti-nNOS (1 500) anti-Bcl2 (1 1000) and anti-Bax (1 500)respectively HRP conjugated secondary antibody was usedfor final detection of the proteins using ECL western blottingdetection kit 120573-actin used as loading control was detectedusing a monoclonal anti-120573-actin peroxidase antibody Thebands were quantified and analyzed using gel densitometrysoftware AlphaImager 2200 The photographs in the figureare representatives of the three western blot repeats

26 Nitric Oxide (NO) Estimation Nitric oxide level wasmeasured by estimating total nitrite (NO

2) and nitrate (NO

3)

content in the tissue extracts as described earlier [13] usingthe method of Sastry et al [34] Briefly tissue fractions(100 120583L) NaNO

2 and KNO

3standards (01mM each) were

mixed separately with 400 120583L of 50mM carbonate buffer(pH 9) For NO

3estimation activated copper-cadmium alloy

(150mg) was added and incubated for an hour at 37∘C Thereaction was stopped using 100 120583L each of 035M NaOHand 120mM zinc sulphate After centrifugation 400 120583Lsupernatant was incubatedwith theGriess reagent 200120583L 1sulphanilamide prepared in 25 H

3PO4and 200120583L 01 N-

(1-naphthyl)-ethylenediamine Absorbance was recorded at545 nm For NO

2estimation similar procedure was followed

except addition of the copper-cadmium alloy

27 Statistical Analysis The data have been expressed asmean plusmn SD For two group comparisons statistical analysiswas performed using unpaired Studentrsquos 119905 test A probabilityof 119875 lt 005 was taken as a significant difference between thegroups Each of the experiments was repeated thrice

3 Results

31 Effect of BM Extract on NR2A and NR2B ExpressionThe combination of constituent NMDAR subunits is evidentto impart unique neurophysiological role of this glutamatereceptor The NR2A dominating combination is known tomediate neuroprotectionwhereas those of NR2B induce neu-ronal death According to Figures 1(a) and 1(b) as comparedto the control rats level of NR2A is found to be significantlyreduced (119875 lt 0001) with a concomitant increase inNR2B level in the cerebellum of the CLF rats (Figures 1(c)and 1(d)) resulting in a significant decline in NR2A2Bratio (Figure 1(e)) However this pattern is observed to berecovered back with a significant enhancement of NR2A(Figures 1(a) and 1(b)) and a decline of NR2B (Figures 1(c)and 1(d)) resulting into attaining a control level NR2A2Bratio (Figure 1(e)) in the cerebellum of the CLF rats treatedwith the BM extract

32 Effect of BM Extract on nNOS and Nitric Oxide (NO)Production Neuronal NOS (nNOS) has a direct molecularlink with NMDAR and therefore it is considered to bethe main determinant of NMDAR activation based down-stream signaling in the postsynaptic neurons Accordinglyoveractivation of nNOS is considered associated with theneuronal changes associated with NMDAR led excitotoxicityAs depicted in Figures 2(a) and 2(b) nNOS expression isobserved to be enhanced significantly (119875 lt 0001) in thecerebellum of the CLF rats as compared to the control grouprats Moreover due to the oral administration of BM extractlevel of this enzyme is observed to be reduced up to thecontrol value (Figures 2(a) and 2(b)) Such a pattern couldcoincide well with the similar changes in the NO level incerebellum of the CLF and BM extract treated CLF rats(Figure 2(c))

33 Effect of BM Extract on the Expression of Bcl2 and BaxBcl2 (antiapoptotic) and Bax (proapoptotic) ratio servesas a rheostat to determine cell susceptibility to apoptosisAccording to Figures 3(a) and 3(c) as compared to the controlgroup rats level of Bcl2 is found to be declined significantly(119875 lt 005) with a concomitant increase in the Bax levelresulting in a significant decline (119875 lt 001) in Bcl2Bax ratio(Figure 3(e)) in cerebellum of the CLF rats However aftertreatment with BM extract the pattern of Bcl2Bax ratio isobserved to regain its normal range in cerebellum of thoseCLF rats (Figure 3(e))

4 Discussion

NMDA receptor overactivation is considered a commonneurochemical event associated with a number of braindysfunctions like epilepsy ischemia drug abuse and HE[23 26 35ndash37] However NMDAR blockage is evident tobe a poor therapeutic target for managing such excitotoxicconditions [25 27] In this background while understandingneurochemical basis of CLF induced HE we observed a clear

4 Evidence-Based Complementary and Alternative Medicine

NR2A

120573-actin

C CLF CLF + BM

(a)

Den

sitom

etric

val

ue (N

R2A

120573-a

ctin

)

000

020

040

060

080

100

120

140

C CLF CLF + BM

lowastlowastlowast

(b)

NR2B

120573-actin

C CLF CLF + BM

(c)

000

020

040

060

080

100

120

140

160

C CLF

D

ensit

omet

ric v

alue

(NR2

B120573

-act

in)

CLF + BM

lowastlowastlowast

(d)

000020040060080100120140160

Den

sitom

etric

val

ue (N

R2A

NR2

B)

C CLF CLF + BM

lowastlowastlowast

(e)

Figure 1 Expression profile of NR2A ((a) and (b)) andNR2B ((c) and (d)) in cerebellum of the CLF and BM extract treated CLF ratsWesternblot analysis was performed as described in the method textThe level of 120573-actin was probed as the loading control In panels (b) (d) and (e)normalized densitometric values of NR2A120573-actin NR2B120573-actin and NR2ANR2B have been presented as mean plusmn SD from three westernblot repeats lowastlowastlowast119875 lt 0001 (control versus CLF rats) 119875 lt 001 119875 lt 0001 (CLF versus CLF + BM rats) C control CLF chronic liverfailure CLF + BM chronic liver failure + Bacopa monnieri extract

shift in the expression of glutamate binding NMDAR subunitfrom a NR2A dominating combination to a NR2B rich com-bination in cerebellum of the CLF rats (Figures 1(a) and 1(c))It is now becoming clearer that NR2A rich NMDAR impartsneuroprotection and that of NR2B dominating combinationinitiates neuronal damage and apoptosis [16 38ndash40] This isbecause NR2B has been reported to show delay in gatingkinetics in comparison to that of NR1NR2A combinationresulting in increased Ca2+ influx and thus rapid activation

of the downstream signaling [41] In the present context asignificant decline in Bcl2 with a concomitant increase in Baxlevel in cerebellum of the CLF rats (Figure 3) also hints for adirect association between NR2B dominating NMDAR com-position and altered neurochemistry of cerebellumof theCLFrats Furthermore alterations in oxidative and nitrosativefactors in the postsynaptic neurons are considered to bethe main downstream mediators of such unusual NMDARactivations [1] and it has been reported that cerebellum shows

Evidence-Based Complementary and Alternative Medicine 5

nNOS

120573-actin

C CLF CLF + BM

(a)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (n

NO

S120573

-act

in)

lowastlowastlowast

C CLF CLF + BM(b)

00

10

20

30

40

50

60

C CLF

lowastlowast

NO

(120583m

olm

g pr

otei

n)

CLF + BM

(c)

Figure 2 Expression profile of nNOS ((a) and (b)) and NO level (c) in cerebellum of the CLF and BM extract treated CLF rats Western blotanalysis of nNOS was performed as described in the text of methods In panel (b) normalized densitometric values of nNOS120573-actin havebeen presented as mean plusmn SD from three western blot repeats lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (control versus CLF rats) 119875 lt 005 119875 lt 0001(CLF versus CLF + BM rats) C control CLF chronic liver failure CLF + BM chronic liver failure + Bacopa monnieri extract

greater susceptibility to undergo oxidative and nitrosativestress during HA and CLF led excitotoxicity [12 13]

It has been described that lesion in cerebellum impairsacquisition of memory consolidation and deranges motorfunctions [10 11] The CLF rats used in this experimenthave recently been reported to show cognitive impairmentand deficit in motor functions as well [2] Since such neu-rochemical alterations leading to neurobehavioural changesare known to emanate from abnormal NMDAR activity [1]it is argued that a shift from a NR2A combination to aNR2B combination of NMDAR in the cerebellum of theCLF rats might be accountable for developing HE associatedsymptoms observed in these CLF rats [2] Moreover keepingaside these explanations the findings provided a basis to alterNMDAR constitution instead of shutting this ion-channeloff as a therapeutic option to bring recovery from the HEsymptoms

To test this hypothesis BM extract was chosen due to thetwo reasons Firstly amidst scarcity of the safer neurophar-macological agents this plant extract is demonstrated toimprove neuronal functions by modulating brain chemistryin many ways [28] Secondly BM is now evident to modulateactivity of the neurotransmitter receptors like serotonergicreceptors during epilepsy [29] and dopaminergic and cholin-ergic signaling in Parkinsonrsquos and Alzheimerrsquos diseases [3031] However report is limited on modulation of NMDAreceptor activity by BMWe observed a remarkable shift froma declined ratio of NR2ANR2B (neurodegeneration sup-portive combination) in the cerebellum of CLF rats towardstheir normal level when these CLF rats were administeredwith BM extract (Figure 1(e)) This finding suggested thatBM is able to alter constitution of the functional NMDARtetramer by differential expression of the two glutamatebinding subunits in cerebellum of the CLF rats Indeed in

6 Evidence-Based Complementary and Alternative Medicine

Bcl2

C CLF CLF + BM

120573-actin

(a)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (B

cl2120573

-act

in)

C CLF CLF + BM

lowast

(b)

Bax

C CLF CLF + BM

120573-actin

(c)

000

020

040

060

080

100

120

140

160

Den

sitom

etric

val

ue (B

ax120573

-act

in)

C CLF CLF + BM

lowastlowast

(d)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (B

cl2B

ax)

C CLF CLF + BM

lowastlowast

(e)

Figure 3 Expression profile of Bcl2 ((a) and (b)) and Bax ((c) and (d)) in cerebellum of the CLF and BM extract treated CLF rats Westernblot analysis of Bcl2 and Bax was performed as described in the text of methods The level of 120573-actin was probed as the loading control Inpanels (b) (d) and (e) normalized densitometric values of Bcl2120573-actin Bax120573-actin and Bcl2Bax ratio respectively have been presentedas mean plusmn SD from three western blot repeats lowast119875 lt 005 lowastlowast119875 lt 001 (control versus CLF rats) 119875 lt 005 119875 lt 001 (CLF versus CLF +BM rats) C control CLF chronic liver failure CLF + BM chronic liver failure + Bacopa monnieri extract

an epilepsy model it has been demonstrated that BM couldalter NR1 gene expression [32] thus suggesting BM as amod-ulator of NMDAR subunit expression A similar finding onmodulation of NR1 by BM was reported in a hypoxia modelalso [42] Importantly such a change in NR1 expression wasfound accountable for modulating glutamatergic signaling incerebellum of those rats [32] Thus it is argued that shiftingfrom a neurodegenerative NR2B overexpression towards

the neuroprotective NR2A level due to the treatment withBME (Figure 1) could also account for preventing down-stream undesirable neurochemical changes in cerebellum ofthe CLF rats

In a number of excitotoxic models including CLF ledHE nNOS activation is considered as the most commonevent after NMDAR activation [13 43] It is initiated by influxof Ca2+ which overactivates this enzyme to produce excess

Evidence-Based Complementary and Alternative Medicine 7

NO in the postsynaptic neurons [44] NO is a moleculeof pleiotropic effects however when produced in excessin the brain cells it uses more than one mechanism toinduce neuronal dysfunction [20 21] A milder increase inNO level is likely to initiate mitochondrial dysfunction ledneuronal apoptosis Ratio of Bcl2 versus Bax is consideredas the most effective regulators of mitochondrial dysfunctionled apoptosis [45] and hence relative levels of both theseproteins are considered as a reliable tool to assay whether acell is likely to undergo internal apoptotic process [46] Incerebellum of the CLF rats a significantly increased level ofnNOS coincides well with a similar increment in NO level(Figure 2) This is consistent with a significant decline in theBcl2Bax ratio (Figure 3) Moreover all these factors werefound to be reversed to regain their normal levels in thecerebellumwhen thoseCLF rats were treatedwith BMextract(Figures 1ndash3) Recent studies have also shown that BM extractdownregulates Bax and upregulates Bcl2 and thus providesneuroprotection in several neurological disease models [47]Thus the findings of Figures 1ndash3 together advocate forconcordant modulation of NMDA receptor constitution andnNOS led apoptotic activation by ethanolic extract of BM(CDRI-08) in cerebellum of the CLF rats

5 Conclusion

Opposing roles of the two main glutamate binding subunitsNR2A and NR2B of NMDA receptor advocate for modu-lation of NMDAR constitution as an effective mechanismto normalize its excitotoxic effects without blocking its ion-channel activity During recent past studies on efficacy ofBacopa monnieri extract have been shown to prevent neu-rodegenerative diseases by modulating neurochemistry ofthe brain cells but with little information about modulationof NMDAR overactivation led excitotoxicity The presentfindings demonstrate that BM extract is able to modulateNMDA receptor signaling by bringing reciprocal changes inthe expression of its two glutamate binding subunits NR2Aand NR2B and thus provide a novel approach to normalizeNMDAR overactivation effects without blocking this ionchannel

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The work was financially supported by DBT Grant (BTPR14049Med303442010) to Surendra Kumar Trigun andthe award of CSIR SRF to Papia Mondal The facilities ofDST-FIST and UGC-CAS programs in the Department ofZoology and BHU-DBT ISLS facilities in Faculty of Scienceare also acknowledged The authors thank Professor HemantSingh Lumen Research Foundation Chennai for providingthe ethanolic extract of Bacopa monnieri (CDRI-08)

References

[1] V Felipo ldquoHyperammonemiardquo inHandbook of Neurochemistryand Molecular Neurobiology Brain and Spinal Cord Trauma Alaztha N Banik and S K Ray Eds pp 43ndash69 Springer BerlinGermany 3rd edition 2009

[2] S Singh and S K Trigun ldquoLow grade cirrhosis inducescognitive impairment and motor dysfunction in rats couldbe a model for minimal hepatic encephalopathyrdquo NeuroscienceLetters vol 559 pp 136ndash140 2014

[3] A T Blei J Cordoba and The Practice Parameters Com-mittee of the American College of Gastroenterology ldquoHepaticencephalopathyrdquo American Journal of Gastroenterology vol 96pp 635ndash643 2001

[4] J S Bajaj J B Wade and A J Sanyal ldquoSpectrum of neurocog-nitive impairment in cirrhosis implications for the assessmentof hepatic encephalopathyrdquoHepatology vol 50 no 6 pp 2014ndash2021 2009

[5] F F Poordad ldquoReview article the burden of hepaticencephalopathyrdquo Alimentary Pharmacology and Therapeuticsvol 25 no 1 pp 3ndash9 2007

[6] J S Bajaj ldquoMinimal hepatic encephalopathy matters in dailyliferdquoWorld Journal of Gastroenterology vol 14 no 23 pp 3609ndash3615 2008

[7] S Mechtcheriakov I W Graziadei A Kugener et al ldquoMotordysfunction in patients with liver cirrhosis impairment ofhandwritingrdquo Journal of Neurology vol 253 no 3 pp 349ndash3562006

[8] S J Gilberstadt H Gilberstadt L Zieve B Buegel R O CollierJr and C J McClain ldquoPsychomotor performance defects incirrhotic patients without overt encephalopathyrdquo Archives ofInternal Medicine vol 140 no 4 pp 519ndash521 1980

[9] V Felipo and R F Butterworth ldquoNeurobiology of ammoniardquoProgress in Neurobiology vol 67 no 4 pp 259ndash279 2002

[10] C C Gandhi R M Kelly R G Wiley and T J WalshldquoImpaired acquisition of a Morris water maze task followingselective destruction of cerebellar purkinje cells with OX7-saporinrdquo Behavioural Brain Research vol 109 no 1 pp 37ndash472000

[11] M G Leggio P Neri A Graziano L Mandolesi M Molinariand L Petrosini ldquoCerebellar contribution to spatial event pro-cessing characterization of procedural learningrdquo ExperimentalBrain Research vol 127 no 1 pp 1ndash11 1999

[12] S Singh R K Koiri and S K Trigun ldquoAcute and chronichyperammonemiamodulate antioxidant enzymes differently incerebral cortex and cerebellumrdquo Neurochemical Research vol33 no 1 pp 103ndash113 2008

[13] S Singh and S K Trigun ldquoActivation of neuronal nitric oxidesynthase in cerebellum of chronic hepatic encephalopathyrats is associated with up-regulation of NADPH-producingpathwayrdquo Cerebellum vol 9 no 3 pp 384ndash397 2010

[14] P Mondal and S K Trigun ldquoPannexin1 as a novel cerebraltarget in pathogenesis of hepatic encephalopathyrdquo MetabolicBrain Disease vol 29 no 4 pp 1007ndash1015 2014

[15] S Cull-Candy S Brickley and M Farrant ldquoNMDA receptorsubunits diversity development and diseaserdquo Current Opinionin Neurobiology vol 11 no 3 pp 327ndash335 2001

[16] Y Liu PW TakM Aarts et al ldquoNMDA receptor subunits havedifferential roles in mediating excitotoxic neuronal death bothin vitro and in vivordquoThe Journal of Neuroscience vol 27 no 11pp 2846ndash2857 2007

8 Evidence-Based Complementary and Alternative Medicine

[17] O A Shipton and O Paulsen ldquoGluN2A and GluN2B subunit-containing NMDA receptors in hippocampal plasticityrdquo Philo-sophical Transactions of the Royal Society B Biological Sciencesvol 369 no 1633 2014

[18] C Montoliu R Rodrigo P Monfort et al ldquoCyclic GMP path-ways in hepatic encephalopathy neurological and therapeuticimplicationsrdquoMetabolic Brain Disease vol 25 no 1 pp 39ndash482010

[19] M V Doucet A Harkin and K K Dev ldquoThe PSD-95nNOScomplex new drugs for depressionrdquo Pharmacology amp Thera-peutics vol 133 no 2 pp 218ndash229 2012

[20] H Prast andA Philippu ldquoNitric oxide asmodulator of neuronalfunctionrdquo Progress in Neurobiology vol 64 no 1 pp 51ndash682001

[21] F X Guix I Uribesalgo M Coma and F J Munoz ldquoThephysiology and pathophysiology of nitric oxide in the brainrdquoProgress in Neurobiology vol 76 no 2 pp 126ndash152 2005

[22] E A Waxman and D R Lynch ldquoN-methyl-D-aspartate recep-tor subtypes multiple roles in excitotoxicity and neurologicaldiseaserdquo Neuroscientist vol 11 no 1 pp 37ndash49 2005

[23] V Felipo ldquoContribution of altered signal transduction asso-ciated to glutamate receptors in brain to the neurologicalalterations of hepatic encephalopathyrdquo World Journal of Gas-troenterology vol 12 no 48 pp 7737ndash7743 2006

[24] M Llansola R Rodrigo P Monfort et al ldquoNMDA receptorsin hyperammonemia and hepatic encephalopathyrdquo MetabolicBrain Disease vol 22 no 3-4 pp 321ndash335 2007

[25] S A Lipton and P A Rosenberg ldquoMechanisms of disease exci-tatory amino acids as a final common pathway for neurologicdisordersrdquoTheNew England Journal of Medicine vol 330 no 9pp 613ndash622 1994

[26] D R Lynch and R P Guttmann ldquoExcitotoxicity perspectivesbased on N-methyl-D-aspartate receptor subtypesrdquo Journal ofPharmacology and Experimental Therapeutics vol 300 no 3pp 717ndash723 2002

[27] C Ikonomidou and L Turski ldquoWhy did NMDA receptorantagonists fail clinical trials for stroke and traumatic braininjuryrdquoThe Lancet Neurology vol 1 no 6 pp 383ndash386 2002

[28] K J Gohil and J J Patel ldquoA review onBacopamonniera currentresearch and future prospectsrdquo International Journal of GreenPharmacy vol 4 no 1 pp 1ndash9 2010

[29] A Krishnakumar P M Abraham J Paul and C S PauloseldquoDown-regulation of cerebellar 5-HT2C receptors inpilocarpine-induced epilepsy in rats therapeutic role ofBacopa monnieri extractrdquo Journal of the Neurological Sciencesvol 284 no 1-2 pp 124ndash128 2009

[30] M Singh V Murthy and C Ramassamy ldquoNeuroprotectivemechanisms of the standardized extract of Bacopa monnierain a paraquatdiquat-mediated acute toxicityrdquo NeurochemistryInternational vol 62 no 5 pp 530ndash539 2013

[31] N Uabundit J Wattanathorn S Mucimapura and K Ingkan-inan ldquoCognitive enhancement and neuroprotective effects ofBacopa monnieri in Alzheimerrsquos disease modelrdquo Journal ofEthnopharmacology vol 127 no 1 pp 26ndash31 2010

[32] R Khan A Krishnakumar and C S Paulose ldquoDecreasedglutamate receptor binding and NMDA R1 gene expression inhippocampus of pilocarpine-induced epileptic rats neuropro-tective role of Bacopa monnieri extractrdquo Epilepsy and Behaviorvol 12 no 1 pp 54ndash60 2008

[33] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[34] K V H Sastry R P Moudgal J Mohan J S Tyagi and G SRao ldquoSpectrophotometric determination of serum nitrite andnitrate by copper-cadmium alloyrdquo Analytical Biochemistry vol306 no 1 pp 79ndash82 2002

[35] D R Lynch and R P Guttmann ldquoNMDA receptor pharma-cology perspectives from molecular biologyrdquo Current DrugTargets vol 2 no 3 pp 215ndash231 2001

[36] L V Kalia S K Kalia and M W Salter ldquoNMDA receptorsin clinical neurology excitatory times aheadrdquo The LancetNeurology vol 7 no 8 pp 742ndash755 2008

[37] A Lau and M Tymianski ldquoGlutamate receptors neurotoxicityand neurodegenerationrdquo Pflugers Archiv European Journal ofPhysiology vol 460 no 2 pp 525ndash542 2010

[38] M M Zeron O Hansson N Chen et al ldquoIncreased sensitivityto N-methyl-D-aspartate receptor-mediated excitotoxicity in amousemodel ofHuntingtonrsquos diseaserdquoNeuron vol 33 no 6 pp849ndash860 2002

[39] A J Williams J R Dave X M Lu G Ling and F C TortellaldquoSelective NR2B NMDA receptor antagonists are protectiveagainst staurosporine-induced apoptosisrdquo European Journal ofPharmacology vol 452 no 1 pp 135ndash136 2002

[40] M Chen T-J Lu X-J Chen et al ldquoDifferential roles of NMDAreceptor subtypes in ischemic neuronal cell death and ischemictolerancerdquo Stroke vol 39 no 11 pp 3042ndash3048 2008

[41] K Erreger S M Dravid T G Banke D J A Wyllie and SF Traynelis ldquoSubunit-specific gating controls rat NR1NR2AandNR1NR2BNMDAchannel kinetics and synaptic signallingprofilesrdquoThe Journal of Physiology vol 563 no 2 pp 345ndash3582005

[42] C S Paulose F Chathu S Reas Khan and A KrishnakumarldquoNeuroprotective role of Bacopa monnieri extract in epilepsyand effect of glucose supplementation during hypoxia gluta-mate receptor gene expressionrdquoNeurochemical Research vol 33no 9 pp 1663ndash1671 2008

[43] V L R Rao R M Audet and R F Butterworth ldquoIncreasednitric oxide synthase activities and L-[3H]arginine uptake inbrain following portacaval anastomosisrdquo Journal of Neurochem-istry vol 65 no 2 pp 677ndash681 1995

[44] P Monfort M-D Munoz A ElAyadi E Kosenko and VFelipo ldquoEffects of hyperammonemia and liver failure on glu-tamatergic neurotransmissionrdquoMetabolic Brain Disease vol 17no 4 pp 237ndash250 2002

[45] C M Snyder E H Shroff J Liu and N S Chandel ldquoNitricoxide induces cell death by regulating anti-apoptotic Bcl2 familymembersrdquo PLoS ONE vol 4 no 9 Article ID e7059 2009

[46] R K Koiri and S K Trigun ldquoDimethyl sulfoxide acti-vates tumor necrosis factor 120572-p53 mediated apoptosis anddown regulates D-fructose-6-phosphate-2-kinase and lactatedehydrogenase-5 in Daltonrsquos Lymphoma in vivordquo LeukemiaResearch vol 35 no 7 pp 950ndash956 2011

[47] R B Thomas S Joy M S Ajayan and C S Paulose ldquoNeuro-protective potential of bacopa monnieri and bacoside a againstdopamine receptor dysfunction in the cerebral cortex of neona-tal hypoglycaemic ratsrdquo Cellular and Molecular Neurobiologyvol 33 no 8 pp 1065ndash1074 2013

Submit your manuscripts athttpwwwhindawicom

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

NMDAR is known to drive the postsynaptic neuron towardsapoptosis during glutamate excitotoxicity [14] Thus regulat-ing NMDAR function by altering its composition withoutpharmacological blockage of the channel could be a uniqueand novel cerebral mechanism to prevent NMDAR overacti-vation led neurological disorders

It is now evident that neuronal nitric oxide synthase(nNOS) via modulating NO level in the brain cells playscritical roles in transmitting NMDAR led neurophysiologicalchanges under different pathophysiological conditions [1819] A threshold level of NO is essential to activate NO-cGMP signaling to maintain NMDAR dependent memoryconsolidation and cognition functions [18] However excessof NO is known to induce apoptosis and neuronal death [2021] Such multimodal roles of NMDAR-nNOS axis in braincells are orchestrated by a molecular link between NMDARand nNOS protein The NR2 subunits of NMDAR complexthrough their tSXV motifs connect with the postsynapticdensity protein-95 (PSD-95) which in turn via its PDZdomain interacts with the nNOS in the postsynaptic neurons[19 22] This constitutes the main mechanism of NMDARactivity led NO production and subsequent changes in theneuronal functions Particularly during HE activation ofNMDAR leads to the increased calcium influx which inturn activates nNOS and thereby overproduces NO in thepostsynaptic neurons [1 9 13] Thus increased glutamate ledNMDAR activation via activation of nNOS is consideredcritically involved in developing HE associated neuropsychi-atric problems in the patientsanimals [13 23 24]

Obviously NMDAR becomes choice of an importanttherapeutic target for the neurobiologists for HE manage-ment [25 26] Some earlier studies conducted in vivo and invitro using NMDAR antagonists indeed demonstrated desir-able results however this approach was found to produceundesirable neurological complications during the clinicaltrials [25 27] This is not surprising as NMDAR activity iscritical for maintaining normal neurophysiology includinghigher order brain functions and memory consolidationmechanisms [17] Therefore instead of blocking NMDARchannel modulation of NMDAR activity by alterations inits functional composition and downstream signaling seemsto be of special scientific merit However this evolvingconcept needs to be examined in the animal models withexcitotoxic neurological problems Since development of HEis related with NMDAR led excitotoxicity [1] and that herbalformulations are now evident to modulate brain chemistryin many ways the present work was undertaken to evaluatewhether Bacopa monnieri (BM) extract a known neuropro-tectant is able tomodulate NMDAR composition and relateddownstream events in cerebellum of the CLF induced HErats

Amongst a goodnumber of herbal drugs availableBacopamonnieri extract has been widely evaluated as a memoryenhancer adaptogenic anti-inflammatory analgesic antipy-retic sedative and antiepileptic agent [28] Also some studiessuggest its neuroprotective roles against epilepsy (neuroexci-totoxic outcome) by modulating serotonergic receptor [29]and against Parkinsonrsquos and Alzheimerrsquos disease via alteringdopaminergic signaling [30] and cholinergic [31] receptors

respectively Moreover molecular mechanisms underlyingthese BM extract effects remain largely unexplored

Importantly though information is limited efficacy ofBM extract has also been shown against glutamate toxicity viamodulating NMDAR1 gene expression and in turn affectingglutamatergic signaling [32] In our previous reports wehave observed a direct association between overexpressionof the constitutive NR1 nNOS activation and enhanced NOproduction in cerebellum of the CLF rats exhibiting HEcharacteristics [2 13 14] Importantly we could also observereciprocal expression of NR2A and NR2B in the cerebellumof those rats (data from this paper) This tempted us toinvestigate whether administration of BME is able to alter thisunusual NR2A2B composition and thus NMDAR-nNOSpathway in the cerebellum of the HE rats

2 Materials and Methods

21 Chemicals Chemicals used were of analytical grade sup-plied by E-Merck and Sisco Research Laboratory (India)Acrylamide NN1015840-methylenebisacrylamide NNN1015840N1015840-te-tramethylethylenediamine (TEMED) phenylmethyl sulpho-nyl fluoride (PMSF) bromophenol blue and Ponceau werepurchased from Sigma-Aldrich USA Primary antibodiesused were procured from the following companies rabbitmonoclonal 120573-actin from Sigma Aldrich rabbit monoclonalanti-NR2B from Invitrogen rabbit monoclonal NR2A fromEpitomics rabbit polyclonal Bcl2 andBax fromCell SignalingTechnology and rabbit polyclonal nNOS from Santa CruzBiotechnology Rabbit and mouse horseradish peroxidase(HRP) conjugated secondary antibodies were obtained fromGenei ECL western blotting detection kit was purchasedfromThermo Scientific

The ethanolic extract of Bacopa monnieri extract (BMCDRI-08) containing 6428 bacoside A and 2711 baco-side B was obtained from the Lumen Research FoundationChennai India

22 Animals Inbred adult female albino rats weighing 150ndash160 g were used in this study The rats were kept in separatecages fed with the recommended diet and maintained atstandard conditions of 12 h light and dark period at roomtemperature (25plusmn2∘C) in an animal houseThe use of animalsfor the present study was approved by the InstitutionalAnimal Care and Use Committee (IACUC) Animal EthicalCommittee (AEC) of the BanarasHinduUniversity Varanasi

23 Induction of Chronic Liver Failure (CLF)HE and Treat-ment Schedule The CLFHE model of neuroexcitotoxicityin adult albino rats was induced by the administration ofthioacetamide (TAA) as standardized previously [2] Forthis rats were randomly divided into three groups with 6rats in each Group A control (C) administered with 09saline ip once daily for 14 days Group B CLFHE groupadministered with 50mgKg bw TAA ip once daily for 14days Group C CLF + Bacopa extract (CLF + BM) Ratsin Group C were orally administered with ethanolic extractof BM extract (CDRI-08 200mgKg bw) suspended in 1

Evidence-Based Complementary and Alternative Medicine 3

gum acacia once daily starting from 8th day onwards till14th day and were administered 4 h after the TAA treatmentThe dose of BM was selected which was able to recover TAAinduced neurobehavioural deficit in the rats All the rats weresacrificed on 15th day The cerebellum was dissected out andstored at minus80∘C for further experiments

24 Preparation of Cerebellar Extracts As described earlier[13] mitochondria free cerebellar extracts were prepared inan extraction medium consisting of 400mM sucrose 1mMEDTA 02mM benzamidine 01mM phenylmethylsulfonylfluoride (PMSF) and 002 heparin The extracts werecentrifuged initially at 12000timesg for 15min and finally at19000timesg for 45min at 4∘C The supernatant obtained wascollected as the cytosolic fractionsThe protein content in thetissue extract wasmeasured by Lowrymethod [33] using BSAas standard

25 Western Blotting As described previously [13] thecytosolic fractions containing 60 120583g proteinlane were sepa-rated on 10 SDS-PAGE and electrotransferred to nitrocel-lulose membrane at 50mA and run overnight at 4∘C Proteintransfer was checked via Ponceau staining The membranewas then placed in a blocking solution of 5 skimmed milkin 1X PBS for 2 h followed by washing in PBS 3 times Themembranes were then separately processed for immunode-tection of NR2A NR2B nNOS Bcl2 and Bax using mono-clonalpolyclonal anti-NR2A (1 1000) anti-NR2B (1 1000)anti-nNOS (1 500) anti-Bcl2 (1 1000) and anti-Bax (1 500)respectively HRP conjugated secondary antibody was usedfor final detection of the proteins using ECL western blottingdetection kit 120573-actin used as loading control was detectedusing a monoclonal anti-120573-actin peroxidase antibody Thebands were quantified and analyzed using gel densitometrysoftware AlphaImager 2200 The photographs in the figureare representatives of the three western blot repeats

26 Nitric Oxide (NO) Estimation Nitric oxide level wasmeasured by estimating total nitrite (NO

2) and nitrate (NO

3)

content in the tissue extracts as described earlier [13] usingthe method of Sastry et al [34] Briefly tissue fractions(100 120583L) NaNO

2 and KNO

3standards (01mM each) were

mixed separately with 400 120583L of 50mM carbonate buffer(pH 9) For NO

3estimation activated copper-cadmium alloy

(150mg) was added and incubated for an hour at 37∘C Thereaction was stopped using 100 120583L each of 035M NaOHand 120mM zinc sulphate After centrifugation 400 120583Lsupernatant was incubatedwith theGriess reagent 200120583L 1sulphanilamide prepared in 25 H

3PO4and 200120583L 01 N-

(1-naphthyl)-ethylenediamine Absorbance was recorded at545 nm For NO

2estimation similar procedure was followed

except addition of the copper-cadmium alloy

27 Statistical Analysis The data have been expressed asmean plusmn SD For two group comparisons statistical analysiswas performed using unpaired Studentrsquos 119905 test A probabilityof 119875 lt 005 was taken as a significant difference between thegroups Each of the experiments was repeated thrice

3 Results

31 Effect of BM Extract on NR2A and NR2B ExpressionThe combination of constituent NMDAR subunits is evidentto impart unique neurophysiological role of this glutamatereceptor The NR2A dominating combination is known tomediate neuroprotectionwhereas those of NR2B induce neu-ronal death According to Figures 1(a) and 1(b) as comparedto the control rats level of NR2A is found to be significantlyreduced (119875 lt 0001) with a concomitant increase inNR2B level in the cerebellum of the CLF rats (Figures 1(c)and 1(d)) resulting in a significant decline in NR2A2Bratio (Figure 1(e)) However this pattern is observed to berecovered back with a significant enhancement of NR2A(Figures 1(a) and 1(b)) and a decline of NR2B (Figures 1(c)and 1(d)) resulting into attaining a control level NR2A2Bratio (Figure 1(e)) in the cerebellum of the CLF rats treatedwith the BM extract

32 Effect of BM Extract on nNOS and Nitric Oxide (NO)Production Neuronal NOS (nNOS) has a direct molecularlink with NMDAR and therefore it is considered to bethe main determinant of NMDAR activation based down-stream signaling in the postsynaptic neurons Accordinglyoveractivation of nNOS is considered associated with theneuronal changes associated with NMDAR led excitotoxicityAs depicted in Figures 2(a) and 2(b) nNOS expression isobserved to be enhanced significantly (119875 lt 0001) in thecerebellum of the CLF rats as compared to the control grouprats Moreover due to the oral administration of BM extractlevel of this enzyme is observed to be reduced up to thecontrol value (Figures 2(a) and 2(b)) Such a pattern couldcoincide well with the similar changes in the NO level incerebellum of the CLF and BM extract treated CLF rats(Figure 2(c))

33 Effect of BM Extract on the Expression of Bcl2 and BaxBcl2 (antiapoptotic) and Bax (proapoptotic) ratio servesas a rheostat to determine cell susceptibility to apoptosisAccording to Figures 3(a) and 3(c) as compared to the controlgroup rats level of Bcl2 is found to be declined significantly(119875 lt 005) with a concomitant increase in the Bax levelresulting in a significant decline (119875 lt 001) in Bcl2Bax ratio(Figure 3(e)) in cerebellum of the CLF rats However aftertreatment with BM extract the pattern of Bcl2Bax ratio isobserved to regain its normal range in cerebellum of thoseCLF rats (Figure 3(e))

4 Discussion

NMDA receptor overactivation is considered a commonneurochemical event associated with a number of braindysfunctions like epilepsy ischemia drug abuse and HE[23 26 35ndash37] However NMDAR blockage is evident tobe a poor therapeutic target for managing such excitotoxicconditions [25 27] In this background while understandingneurochemical basis of CLF induced HE we observed a clear

4 Evidence-Based Complementary and Alternative Medicine

NR2A

120573-actin

C CLF CLF + BM

(a)

Den

sitom

etric

val

ue (N

R2A

120573-a

ctin

)

000

020

040

060

080

100

120

140

C CLF CLF + BM

lowastlowastlowast

(b)

NR2B

120573-actin

C CLF CLF + BM

(c)

000

020

040

060

080

100

120

140

160

C CLF

D

ensit

omet

ric v

alue

(NR2

B120573

-act

in)

CLF + BM

lowastlowastlowast

(d)

000020040060080100120140160

Den

sitom

etric

val

ue (N

R2A

NR2

B)

C CLF CLF + BM

lowastlowastlowast

(e)

Figure 1 Expression profile of NR2A ((a) and (b)) andNR2B ((c) and (d)) in cerebellum of the CLF and BM extract treated CLF ratsWesternblot analysis was performed as described in the method textThe level of 120573-actin was probed as the loading control In panels (b) (d) and (e)normalized densitometric values of NR2A120573-actin NR2B120573-actin and NR2ANR2B have been presented as mean plusmn SD from three westernblot repeats lowastlowastlowast119875 lt 0001 (control versus CLF rats) 119875 lt 001 119875 lt 0001 (CLF versus CLF + BM rats) C control CLF chronic liverfailure CLF + BM chronic liver failure + Bacopa monnieri extract

shift in the expression of glutamate binding NMDAR subunitfrom a NR2A dominating combination to a NR2B rich com-bination in cerebellum of the CLF rats (Figures 1(a) and 1(c))It is now becoming clearer that NR2A rich NMDAR impartsneuroprotection and that of NR2B dominating combinationinitiates neuronal damage and apoptosis [16 38ndash40] This isbecause NR2B has been reported to show delay in gatingkinetics in comparison to that of NR1NR2A combinationresulting in increased Ca2+ influx and thus rapid activation

of the downstream signaling [41] In the present context asignificant decline in Bcl2 with a concomitant increase in Baxlevel in cerebellum of the CLF rats (Figure 3) also hints for adirect association between NR2B dominating NMDAR com-position and altered neurochemistry of cerebellumof theCLFrats Furthermore alterations in oxidative and nitrosativefactors in the postsynaptic neurons are considered to bethe main downstream mediators of such unusual NMDARactivations [1] and it has been reported that cerebellum shows

Evidence-Based Complementary and Alternative Medicine 5

nNOS

120573-actin

C CLF CLF + BM

(a)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (n

NO

S120573

-act

in)

lowastlowastlowast

C CLF CLF + BM(b)

00

10

20

30

40

50

60

C CLF

lowastlowast

NO

(120583m

olm

g pr

otei

n)

CLF + BM

(c)

Figure 2 Expression profile of nNOS ((a) and (b)) and NO level (c) in cerebellum of the CLF and BM extract treated CLF rats Western blotanalysis of nNOS was performed as described in the text of methods In panel (b) normalized densitometric values of nNOS120573-actin havebeen presented as mean plusmn SD from three western blot repeats lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (control versus CLF rats) 119875 lt 005 119875 lt 0001(CLF versus CLF + BM rats) C control CLF chronic liver failure CLF + BM chronic liver failure + Bacopa monnieri extract

greater susceptibility to undergo oxidative and nitrosativestress during HA and CLF led excitotoxicity [12 13]

It has been described that lesion in cerebellum impairsacquisition of memory consolidation and deranges motorfunctions [10 11] The CLF rats used in this experimenthave recently been reported to show cognitive impairmentand deficit in motor functions as well [2] Since such neu-rochemical alterations leading to neurobehavioural changesare known to emanate from abnormal NMDAR activity [1]it is argued that a shift from a NR2A combination to aNR2B combination of NMDAR in the cerebellum of theCLF rats might be accountable for developing HE associatedsymptoms observed in these CLF rats [2] Moreover keepingaside these explanations the findings provided a basis to alterNMDAR constitution instead of shutting this ion-channeloff as a therapeutic option to bring recovery from the HEsymptoms

To test this hypothesis BM extract was chosen due to thetwo reasons Firstly amidst scarcity of the safer neurophar-macological agents this plant extract is demonstrated toimprove neuronal functions by modulating brain chemistryin many ways [28] Secondly BM is now evident to modulateactivity of the neurotransmitter receptors like serotonergicreceptors during epilepsy [29] and dopaminergic and cholin-ergic signaling in Parkinsonrsquos and Alzheimerrsquos diseases [3031] However report is limited on modulation of NMDAreceptor activity by BMWe observed a remarkable shift froma declined ratio of NR2ANR2B (neurodegeneration sup-portive combination) in the cerebellum of CLF rats towardstheir normal level when these CLF rats were administeredwith BM extract (Figure 1(e)) This finding suggested thatBM is able to alter constitution of the functional NMDARtetramer by differential expression of the two glutamatebinding subunits in cerebellum of the CLF rats Indeed in

6 Evidence-Based Complementary and Alternative Medicine

Bcl2

C CLF CLF + BM

120573-actin

(a)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (B

cl2120573

-act

in)

C CLF CLF + BM

lowast

(b)

Bax

C CLF CLF + BM

120573-actin

(c)

000

020

040

060

080

100

120

140

160

Den

sitom

etric

val

ue (B

ax120573

-act

in)

C CLF CLF + BM

lowastlowast

(d)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (B

cl2B

ax)

C CLF CLF + BM

lowastlowast

(e)

Figure 3 Expression profile of Bcl2 ((a) and (b)) and Bax ((c) and (d)) in cerebellum of the CLF and BM extract treated CLF rats Westernblot analysis of Bcl2 and Bax was performed as described in the text of methods The level of 120573-actin was probed as the loading control Inpanels (b) (d) and (e) normalized densitometric values of Bcl2120573-actin Bax120573-actin and Bcl2Bax ratio respectively have been presentedas mean plusmn SD from three western blot repeats lowast119875 lt 005 lowastlowast119875 lt 001 (control versus CLF rats) 119875 lt 005 119875 lt 001 (CLF versus CLF +BM rats) C control CLF chronic liver failure CLF + BM chronic liver failure + Bacopa monnieri extract

an epilepsy model it has been demonstrated that BM couldalter NR1 gene expression [32] thus suggesting BM as amod-ulator of NMDAR subunit expression A similar finding onmodulation of NR1 by BM was reported in a hypoxia modelalso [42] Importantly such a change in NR1 expression wasfound accountable for modulating glutamatergic signaling incerebellum of those rats [32] Thus it is argued that shiftingfrom a neurodegenerative NR2B overexpression towards

the neuroprotective NR2A level due to the treatment withBME (Figure 1) could also account for preventing down-stream undesirable neurochemical changes in cerebellum ofthe CLF rats

In a number of excitotoxic models including CLF ledHE nNOS activation is considered as the most commonevent after NMDAR activation [13 43] It is initiated by influxof Ca2+ which overactivates this enzyme to produce excess

Evidence-Based Complementary and Alternative Medicine 7

NO in the postsynaptic neurons [44] NO is a moleculeof pleiotropic effects however when produced in excessin the brain cells it uses more than one mechanism toinduce neuronal dysfunction [20 21] A milder increase inNO level is likely to initiate mitochondrial dysfunction ledneuronal apoptosis Ratio of Bcl2 versus Bax is consideredas the most effective regulators of mitochondrial dysfunctionled apoptosis [45] and hence relative levels of both theseproteins are considered as a reliable tool to assay whether acell is likely to undergo internal apoptotic process [46] Incerebellum of the CLF rats a significantly increased level ofnNOS coincides well with a similar increment in NO level(Figure 2) This is consistent with a significant decline in theBcl2Bax ratio (Figure 3) Moreover all these factors werefound to be reversed to regain their normal levels in thecerebellumwhen thoseCLF rats were treatedwith BMextract(Figures 1ndash3) Recent studies have also shown that BM extractdownregulates Bax and upregulates Bcl2 and thus providesneuroprotection in several neurological disease models [47]Thus the findings of Figures 1ndash3 together advocate forconcordant modulation of NMDA receptor constitution andnNOS led apoptotic activation by ethanolic extract of BM(CDRI-08) in cerebellum of the CLF rats

5 Conclusion

Opposing roles of the two main glutamate binding subunitsNR2A and NR2B of NMDA receptor advocate for modu-lation of NMDAR constitution as an effective mechanismto normalize its excitotoxic effects without blocking its ion-channel activity During recent past studies on efficacy ofBacopa monnieri extract have been shown to prevent neu-rodegenerative diseases by modulating neurochemistry ofthe brain cells but with little information about modulationof NMDAR overactivation led excitotoxicity The presentfindings demonstrate that BM extract is able to modulateNMDA receptor signaling by bringing reciprocal changes inthe expression of its two glutamate binding subunits NR2Aand NR2B and thus provide a novel approach to normalizeNMDAR overactivation effects without blocking this ionchannel

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The work was financially supported by DBT Grant (BTPR14049Med303442010) to Surendra Kumar Trigun andthe award of CSIR SRF to Papia Mondal The facilities ofDST-FIST and UGC-CAS programs in the Department ofZoology and BHU-DBT ISLS facilities in Faculty of Scienceare also acknowledged The authors thank Professor HemantSingh Lumen Research Foundation Chennai for providingthe ethanolic extract of Bacopa monnieri (CDRI-08)

References

[1] V Felipo ldquoHyperammonemiardquo inHandbook of Neurochemistryand Molecular Neurobiology Brain and Spinal Cord Trauma Alaztha N Banik and S K Ray Eds pp 43ndash69 Springer BerlinGermany 3rd edition 2009

[2] S Singh and S K Trigun ldquoLow grade cirrhosis inducescognitive impairment and motor dysfunction in rats couldbe a model for minimal hepatic encephalopathyrdquo NeuroscienceLetters vol 559 pp 136ndash140 2014

[3] A T Blei J Cordoba and The Practice Parameters Com-mittee of the American College of Gastroenterology ldquoHepaticencephalopathyrdquo American Journal of Gastroenterology vol 96pp 635ndash643 2001

[4] J S Bajaj J B Wade and A J Sanyal ldquoSpectrum of neurocog-nitive impairment in cirrhosis implications for the assessmentof hepatic encephalopathyrdquoHepatology vol 50 no 6 pp 2014ndash2021 2009

[5] F F Poordad ldquoReview article the burden of hepaticencephalopathyrdquo Alimentary Pharmacology and Therapeuticsvol 25 no 1 pp 3ndash9 2007

[6] J S Bajaj ldquoMinimal hepatic encephalopathy matters in dailyliferdquoWorld Journal of Gastroenterology vol 14 no 23 pp 3609ndash3615 2008

[7] S Mechtcheriakov I W Graziadei A Kugener et al ldquoMotordysfunction in patients with liver cirrhosis impairment ofhandwritingrdquo Journal of Neurology vol 253 no 3 pp 349ndash3562006

[8] S J Gilberstadt H Gilberstadt L Zieve B Buegel R O CollierJr and C J McClain ldquoPsychomotor performance defects incirrhotic patients without overt encephalopathyrdquo Archives ofInternal Medicine vol 140 no 4 pp 519ndash521 1980

[9] V Felipo and R F Butterworth ldquoNeurobiology of ammoniardquoProgress in Neurobiology vol 67 no 4 pp 259ndash279 2002

[10] C C Gandhi R M Kelly R G Wiley and T J WalshldquoImpaired acquisition of a Morris water maze task followingselective destruction of cerebellar purkinje cells with OX7-saporinrdquo Behavioural Brain Research vol 109 no 1 pp 37ndash472000

[11] M G Leggio P Neri A Graziano L Mandolesi M Molinariand L Petrosini ldquoCerebellar contribution to spatial event pro-cessing characterization of procedural learningrdquo ExperimentalBrain Research vol 127 no 1 pp 1ndash11 1999

[12] S Singh R K Koiri and S K Trigun ldquoAcute and chronichyperammonemiamodulate antioxidant enzymes differently incerebral cortex and cerebellumrdquo Neurochemical Research vol33 no 1 pp 103ndash113 2008

[13] S Singh and S K Trigun ldquoActivation of neuronal nitric oxidesynthase in cerebellum of chronic hepatic encephalopathyrats is associated with up-regulation of NADPH-producingpathwayrdquo Cerebellum vol 9 no 3 pp 384ndash397 2010

[14] P Mondal and S K Trigun ldquoPannexin1 as a novel cerebraltarget in pathogenesis of hepatic encephalopathyrdquo MetabolicBrain Disease vol 29 no 4 pp 1007ndash1015 2014

[15] S Cull-Candy S Brickley and M Farrant ldquoNMDA receptorsubunits diversity development and diseaserdquo Current Opinionin Neurobiology vol 11 no 3 pp 327ndash335 2001

[16] Y Liu PW TakM Aarts et al ldquoNMDA receptor subunits havedifferential roles in mediating excitotoxic neuronal death bothin vitro and in vivordquoThe Journal of Neuroscience vol 27 no 11pp 2846ndash2857 2007

8 Evidence-Based Complementary and Alternative Medicine

[17] O A Shipton and O Paulsen ldquoGluN2A and GluN2B subunit-containing NMDA receptors in hippocampal plasticityrdquo Philo-sophical Transactions of the Royal Society B Biological Sciencesvol 369 no 1633 2014

[18] C Montoliu R Rodrigo P Monfort et al ldquoCyclic GMP path-ways in hepatic encephalopathy neurological and therapeuticimplicationsrdquoMetabolic Brain Disease vol 25 no 1 pp 39ndash482010

[19] M V Doucet A Harkin and K K Dev ldquoThe PSD-95nNOScomplex new drugs for depressionrdquo Pharmacology amp Thera-peutics vol 133 no 2 pp 218ndash229 2012

[20] H Prast andA Philippu ldquoNitric oxide asmodulator of neuronalfunctionrdquo Progress in Neurobiology vol 64 no 1 pp 51ndash682001

[21] F X Guix I Uribesalgo M Coma and F J Munoz ldquoThephysiology and pathophysiology of nitric oxide in the brainrdquoProgress in Neurobiology vol 76 no 2 pp 126ndash152 2005

[22] E A Waxman and D R Lynch ldquoN-methyl-D-aspartate recep-tor subtypes multiple roles in excitotoxicity and neurologicaldiseaserdquo Neuroscientist vol 11 no 1 pp 37ndash49 2005

[23] V Felipo ldquoContribution of altered signal transduction asso-ciated to glutamate receptors in brain to the neurologicalalterations of hepatic encephalopathyrdquo World Journal of Gas-troenterology vol 12 no 48 pp 7737ndash7743 2006

[24] M Llansola R Rodrigo P Monfort et al ldquoNMDA receptorsin hyperammonemia and hepatic encephalopathyrdquo MetabolicBrain Disease vol 22 no 3-4 pp 321ndash335 2007

[25] S A Lipton and P A Rosenberg ldquoMechanisms of disease exci-tatory amino acids as a final common pathway for neurologicdisordersrdquoTheNew England Journal of Medicine vol 330 no 9pp 613ndash622 1994

[26] D R Lynch and R P Guttmann ldquoExcitotoxicity perspectivesbased on N-methyl-D-aspartate receptor subtypesrdquo Journal ofPharmacology and Experimental Therapeutics vol 300 no 3pp 717ndash723 2002

[27] C Ikonomidou and L Turski ldquoWhy did NMDA receptorantagonists fail clinical trials for stroke and traumatic braininjuryrdquoThe Lancet Neurology vol 1 no 6 pp 383ndash386 2002

[28] K J Gohil and J J Patel ldquoA review onBacopamonniera currentresearch and future prospectsrdquo International Journal of GreenPharmacy vol 4 no 1 pp 1ndash9 2010

[29] A Krishnakumar P M Abraham J Paul and C S PauloseldquoDown-regulation of cerebellar 5-HT2C receptors inpilocarpine-induced epilepsy in rats therapeutic role ofBacopa monnieri extractrdquo Journal of the Neurological Sciencesvol 284 no 1-2 pp 124ndash128 2009

[30] M Singh V Murthy and C Ramassamy ldquoNeuroprotectivemechanisms of the standardized extract of Bacopa monnierain a paraquatdiquat-mediated acute toxicityrdquo NeurochemistryInternational vol 62 no 5 pp 530ndash539 2013

[31] N Uabundit J Wattanathorn S Mucimapura and K Ingkan-inan ldquoCognitive enhancement and neuroprotective effects ofBacopa monnieri in Alzheimerrsquos disease modelrdquo Journal ofEthnopharmacology vol 127 no 1 pp 26ndash31 2010

[32] R Khan A Krishnakumar and C S Paulose ldquoDecreasedglutamate receptor binding and NMDA R1 gene expression inhippocampus of pilocarpine-induced epileptic rats neuropro-tective role of Bacopa monnieri extractrdquo Epilepsy and Behaviorvol 12 no 1 pp 54ndash60 2008

[33] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[34] K V H Sastry R P Moudgal J Mohan J S Tyagi and G SRao ldquoSpectrophotometric determination of serum nitrite andnitrate by copper-cadmium alloyrdquo Analytical Biochemistry vol306 no 1 pp 79ndash82 2002

[35] D R Lynch and R P Guttmann ldquoNMDA receptor pharma-cology perspectives from molecular biologyrdquo Current DrugTargets vol 2 no 3 pp 215ndash231 2001

[36] L V Kalia S K Kalia and M W Salter ldquoNMDA receptorsin clinical neurology excitatory times aheadrdquo The LancetNeurology vol 7 no 8 pp 742ndash755 2008

[37] A Lau and M Tymianski ldquoGlutamate receptors neurotoxicityand neurodegenerationrdquo Pflugers Archiv European Journal ofPhysiology vol 460 no 2 pp 525ndash542 2010

[38] M M Zeron O Hansson N Chen et al ldquoIncreased sensitivityto N-methyl-D-aspartate receptor-mediated excitotoxicity in amousemodel ofHuntingtonrsquos diseaserdquoNeuron vol 33 no 6 pp849ndash860 2002

[39] A J Williams J R Dave X M Lu G Ling and F C TortellaldquoSelective NR2B NMDA receptor antagonists are protectiveagainst staurosporine-induced apoptosisrdquo European Journal ofPharmacology vol 452 no 1 pp 135ndash136 2002

[40] M Chen T-J Lu X-J Chen et al ldquoDifferential roles of NMDAreceptor subtypes in ischemic neuronal cell death and ischemictolerancerdquo Stroke vol 39 no 11 pp 3042ndash3048 2008

[41] K Erreger S M Dravid T G Banke D J A Wyllie and SF Traynelis ldquoSubunit-specific gating controls rat NR1NR2AandNR1NR2BNMDAchannel kinetics and synaptic signallingprofilesrdquoThe Journal of Physiology vol 563 no 2 pp 345ndash3582005

[42] C S Paulose F Chathu S Reas Khan and A KrishnakumarldquoNeuroprotective role of Bacopa monnieri extract in epilepsyand effect of glucose supplementation during hypoxia gluta-mate receptor gene expressionrdquoNeurochemical Research vol 33no 9 pp 1663ndash1671 2008

[43] V L R Rao R M Audet and R F Butterworth ldquoIncreasednitric oxide synthase activities and L-[3H]arginine uptake inbrain following portacaval anastomosisrdquo Journal of Neurochem-istry vol 65 no 2 pp 677ndash681 1995

[44] P Monfort M-D Munoz A ElAyadi E Kosenko and VFelipo ldquoEffects of hyperammonemia and liver failure on glu-tamatergic neurotransmissionrdquoMetabolic Brain Disease vol 17no 4 pp 237ndash250 2002

[45] C M Snyder E H Shroff J Liu and N S Chandel ldquoNitricoxide induces cell death by regulating anti-apoptotic Bcl2 familymembersrdquo PLoS ONE vol 4 no 9 Article ID e7059 2009

[46] R K Koiri and S K Trigun ldquoDimethyl sulfoxide acti-vates tumor necrosis factor 120572-p53 mediated apoptosis anddown regulates D-fructose-6-phosphate-2-kinase and lactatedehydrogenase-5 in Daltonrsquos Lymphoma in vivordquo LeukemiaResearch vol 35 no 7 pp 950ndash956 2011

[47] R B Thomas S Joy M S Ajayan and C S Paulose ldquoNeuro-protective potential of bacopa monnieri and bacoside a againstdopamine receptor dysfunction in the cerebral cortex of neona-tal hypoglycaemic ratsrdquo Cellular and Molecular Neurobiologyvol 33 no 8 pp 1065ndash1074 2013

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Research and TreatmentAIDS

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

gum acacia once daily starting from 8th day onwards till14th day and were administered 4 h after the TAA treatmentThe dose of BM was selected which was able to recover TAAinduced neurobehavioural deficit in the rats All the rats weresacrificed on 15th day The cerebellum was dissected out andstored at minus80∘C for further experiments

24 Preparation of Cerebellar Extracts As described earlier[13] mitochondria free cerebellar extracts were prepared inan extraction medium consisting of 400mM sucrose 1mMEDTA 02mM benzamidine 01mM phenylmethylsulfonylfluoride (PMSF) and 002 heparin The extracts werecentrifuged initially at 12000timesg for 15min and finally at19000timesg for 45min at 4∘C The supernatant obtained wascollected as the cytosolic fractionsThe protein content in thetissue extract wasmeasured by Lowrymethod [33] using BSAas standard

25 Western Blotting As described previously [13] thecytosolic fractions containing 60 120583g proteinlane were sepa-rated on 10 SDS-PAGE and electrotransferred to nitrocel-lulose membrane at 50mA and run overnight at 4∘C Proteintransfer was checked via Ponceau staining The membranewas then placed in a blocking solution of 5 skimmed milkin 1X PBS for 2 h followed by washing in PBS 3 times Themembranes were then separately processed for immunode-tection of NR2A NR2B nNOS Bcl2 and Bax using mono-clonalpolyclonal anti-NR2A (1 1000) anti-NR2B (1 1000)anti-nNOS (1 500) anti-Bcl2 (1 1000) and anti-Bax (1 500)respectively HRP conjugated secondary antibody was usedfor final detection of the proteins using ECL western blottingdetection kit 120573-actin used as loading control was detectedusing a monoclonal anti-120573-actin peroxidase antibody Thebands were quantified and analyzed using gel densitometrysoftware AlphaImager 2200 The photographs in the figureare representatives of the three western blot repeats

26 Nitric Oxide (NO) Estimation Nitric oxide level wasmeasured by estimating total nitrite (NO

2) and nitrate (NO

3)

content in the tissue extracts as described earlier [13] usingthe method of Sastry et al [34] Briefly tissue fractions(100 120583L) NaNO

2 and KNO

3standards (01mM each) were

mixed separately with 400 120583L of 50mM carbonate buffer(pH 9) For NO

3estimation activated copper-cadmium alloy

(150mg) was added and incubated for an hour at 37∘C Thereaction was stopped using 100 120583L each of 035M NaOHand 120mM zinc sulphate After centrifugation 400 120583Lsupernatant was incubatedwith theGriess reagent 200120583L 1sulphanilamide prepared in 25 H

3PO4and 200120583L 01 N-

(1-naphthyl)-ethylenediamine Absorbance was recorded at545 nm For NO

2estimation similar procedure was followed

except addition of the copper-cadmium alloy

27 Statistical Analysis The data have been expressed asmean plusmn SD For two group comparisons statistical analysiswas performed using unpaired Studentrsquos 119905 test A probabilityof 119875 lt 005 was taken as a significant difference between thegroups Each of the experiments was repeated thrice

3 Results

31 Effect of BM Extract on NR2A and NR2B ExpressionThe combination of constituent NMDAR subunits is evidentto impart unique neurophysiological role of this glutamatereceptor The NR2A dominating combination is known tomediate neuroprotectionwhereas those of NR2B induce neu-ronal death According to Figures 1(a) and 1(b) as comparedto the control rats level of NR2A is found to be significantlyreduced (119875 lt 0001) with a concomitant increase inNR2B level in the cerebellum of the CLF rats (Figures 1(c)and 1(d)) resulting in a significant decline in NR2A2Bratio (Figure 1(e)) However this pattern is observed to berecovered back with a significant enhancement of NR2A(Figures 1(a) and 1(b)) and a decline of NR2B (Figures 1(c)and 1(d)) resulting into attaining a control level NR2A2Bratio (Figure 1(e)) in the cerebellum of the CLF rats treatedwith the BM extract

32 Effect of BM Extract on nNOS and Nitric Oxide (NO)Production Neuronal NOS (nNOS) has a direct molecularlink with NMDAR and therefore it is considered to bethe main determinant of NMDAR activation based down-stream signaling in the postsynaptic neurons Accordinglyoveractivation of nNOS is considered associated with theneuronal changes associated with NMDAR led excitotoxicityAs depicted in Figures 2(a) and 2(b) nNOS expression isobserved to be enhanced significantly (119875 lt 0001) in thecerebellum of the CLF rats as compared to the control grouprats Moreover due to the oral administration of BM extractlevel of this enzyme is observed to be reduced up to thecontrol value (Figures 2(a) and 2(b)) Such a pattern couldcoincide well with the similar changes in the NO level incerebellum of the CLF and BM extract treated CLF rats(Figure 2(c))

33 Effect of BM Extract on the Expression of Bcl2 and BaxBcl2 (antiapoptotic) and Bax (proapoptotic) ratio servesas a rheostat to determine cell susceptibility to apoptosisAccording to Figures 3(a) and 3(c) as compared to the controlgroup rats level of Bcl2 is found to be declined significantly(119875 lt 005) with a concomitant increase in the Bax levelresulting in a significant decline (119875 lt 001) in Bcl2Bax ratio(Figure 3(e)) in cerebellum of the CLF rats However aftertreatment with BM extract the pattern of Bcl2Bax ratio isobserved to regain its normal range in cerebellum of thoseCLF rats (Figure 3(e))

4 Discussion

NMDA receptor overactivation is considered a commonneurochemical event associated with a number of braindysfunctions like epilepsy ischemia drug abuse and HE[23 26 35ndash37] However NMDAR blockage is evident tobe a poor therapeutic target for managing such excitotoxicconditions [25 27] In this background while understandingneurochemical basis of CLF induced HE we observed a clear

4 Evidence-Based Complementary and Alternative Medicine

NR2A

120573-actin

C CLF CLF + BM

(a)

Den

sitom

etric

val

ue (N

R2A

120573-a

ctin

)

000

020

040

060

080

100

120

140

C CLF CLF + BM

lowastlowastlowast

(b)

NR2B

120573-actin

C CLF CLF + BM

(c)

000

020

040

060

080

100

120

140

160

C CLF

D

ensit

omet

ric v

alue

(NR2

B120573

-act

in)

CLF + BM

lowastlowastlowast

(d)

000020040060080100120140160

Den

sitom

etric

val

ue (N

R2A

NR2

B)

C CLF CLF + BM

lowastlowastlowast

(e)

Figure 1 Expression profile of NR2A ((a) and (b)) andNR2B ((c) and (d)) in cerebellum of the CLF and BM extract treated CLF ratsWesternblot analysis was performed as described in the method textThe level of 120573-actin was probed as the loading control In panels (b) (d) and (e)normalized densitometric values of NR2A120573-actin NR2B120573-actin and NR2ANR2B have been presented as mean plusmn SD from three westernblot repeats lowastlowastlowast119875 lt 0001 (control versus CLF rats) 119875 lt 001 119875 lt 0001 (CLF versus CLF + BM rats) C control CLF chronic liverfailure CLF + BM chronic liver failure + Bacopa monnieri extract

shift in the expression of glutamate binding NMDAR subunitfrom a NR2A dominating combination to a NR2B rich com-bination in cerebellum of the CLF rats (Figures 1(a) and 1(c))It is now becoming clearer that NR2A rich NMDAR impartsneuroprotection and that of NR2B dominating combinationinitiates neuronal damage and apoptosis [16 38ndash40] This isbecause NR2B has been reported to show delay in gatingkinetics in comparison to that of NR1NR2A combinationresulting in increased Ca2+ influx and thus rapid activation

of the downstream signaling [41] In the present context asignificant decline in Bcl2 with a concomitant increase in Baxlevel in cerebellum of the CLF rats (Figure 3) also hints for adirect association between NR2B dominating NMDAR com-position and altered neurochemistry of cerebellumof theCLFrats Furthermore alterations in oxidative and nitrosativefactors in the postsynaptic neurons are considered to bethe main downstream mediators of such unusual NMDARactivations [1] and it has been reported that cerebellum shows

Evidence-Based Complementary and Alternative Medicine 5

nNOS

120573-actin

C CLF CLF + BM

(a)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (n

NO

S120573

-act

in)

lowastlowastlowast

C CLF CLF + BM(b)

00

10

20

30

40

50

60

C CLF

lowastlowast

NO

(120583m

olm

g pr

otei

n)

CLF + BM

(c)

Figure 2 Expression profile of nNOS ((a) and (b)) and NO level (c) in cerebellum of the CLF and BM extract treated CLF rats Western blotanalysis of nNOS was performed as described in the text of methods In panel (b) normalized densitometric values of nNOS120573-actin havebeen presented as mean plusmn SD from three western blot repeats lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (control versus CLF rats) 119875 lt 005 119875 lt 0001(CLF versus CLF + BM rats) C control CLF chronic liver failure CLF + BM chronic liver failure + Bacopa monnieri extract

greater susceptibility to undergo oxidative and nitrosativestress during HA and CLF led excitotoxicity [12 13]

It has been described that lesion in cerebellum impairsacquisition of memory consolidation and deranges motorfunctions [10 11] The CLF rats used in this experimenthave recently been reported to show cognitive impairmentand deficit in motor functions as well [2] Since such neu-rochemical alterations leading to neurobehavioural changesare known to emanate from abnormal NMDAR activity [1]it is argued that a shift from a NR2A combination to aNR2B combination of NMDAR in the cerebellum of theCLF rats might be accountable for developing HE associatedsymptoms observed in these CLF rats [2] Moreover keepingaside these explanations the findings provided a basis to alterNMDAR constitution instead of shutting this ion-channeloff as a therapeutic option to bring recovery from the HEsymptoms

To test this hypothesis BM extract was chosen due to thetwo reasons Firstly amidst scarcity of the safer neurophar-macological agents this plant extract is demonstrated toimprove neuronal functions by modulating brain chemistryin many ways [28] Secondly BM is now evident to modulateactivity of the neurotransmitter receptors like serotonergicreceptors during epilepsy [29] and dopaminergic and cholin-ergic signaling in Parkinsonrsquos and Alzheimerrsquos diseases [3031] However report is limited on modulation of NMDAreceptor activity by BMWe observed a remarkable shift froma declined ratio of NR2ANR2B (neurodegeneration sup-portive combination) in the cerebellum of CLF rats towardstheir normal level when these CLF rats were administeredwith BM extract (Figure 1(e)) This finding suggested thatBM is able to alter constitution of the functional NMDARtetramer by differential expression of the two glutamatebinding subunits in cerebellum of the CLF rats Indeed in

6 Evidence-Based Complementary and Alternative Medicine

Bcl2

C CLF CLF + BM

120573-actin

(a)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (B

cl2120573

-act

in)

C CLF CLF + BM

lowast

(b)

Bax

C CLF CLF + BM

120573-actin

(c)

000

020

040

060

080

100

120

140

160

Den

sitom

etric

val

ue (B

ax120573

-act

in)

C CLF CLF + BM

lowastlowast

(d)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (B

cl2B

ax)

C CLF CLF + BM

lowastlowast

(e)

Figure 3 Expression profile of Bcl2 ((a) and (b)) and Bax ((c) and (d)) in cerebellum of the CLF and BM extract treated CLF rats Westernblot analysis of Bcl2 and Bax was performed as described in the text of methods The level of 120573-actin was probed as the loading control Inpanels (b) (d) and (e) normalized densitometric values of Bcl2120573-actin Bax120573-actin and Bcl2Bax ratio respectively have been presentedas mean plusmn SD from three western blot repeats lowast119875 lt 005 lowastlowast119875 lt 001 (control versus CLF rats) 119875 lt 005 119875 lt 001 (CLF versus CLF +BM rats) C control CLF chronic liver failure CLF + BM chronic liver failure + Bacopa monnieri extract

an epilepsy model it has been demonstrated that BM couldalter NR1 gene expression [32] thus suggesting BM as amod-ulator of NMDAR subunit expression A similar finding onmodulation of NR1 by BM was reported in a hypoxia modelalso [42] Importantly such a change in NR1 expression wasfound accountable for modulating glutamatergic signaling incerebellum of those rats [32] Thus it is argued that shiftingfrom a neurodegenerative NR2B overexpression towards

the neuroprotective NR2A level due to the treatment withBME (Figure 1) could also account for preventing down-stream undesirable neurochemical changes in cerebellum ofthe CLF rats

In a number of excitotoxic models including CLF ledHE nNOS activation is considered as the most commonevent after NMDAR activation [13 43] It is initiated by influxof Ca2+ which overactivates this enzyme to produce excess

Evidence-Based Complementary and Alternative Medicine 7

NO in the postsynaptic neurons [44] NO is a moleculeof pleiotropic effects however when produced in excessin the brain cells it uses more than one mechanism toinduce neuronal dysfunction [20 21] A milder increase inNO level is likely to initiate mitochondrial dysfunction ledneuronal apoptosis Ratio of Bcl2 versus Bax is consideredas the most effective regulators of mitochondrial dysfunctionled apoptosis [45] and hence relative levels of both theseproteins are considered as a reliable tool to assay whether acell is likely to undergo internal apoptotic process [46] Incerebellum of the CLF rats a significantly increased level ofnNOS coincides well with a similar increment in NO level(Figure 2) This is consistent with a significant decline in theBcl2Bax ratio (Figure 3) Moreover all these factors werefound to be reversed to regain their normal levels in thecerebellumwhen thoseCLF rats were treatedwith BMextract(Figures 1ndash3) Recent studies have also shown that BM extractdownregulates Bax and upregulates Bcl2 and thus providesneuroprotection in several neurological disease models [47]Thus the findings of Figures 1ndash3 together advocate forconcordant modulation of NMDA receptor constitution andnNOS led apoptotic activation by ethanolic extract of BM(CDRI-08) in cerebellum of the CLF rats

5 Conclusion

Opposing roles of the two main glutamate binding subunitsNR2A and NR2B of NMDA receptor advocate for modu-lation of NMDAR constitution as an effective mechanismto normalize its excitotoxic effects without blocking its ion-channel activity During recent past studies on efficacy ofBacopa monnieri extract have been shown to prevent neu-rodegenerative diseases by modulating neurochemistry ofthe brain cells but with little information about modulationof NMDAR overactivation led excitotoxicity The presentfindings demonstrate that BM extract is able to modulateNMDA receptor signaling by bringing reciprocal changes inthe expression of its two glutamate binding subunits NR2Aand NR2B and thus provide a novel approach to normalizeNMDAR overactivation effects without blocking this ionchannel

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The work was financially supported by DBT Grant (BTPR14049Med303442010) to Surendra Kumar Trigun andthe award of CSIR SRF to Papia Mondal The facilities ofDST-FIST and UGC-CAS programs in the Department ofZoology and BHU-DBT ISLS facilities in Faculty of Scienceare also acknowledged The authors thank Professor HemantSingh Lumen Research Foundation Chennai for providingthe ethanolic extract of Bacopa monnieri (CDRI-08)

References

[1] V Felipo ldquoHyperammonemiardquo inHandbook of Neurochemistryand Molecular Neurobiology Brain and Spinal Cord Trauma Alaztha N Banik and S K Ray Eds pp 43ndash69 Springer BerlinGermany 3rd edition 2009

[2] S Singh and S K Trigun ldquoLow grade cirrhosis inducescognitive impairment and motor dysfunction in rats couldbe a model for minimal hepatic encephalopathyrdquo NeuroscienceLetters vol 559 pp 136ndash140 2014

[3] A T Blei J Cordoba and The Practice Parameters Com-mittee of the American College of Gastroenterology ldquoHepaticencephalopathyrdquo American Journal of Gastroenterology vol 96pp 635ndash643 2001

[4] J S Bajaj J B Wade and A J Sanyal ldquoSpectrum of neurocog-nitive impairment in cirrhosis implications for the assessmentof hepatic encephalopathyrdquoHepatology vol 50 no 6 pp 2014ndash2021 2009

[5] F F Poordad ldquoReview article the burden of hepaticencephalopathyrdquo Alimentary Pharmacology and Therapeuticsvol 25 no 1 pp 3ndash9 2007

[6] J S Bajaj ldquoMinimal hepatic encephalopathy matters in dailyliferdquoWorld Journal of Gastroenterology vol 14 no 23 pp 3609ndash3615 2008

[7] S Mechtcheriakov I W Graziadei A Kugener et al ldquoMotordysfunction in patients with liver cirrhosis impairment ofhandwritingrdquo Journal of Neurology vol 253 no 3 pp 349ndash3562006

[8] S J Gilberstadt H Gilberstadt L Zieve B Buegel R O CollierJr and C J McClain ldquoPsychomotor performance defects incirrhotic patients without overt encephalopathyrdquo Archives ofInternal Medicine vol 140 no 4 pp 519ndash521 1980

[9] V Felipo and R F Butterworth ldquoNeurobiology of ammoniardquoProgress in Neurobiology vol 67 no 4 pp 259ndash279 2002

[10] C C Gandhi R M Kelly R G Wiley and T J WalshldquoImpaired acquisition of a Morris water maze task followingselective destruction of cerebellar purkinje cells with OX7-saporinrdquo Behavioural Brain Research vol 109 no 1 pp 37ndash472000

[11] M G Leggio P Neri A Graziano L Mandolesi M Molinariand L Petrosini ldquoCerebellar contribution to spatial event pro-cessing characterization of procedural learningrdquo ExperimentalBrain Research vol 127 no 1 pp 1ndash11 1999

[12] S Singh R K Koiri and S K Trigun ldquoAcute and chronichyperammonemiamodulate antioxidant enzymes differently incerebral cortex and cerebellumrdquo Neurochemical Research vol33 no 1 pp 103ndash113 2008

[13] S Singh and S K Trigun ldquoActivation of neuronal nitric oxidesynthase in cerebellum of chronic hepatic encephalopathyrats is associated with up-regulation of NADPH-producingpathwayrdquo Cerebellum vol 9 no 3 pp 384ndash397 2010

[14] P Mondal and S K Trigun ldquoPannexin1 as a novel cerebraltarget in pathogenesis of hepatic encephalopathyrdquo MetabolicBrain Disease vol 29 no 4 pp 1007ndash1015 2014

[15] S Cull-Candy S Brickley and M Farrant ldquoNMDA receptorsubunits diversity development and diseaserdquo Current Opinionin Neurobiology vol 11 no 3 pp 327ndash335 2001

[16] Y Liu PW TakM Aarts et al ldquoNMDA receptor subunits havedifferential roles in mediating excitotoxic neuronal death bothin vitro and in vivordquoThe Journal of Neuroscience vol 27 no 11pp 2846ndash2857 2007

8 Evidence-Based Complementary and Alternative Medicine

[17] O A Shipton and O Paulsen ldquoGluN2A and GluN2B subunit-containing NMDA receptors in hippocampal plasticityrdquo Philo-sophical Transactions of the Royal Society B Biological Sciencesvol 369 no 1633 2014

[18] C Montoliu R Rodrigo P Monfort et al ldquoCyclic GMP path-ways in hepatic encephalopathy neurological and therapeuticimplicationsrdquoMetabolic Brain Disease vol 25 no 1 pp 39ndash482010

[19] M V Doucet A Harkin and K K Dev ldquoThe PSD-95nNOScomplex new drugs for depressionrdquo Pharmacology amp Thera-peutics vol 133 no 2 pp 218ndash229 2012

[20] H Prast andA Philippu ldquoNitric oxide asmodulator of neuronalfunctionrdquo Progress in Neurobiology vol 64 no 1 pp 51ndash682001

[21] F X Guix I Uribesalgo M Coma and F J Munoz ldquoThephysiology and pathophysiology of nitric oxide in the brainrdquoProgress in Neurobiology vol 76 no 2 pp 126ndash152 2005

[22] E A Waxman and D R Lynch ldquoN-methyl-D-aspartate recep-tor subtypes multiple roles in excitotoxicity and neurologicaldiseaserdquo Neuroscientist vol 11 no 1 pp 37ndash49 2005

[23] V Felipo ldquoContribution of altered signal transduction asso-ciated to glutamate receptors in brain to the neurologicalalterations of hepatic encephalopathyrdquo World Journal of Gas-troenterology vol 12 no 48 pp 7737ndash7743 2006

[24] M Llansola R Rodrigo P Monfort et al ldquoNMDA receptorsin hyperammonemia and hepatic encephalopathyrdquo MetabolicBrain Disease vol 22 no 3-4 pp 321ndash335 2007

[25] S A Lipton and P A Rosenberg ldquoMechanisms of disease exci-tatory amino acids as a final common pathway for neurologicdisordersrdquoTheNew England Journal of Medicine vol 330 no 9pp 613ndash622 1994

[26] D R Lynch and R P Guttmann ldquoExcitotoxicity perspectivesbased on N-methyl-D-aspartate receptor subtypesrdquo Journal ofPharmacology and Experimental Therapeutics vol 300 no 3pp 717ndash723 2002

[27] C Ikonomidou and L Turski ldquoWhy did NMDA receptorantagonists fail clinical trials for stroke and traumatic braininjuryrdquoThe Lancet Neurology vol 1 no 6 pp 383ndash386 2002

[28] K J Gohil and J J Patel ldquoA review onBacopamonniera currentresearch and future prospectsrdquo International Journal of GreenPharmacy vol 4 no 1 pp 1ndash9 2010

[29] A Krishnakumar P M Abraham J Paul and C S PauloseldquoDown-regulation of cerebellar 5-HT2C receptors inpilocarpine-induced epilepsy in rats therapeutic role ofBacopa monnieri extractrdquo Journal of the Neurological Sciencesvol 284 no 1-2 pp 124ndash128 2009

[30] M Singh V Murthy and C Ramassamy ldquoNeuroprotectivemechanisms of the standardized extract of Bacopa monnierain a paraquatdiquat-mediated acute toxicityrdquo NeurochemistryInternational vol 62 no 5 pp 530ndash539 2013

[31] N Uabundit J Wattanathorn S Mucimapura and K Ingkan-inan ldquoCognitive enhancement and neuroprotective effects ofBacopa monnieri in Alzheimerrsquos disease modelrdquo Journal ofEthnopharmacology vol 127 no 1 pp 26ndash31 2010

[32] R Khan A Krishnakumar and C S Paulose ldquoDecreasedglutamate receptor binding and NMDA R1 gene expression inhippocampus of pilocarpine-induced epileptic rats neuropro-tective role of Bacopa monnieri extractrdquo Epilepsy and Behaviorvol 12 no 1 pp 54ndash60 2008

[33] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[34] K V H Sastry R P Moudgal J Mohan J S Tyagi and G SRao ldquoSpectrophotometric determination of serum nitrite andnitrate by copper-cadmium alloyrdquo Analytical Biochemistry vol306 no 1 pp 79ndash82 2002

[35] D R Lynch and R P Guttmann ldquoNMDA receptor pharma-cology perspectives from molecular biologyrdquo Current DrugTargets vol 2 no 3 pp 215ndash231 2001

[36] L V Kalia S K Kalia and M W Salter ldquoNMDA receptorsin clinical neurology excitatory times aheadrdquo The LancetNeurology vol 7 no 8 pp 742ndash755 2008

[37] A Lau and M Tymianski ldquoGlutamate receptors neurotoxicityand neurodegenerationrdquo Pflugers Archiv European Journal ofPhysiology vol 460 no 2 pp 525ndash542 2010

[38] M M Zeron O Hansson N Chen et al ldquoIncreased sensitivityto N-methyl-D-aspartate receptor-mediated excitotoxicity in amousemodel ofHuntingtonrsquos diseaserdquoNeuron vol 33 no 6 pp849ndash860 2002

[39] A J Williams J R Dave X M Lu G Ling and F C TortellaldquoSelective NR2B NMDA receptor antagonists are protectiveagainst staurosporine-induced apoptosisrdquo European Journal ofPharmacology vol 452 no 1 pp 135ndash136 2002

[40] M Chen T-J Lu X-J Chen et al ldquoDifferential roles of NMDAreceptor subtypes in ischemic neuronal cell death and ischemictolerancerdquo Stroke vol 39 no 11 pp 3042ndash3048 2008

[41] K Erreger S M Dravid T G Banke D J A Wyllie and SF Traynelis ldquoSubunit-specific gating controls rat NR1NR2AandNR1NR2BNMDAchannel kinetics and synaptic signallingprofilesrdquoThe Journal of Physiology vol 563 no 2 pp 345ndash3582005

[42] C S Paulose F Chathu S Reas Khan and A KrishnakumarldquoNeuroprotective role of Bacopa monnieri extract in epilepsyand effect of glucose supplementation during hypoxia gluta-mate receptor gene expressionrdquoNeurochemical Research vol 33no 9 pp 1663ndash1671 2008

[43] V L R Rao R M Audet and R F Butterworth ldquoIncreasednitric oxide synthase activities and L-[3H]arginine uptake inbrain following portacaval anastomosisrdquo Journal of Neurochem-istry vol 65 no 2 pp 677ndash681 1995

[44] P Monfort M-D Munoz A ElAyadi E Kosenko and VFelipo ldquoEffects of hyperammonemia and liver failure on glu-tamatergic neurotransmissionrdquoMetabolic Brain Disease vol 17no 4 pp 237ndash250 2002

[45] C M Snyder E H Shroff J Liu and N S Chandel ldquoNitricoxide induces cell death by regulating anti-apoptotic Bcl2 familymembersrdquo PLoS ONE vol 4 no 9 Article ID e7059 2009

[46] R K Koiri and S K Trigun ldquoDimethyl sulfoxide acti-vates tumor necrosis factor 120572-p53 mediated apoptosis anddown regulates D-fructose-6-phosphate-2-kinase and lactatedehydrogenase-5 in Daltonrsquos Lymphoma in vivordquo LeukemiaResearch vol 35 no 7 pp 950ndash956 2011

[47] R B Thomas S Joy M S Ajayan and C S Paulose ldquoNeuro-protective potential of bacopa monnieri and bacoside a againstdopamine receptor dysfunction in the cerebral cortex of neona-tal hypoglycaemic ratsrdquo Cellular and Molecular Neurobiologyvol 33 no 8 pp 1065ndash1074 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

NR2A

120573-actin

C CLF CLF + BM

(a)

Den

sitom

etric

val

ue (N

R2A

120573-a

ctin

)

000

020

040

060

080

100

120

140

C CLF CLF + BM

lowastlowastlowast

(b)

NR2B

120573-actin

C CLF CLF + BM

(c)

000

020

040

060

080

100

120

140

160

C CLF

D

ensit

omet

ric v

alue

(NR2

B120573

-act

in)

CLF + BM

lowastlowastlowast

(d)

000020040060080100120140160

Den

sitom

etric

val

ue (N

R2A

NR2

B)

C CLF CLF + BM

lowastlowastlowast

(e)

Figure 1 Expression profile of NR2A ((a) and (b)) andNR2B ((c) and (d)) in cerebellum of the CLF and BM extract treated CLF ratsWesternblot analysis was performed as described in the method textThe level of 120573-actin was probed as the loading control In panels (b) (d) and (e)normalized densitometric values of NR2A120573-actin NR2B120573-actin and NR2ANR2B have been presented as mean plusmn SD from three westernblot repeats lowastlowastlowast119875 lt 0001 (control versus CLF rats) 119875 lt 001 119875 lt 0001 (CLF versus CLF + BM rats) C control CLF chronic liverfailure CLF + BM chronic liver failure + Bacopa monnieri extract

shift in the expression of glutamate binding NMDAR subunitfrom a NR2A dominating combination to a NR2B rich com-bination in cerebellum of the CLF rats (Figures 1(a) and 1(c))It is now becoming clearer that NR2A rich NMDAR impartsneuroprotection and that of NR2B dominating combinationinitiates neuronal damage and apoptosis [16 38ndash40] This isbecause NR2B has been reported to show delay in gatingkinetics in comparison to that of NR1NR2A combinationresulting in increased Ca2+ influx and thus rapid activation

of the downstream signaling [41] In the present context asignificant decline in Bcl2 with a concomitant increase in Baxlevel in cerebellum of the CLF rats (Figure 3) also hints for adirect association between NR2B dominating NMDAR com-position and altered neurochemistry of cerebellumof theCLFrats Furthermore alterations in oxidative and nitrosativefactors in the postsynaptic neurons are considered to bethe main downstream mediators of such unusual NMDARactivations [1] and it has been reported that cerebellum shows

Evidence-Based Complementary and Alternative Medicine 5

nNOS

120573-actin

C CLF CLF + BM

(a)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (n

NO

S120573

-act

in)

lowastlowastlowast

C CLF CLF + BM(b)

00

10

20

30

40

50

60

C CLF

lowastlowast

NO

(120583m

olm

g pr

otei

n)

CLF + BM

(c)

Figure 2 Expression profile of nNOS ((a) and (b)) and NO level (c) in cerebellum of the CLF and BM extract treated CLF rats Western blotanalysis of nNOS was performed as described in the text of methods In panel (b) normalized densitometric values of nNOS120573-actin havebeen presented as mean plusmn SD from three western blot repeats lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (control versus CLF rats) 119875 lt 005 119875 lt 0001(CLF versus CLF + BM rats) C control CLF chronic liver failure CLF + BM chronic liver failure + Bacopa monnieri extract

greater susceptibility to undergo oxidative and nitrosativestress during HA and CLF led excitotoxicity [12 13]

It has been described that lesion in cerebellum impairsacquisition of memory consolidation and deranges motorfunctions [10 11] The CLF rats used in this experimenthave recently been reported to show cognitive impairmentand deficit in motor functions as well [2] Since such neu-rochemical alterations leading to neurobehavioural changesare known to emanate from abnormal NMDAR activity [1]it is argued that a shift from a NR2A combination to aNR2B combination of NMDAR in the cerebellum of theCLF rats might be accountable for developing HE associatedsymptoms observed in these CLF rats [2] Moreover keepingaside these explanations the findings provided a basis to alterNMDAR constitution instead of shutting this ion-channeloff as a therapeutic option to bring recovery from the HEsymptoms

To test this hypothesis BM extract was chosen due to thetwo reasons Firstly amidst scarcity of the safer neurophar-macological agents this plant extract is demonstrated toimprove neuronal functions by modulating brain chemistryin many ways [28] Secondly BM is now evident to modulateactivity of the neurotransmitter receptors like serotonergicreceptors during epilepsy [29] and dopaminergic and cholin-ergic signaling in Parkinsonrsquos and Alzheimerrsquos diseases [3031] However report is limited on modulation of NMDAreceptor activity by BMWe observed a remarkable shift froma declined ratio of NR2ANR2B (neurodegeneration sup-portive combination) in the cerebellum of CLF rats towardstheir normal level when these CLF rats were administeredwith BM extract (Figure 1(e)) This finding suggested thatBM is able to alter constitution of the functional NMDARtetramer by differential expression of the two glutamatebinding subunits in cerebellum of the CLF rats Indeed in

6 Evidence-Based Complementary and Alternative Medicine

Bcl2

C CLF CLF + BM

120573-actin

(a)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (B

cl2120573

-act

in)

C CLF CLF + BM

lowast

(b)

Bax

C CLF CLF + BM

120573-actin

(c)

000

020

040

060

080

100

120

140

160

Den

sitom

etric

val

ue (B

ax120573

-act

in)

C CLF CLF + BM

lowastlowast

(d)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (B

cl2B

ax)

C CLF CLF + BM

lowastlowast

(e)

Figure 3 Expression profile of Bcl2 ((a) and (b)) and Bax ((c) and (d)) in cerebellum of the CLF and BM extract treated CLF rats Westernblot analysis of Bcl2 and Bax was performed as described in the text of methods The level of 120573-actin was probed as the loading control Inpanels (b) (d) and (e) normalized densitometric values of Bcl2120573-actin Bax120573-actin and Bcl2Bax ratio respectively have been presentedas mean plusmn SD from three western blot repeats lowast119875 lt 005 lowastlowast119875 lt 001 (control versus CLF rats) 119875 lt 005 119875 lt 001 (CLF versus CLF +BM rats) C control CLF chronic liver failure CLF + BM chronic liver failure + Bacopa monnieri extract

an epilepsy model it has been demonstrated that BM couldalter NR1 gene expression [32] thus suggesting BM as amod-ulator of NMDAR subunit expression A similar finding onmodulation of NR1 by BM was reported in a hypoxia modelalso [42] Importantly such a change in NR1 expression wasfound accountable for modulating glutamatergic signaling incerebellum of those rats [32] Thus it is argued that shiftingfrom a neurodegenerative NR2B overexpression towards

the neuroprotective NR2A level due to the treatment withBME (Figure 1) could also account for preventing down-stream undesirable neurochemical changes in cerebellum ofthe CLF rats

In a number of excitotoxic models including CLF ledHE nNOS activation is considered as the most commonevent after NMDAR activation [13 43] It is initiated by influxof Ca2+ which overactivates this enzyme to produce excess

Evidence-Based Complementary and Alternative Medicine 7

NO in the postsynaptic neurons [44] NO is a moleculeof pleiotropic effects however when produced in excessin the brain cells it uses more than one mechanism toinduce neuronal dysfunction [20 21] A milder increase inNO level is likely to initiate mitochondrial dysfunction ledneuronal apoptosis Ratio of Bcl2 versus Bax is consideredas the most effective regulators of mitochondrial dysfunctionled apoptosis [45] and hence relative levels of both theseproteins are considered as a reliable tool to assay whether acell is likely to undergo internal apoptotic process [46] Incerebellum of the CLF rats a significantly increased level ofnNOS coincides well with a similar increment in NO level(Figure 2) This is consistent with a significant decline in theBcl2Bax ratio (Figure 3) Moreover all these factors werefound to be reversed to regain their normal levels in thecerebellumwhen thoseCLF rats were treatedwith BMextract(Figures 1ndash3) Recent studies have also shown that BM extractdownregulates Bax and upregulates Bcl2 and thus providesneuroprotection in several neurological disease models [47]Thus the findings of Figures 1ndash3 together advocate forconcordant modulation of NMDA receptor constitution andnNOS led apoptotic activation by ethanolic extract of BM(CDRI-08) in cerebellum of the CLF rats

5 Conclusion

Opposing roles of the two main glutamate binding subunitsNR2A and NR2B of NMDA receptor advocate for modu-lation of NMDAR constitution as an effective mechanismto normalize its excitotoxic effects without blocking its ion-channel activity During recent past studies on efficacy ofBacopa monnieri extract have been shown to prevent neu-rodegenerative diseases by modulating neurochemistry ofthe brain cells but with little information about modulationof NMDAR overactivation led excitotoxicity The presentfindings demonstrate that BM extract is able to modulateNMDA receptor signaling by bringing reciprocal changes inthe expression of its two glutamate binding subunits NR2Aand NR2B and thus provide a novel approach to normalizeNMDAR overactivation effects without blocking this ionchannel

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The work was financially supported by DBT Grant (BTPR14049Med303442010) to Surendra Kumar Trigun andthe award of CSIR SRF to Papia Mondal The facilities ofDST-FIST and UGC-CAS programs in the Department ofZoology and BHU-DBT ISLS facilities in Faculty of Scienceare also acknowledged The authors thank Professor HemantSingh Lumen Research Foundation Chennai for providingthe ethanolic extract of Bacopa monnieri (CDRI-08)

References

[1] V Felipo ldquoHyperammonemiardquo inHandbook of Neurochemistryand Molecular Neurobiology Brain and Spinal Cord Trauma Alaztha N Banik and S K Ray Eds pp 43ndash69 Springer BerlinGermany 3rd edition 2009

[2] S Singh and S K Trigun ldquoLow grade cirrhosis inducescognitive impairment and motor dysfunction in rats couldbe a model for minimal hepatic encephalopathyrdquo NeuroscienceLetters vol 559 pp 136ndash140 2014

[3] A T Blei J Cordoba and The Practice Parameters Com-mittee of the American College of Gastroenterology ldquoHepaticencephalopathyrdquo American Journal of Gastroenterology vol 96pp 635ndash643 2001

[4] J S Bajaj J B Wade and A J Sanyal ldquoSpectrum of neurocog-nitive impairment in cirrhosis implications for the assessmentof hepatic encephalopathyrdquoHepatology vol 50 no 6 pp 2014ndash2021 2009

[5] F F Poordad ldquoReview article the burden of hepaticencephalopathyrdquo Alimentary Pharmacology and Therapeuticsvol 25 no 1 pp 3ndash9 2007

[6] J S Bajaj ldquoMinimal hepatic encephalopathy matters in dailyliferdquoWorld Journal of Gastroenterology vol 14 no 23 pp 3609ndash3615 2008

[7] S Mechtcheriakov I W Graziadei A Kugener et al ldquoMotordysfunction in patients with liver cirrhosis impairment ofhandwritingrdquo Journal of Neurology vol 253 no 3 pp 349ndash3562006

[8] S J Gilberstadt H Gilberstadt L Zieve B Buegel R O CollierJr and C J McClain ldquoPsychomotor performance defects incirrhotic patients without overt encephalopathyrdquo Archives ofInternal Medicine vol 140 no 4 pp 519ndash521 1980

[9] V Felipo and R F Butterworth ldquoNeurobiology of ammoniardquoProgress in Neurobiology vol 67 no 4 pp 259ndash279 2002

[10] C C Gandhi R M Kelly R G Wiley and T J WalshldquoImpaired acquisition of a Morris water maze task followingselective destruction of cerebellar purkinje cells with OX7-saporinrdquo Behavioural Brain Research vol 109 no 1 pp 37ndash472000

[11] M G Leggio P Neri A Graziano L Mandolesi M Molinariand L Petrosini ldquoCerebellar contribution to spatial event pro-cessing characterization of procedural learningrdquo ExperimentalBrain Research vol 127 no 1 pp 1ndash11 1999

[12] S Singh R K Koiri and S K Trigun ldquoAcute and chronichyperammonemiamodulate antioxidant enzymes differently incerebral cortex and cerebellumrdquo Neurochemical Research vol33 no 1 pp 103ndash113 2008

[13] S Singh and S K Trigun ldquoActivation of neuronal nitric oxidesynthase in cerebellum of chronic hepatic encephalopathyrats is associated with up-regulation of NADPH-producingpathwayrdquo Cerebellum vol 9 no 3 pp 384ndash397 2010

[14] P Mondal and S K Trigun ldquoPannexin1 as a novel cerebraltarget in pathogenesis of hepatic encephalopathyrdquo MetabolicBrain Disease vol 29 no 4 pp 1007ndash1015 2014

[15] S Cull-Candy S Brickley and M Farrant ldquoNMDA receptorsubunits diversity development and diseaserdquo Current Opinionin Neurobiology vol 11 no 3 pp 327ndash335 2001

[16] Y Liu PW TakM Aarts et al ldquoNMDA receptor subunits havedifferential roles in mediating excitotoxic neuronal death bothin vitro and in vivordquoThe Journal of Neuroscience vol 27 no 11pp 2846ndash2857 2007

8 Evidence-Based Complementary and Alternative Medicine

[17] O A Shipton and O Paulsen ldquoGluN2A and GluN2B subunit-containing NMDA receptors in hippocampal plasticityrdquo Philo-sophical Transactions of the Royal Society B Biological Sciencesvol 369 no 1633 2014

[18] C Montoliu R Rodrigo P Monfort et al ldquoCyclic GMP path-ways in hepatic encephalopathy neurological and therapeuticimplicationsrdquoMetabolic Brain Disease vol 25 no 1 pp 39ndash482010

[19] M V Doucet A Harkin and K K Dev ldquoThe PSD-95nNOScomplex new drugs for depressionrdquo Pharmacology amp Thera-peutics vol 133 no 2 pp 218ndash229 2012

[20] H Prast andA Philippu ldquoNitric oxide asmodulator of neuronalfunctionrdquo Progress in Neurobiology vol 64 no 1 pp 51ndash682001

[21] F X Guix I Uribesalgo M Coma and F J Munoz ldquoThephysiology and pathophysiology of nitric oxide in the brainrdquoProgress in Neurobiology vol 76 no 2 pp 126ndash152 2005

[22] E A Waxman and D R Lynch ldquoN-methyl-D-aspartate recep-tor subtypes multiple roles in excitotoxicity and neurologicaldiseaserdquo Neuroscientist vol 11 no 1 pp 37ndash49 2005

[23] V Felipo ldquoContribution of altered signal transduction asso-ciated to glutamate receptors in brain to the neurologicalalterations of hepatic encephalopathyrdquo World Journal of Gas-troenterology vol 12 no 48 pp 7737ndash7743 2006

[24] M Llansola R Rodrigo P Monfort et al ldquoNMDA receptorsin hyperammonemia and hepatic encephalopathyrdquo MetabolicBrain Disease vol 22 no 3-4 pp 321ndash335 2007

[25] S A Lipton and P A Rosenberg ldquoMechanisms of disease exci-tatory amino acids as a final common pathway for neurologicdisordersrdquoTheNew England Journal of Medicine vol 330 no 9pp 613ndash622 1994

[26] D R Lynch and R P Guttmann ldquoExcitotoxicity perspectivesbased on N-methyl-D-aspartate receptor subtypesrdquo Journal ofPharmacology and Experimental Therapeutics vol 300 no 3pp 717ndash723 2002

[27] C Ikonomidou and L Turski ldquoWhy did NMDA receptorantagonists fail clinical trials for stroke and traumatic braininjuryrdquoThe Lancet Neurology vol 1 no 6 pp 383ndash386 2002

[28] K J Gohil and J J Patel ldquoA review onBacopamonniera currentresearch and future prospectsrdquo International Journal of GreenPharmacy vol 4 no 1 pp 1ndash9 2010

[29] A Krishnakumar P M Abraham J Paul and C S PauloseldquoDown-regulation of cerebellar 5-HT2C receptors inpilocarpine-induced epilepsy in rats therapeutic role ofBacopa monnieri extractrdquo Journal of the Neurological Sciencesvol 284 no 1-2 pp 124ndash128 2009

[30] M Singh V Murthy and C Ramassamy ldquoNeuroprotectivemechanisms of the standardized extract of Bacopa monnierain a paraquatdiquat-mediated acute toxicityrdquo NeurochemistryInternational vol 62 no 5 pp 530ndash539 2013

[31] N Uabundit J Wattanathorn S Mucimapura and K Ingkan-inan ldquoCognitive enhancement and neuroprotective effects ofBacopa monnieri in Alzheimerrsquos disease modelrdquo Journal ofEthnopharmacology vol 127 no 1 pp 26ndash31 2010

[32] R Khan A Krishnakumar and C S Paulose ldquoDecreasedglutamate receptor binding and NMDA R1 gene expression inhippocampus of pilocarpine-induced epileptic rats neuropro-tective role of Bacopa monnieri extractrdquo Epilepsy and Behaviorvol 12 no 1 pp 54ndash60 2008

[33] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[34] K V H Sastry R P Moudgal J Mohan J S Tyagi and G SRao ldquoSpectrophotometric determination of serum nitrite andnitrate by copper-cadmium alloyrdquo Analytical Biochemistry vol306 no 1 pp 79ndash82 2002

[35] D R Lynch and R P Guttmann ldquoNMDA receptor pharma-cology perspectives from molecular biologyrdquo Current DrugTargets vol 2 no 3 pp 215ndash231 2001

[36] L V Kalia S K Kalia and M W Salter ldquoNMDA receptorsin clinical neurology excitatory times aheadrdquo The LancetNeurology vol 7 no 8 pp 742ndash755 2008

[37] A Lau and M Tymianski ldquoGlutamate receptors neurotoxicityand neurodegenerationrdquo Pflugers Archiv European Journal ofPhysiology vol 460 no 2 pp 525ndash542 2010

[38] M M Zeron O Hansson N Chen et al ldquoIncreased sensitivityto N-methyl-D-aspartate receptor-mediated excitotoxicity in amousemodel ofHuntingtonrsquos diseaserdquoNeuron vol 33 no 6 pp849ndash860 2002

[39] A J Williams J R Dave X M Lu G Ling and F C TortellaldquoSelective NR2B NMDA receptor antagonists are protectiveagainst staurosporine-induced apoptosisrdquo European Journal ofPharmacology vol 452 no 1 pp 135ndash136 2002

[40] M Chen T-J Lu X-J Chen et al ldquoDifferential roles of NMDAreceptor subtypes in ischemic neuronal cell death and ischemictolerancerdquo Stroke vol 39 no 11 pp 3042ndash3048 2008

[41] K Erreger S M Dravid T G Banke D J A Wyllie and SF Traynelis ldquoSubunit-specific gating controls rat NR1NR2AandNR1NR2BNMDAchannel kinetics and synaptic signallingprofilesrdquoThe Journal of Physiology vol 563 no 2 pp 345ndash3582005

[42] C S Paulose F Chathu S Reas Khan and A KrishnakumarldquoNeuroprotective role of Bacopa monnieri extract in epilepsyand effect of glucose supplementation during hypoxia gluta-mate receptor gene expressionrdquoNeurochemical Research vol 33no 9 pp 1663ndash1671 2008

[43] V L R Rao R M Audet and R F Butterworth ldquoIncreasednitric oxide synthase activities and L-[3H]arginine uptake inbrain following portacaval anastomosisrdquo Journal of Neurochem-istry vol 65 no 2 pp 677ndash681 1995

[44] P Monfort M-D Munoz A ElAyadi E Kosenko and VFelipo ldquoEffects of hyperammonemia and liver failure on glu-tamatergic neurotransmissionrdquoMetabolic Brain Disease vol 17no 4 pp 237ndash250 2002

[45] C M Snyder E H Shroff J Liu and N S Chandel ldquoNitricoxide induces cell death by regulating anti-apoptotic Bcl2 familymembersrdquo PLoS ONE vol 4 no 9 Article ID e7059 2009

[46] R K Koiri and S K Trigun ldquoDimethyl sulfoxide acti-vates tumor necrosis factor 120572-p53 mediated apoptosis anddown regulates D-fructose-6-phosphate-2-kinase and lactatedehydrogenase-5 in Daltonrsquos Lymphoma in vivordquo LeukemiaResearch vol 35 no 7 pp 950ndash956 2011

[47] R B Thomas S Joy M S Ajayan and C S Paulose ldquoNeuro-protective potential of bacopa monnieri and bacoside a againstdopamine receptor dysfunction in the cerebral cortex of neona-tal hypoglycaemic ratsrdquo Cellular and Molecular Neurobiologyvol 33 no 8 pp 1065ndash1074 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

nNOS

120573-actin

C CLF CLF + BM

(a)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (n

NO

S120573

-act

in)

lowastlowastlowast

C CLF CLF + BM(b)

00

10

20

30

40

50

60

C CLF

lowastlowast

NO

(120583m

olm

g pr

otei

n)

CLF + BM

(c)

Figure 2 Expression profile of nNOS ((a) and (b)) and NO level (c) in cerebellum of the CLF and BM extract treated CLF rats Western blotanalysis of nNOS was performed as described in the text of methods In panel (b) normalized densitometric values of nNOS120573-actin havebeen presented as mean plusmn SD from three western blot repeats lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 (control versus CLF rats) 119875 lt 005 119875 lt 0001(CLF versus CLF + BM rats) C control CLF chronic liver failure CLF + BM chronic liver failure + Bacopa monnieri extract

greater susceptibility to undergo oxidative and nitrosativestress during HA and CLF led excitotoxicity [12 13]

It has been described that lesion in cerebellum impairsacquisition of memory consolidation and deranges motorfunctions [10 11] The CLF rats used in this experimenthave recently been reported to show cognitive impairmentand deficit in motor functions as well [2] Since such neu-rochemical alterations leading to neurobehavioural changesare known to emanate from abnormal NMDAR activity [1]it is argued that a shift from a NR2A combination to aNR2B combination of NMDAR in the cerebellum of theCLF rats might be accountable for developing HE associatedsymptoms observed in these CLF rats [2] Moreover keepingaside these explanations the findings provided a basis to alterNMDAR constitution instead of shutting this ion-channeloff as a therapeutic option to bring recovery from the HEsymptoms

To test this hypothesis BM extract was chosen due to thetwo reasons Firstly amidst scarcity of the safer neurophar-macological agents this plant extract is demonstrated toimprove neuronal functions by modulating brain chemistryin many ways [28] Secondly BM is now evident to modulateactivity of the neurotransmitter receptors like serotonergicreceptors during epilepsy [29] and dopaminergic and cholin-ergic signaling in Parkinsonrsquos and Alzheimerrsquos diseases [3031] However report is limited on modulation of NMDAreceptor activity by BMWe observed a remarkable shift froma declined ratio of NR2ANR2B (neurodegeneration sup-portive combination) in the cerebellum of CLF rats towardstheir normal level when these CLF rats were administeredwith BM extract (Figure 1(e)) This finding suggested thatBM is able to alter constitution of the functional NMDARtetramer by differential expression of the two glutamatebinding subunits in cerebellum of the CLF rats Indeed in

6 Evidence-Based Complementary and Alternative Medicine

Bcl2

C CLF CLF + BM

120573-actin

(a)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (B

cl2120573

-act

in)

C CLF CLF + BM

lowast

(b)

Bax

C CLF CLF + BM

120573-actin

(c)

000

020

040

060

080

100

120

140

160

Den

sitom

etric

val

ue (B

ax120573

-act

in)

C CLF CLF + BM

lowastlowast

(d)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (B

cl2B

ax)

C CLF CLF + BM

lowastlowast

(e)

Figure 3 Expression profile of Bcl2 ((a) and (b)) and Bax ((c) and (d)) in cerebellum of the CLF and BM extract treated CLF rats Westernblot analysis of Bcl2 and Bax was performed as described in the text of methods The level of 120573-actin was probed as the loading control Inpanels (b) (d) and (e) normalized densitometric values of Bcl2120573-actin Bax120573-actin and Bcl2Bax ratio respectively have been presentedas mean plusmn SD from three western blot repeats lowast119875 lt 005 lowastlowast119875 lt 001 (control versus CLF rats) 119875 lt 005 119875 lt 001 (CLF versus CLF +BM rats) C control CLF chronic liver failure CLF + BM chronic liver failure + Bacopa monnieri extract

an epilepsy model it has been demonstrated that BM couldalter NR1 gene expression [32] thus suggesting BM as amod-ulator of NMDAR subunit expression A similar finding onmodulation of NR1 by BM was reported in a hypoxia modelalso [42] Importantly such a change in NR1 expression wasfound accountable for modulating glutamatergic signaling incerebellum of those rats [32] Thus it is argued that shiftingfrom a neurodegenerative NR2B overexpression towards

the neuroprotective NR2A level due to the treatment withBME (Figure 1) could also account for preventing down-stream undesirable neurochemical changes in cerebellum ofthe CLF rats

In a number of excitotoxic models including CLF ledHE nNOS activation is considered as the most commonevent after NMDAR activation [13 43] It is initiated by influxof Ca2+ which overactivates this enzyme to produce excess

Evidence-Based Complementary and Alternative Medicine 7

NO in the postsynaptic neurons [44] NO is a moleculeof pleiotropic effects however when produced in excessin the brain cells it uses more than one mechanism toinduce neuronal dysfunction [20 21] A milder increase inNO level is likely to initiate mitochondrial dysfunction ledneuronal apoptosis Ratio of Bcl2 versus Bax is consideredas the most effective regulators of mitochondrial dysfunctionled apoptosis [45] and hence relative levels of both theseproteins are considered as a reliable tool to assay whether acell is likely to undergo internal apoptotic process [46] Incerebellum of the CLF rats a significantly increased level ofnNOS coincides well with a similar increment in NO level(Figure 2) This is consistent with a significant decline in theBcl2Bax ratio (Figure 3) Moreover all these factors werefound to be reversed to regain their normal levels in thecerebellumwhen thoseCLF rats were treatedwith BMextract(Figures 1ndash3) Recent studies have also shown that BM extractdownregulates Bax and upregulates Bcl2 and thus providesneuroprotection in several neurological disease models [47]Thus the findings of Figures 1ndash3 together advocate forconcordant modulation of NMDA receptor constitution andnNOS led apoptotic activation by ethanolic extract of BM(CDRI-08) in cerebellum of the CLF rats

5 Conclusion

Opposing roles of the two main glutamate binding subunitsNR2A and NR2B of NMDA receptor advocate for modu-lation of NMDAR constitution as an effective mechanismto normalize its excitotoxic effects without blocking its ion-channel activity During recent past studies on efficacy ofBacopa monnieri extract have been shown to prevent neu-rodegenerative diseases by modulating neurochemistry ofthe brain cells but with little information about modulationof NMDAR overactivation led excitotoxicity The presentfindings demonstrate that BM extract is able to modulateNMDA receptor signaling by bringing reciprocal changes inthe expression of its two glutamate binding subunits NR2Aand NR2B and thus provide a novel approach to normalizeNMDAR overactivation effects without blocking this ionchannel

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The work was financially supported by DBT Grant (BTPR14049Med303442010) to Surendra Kumar Trigun andthe award of CSIR SRF to Papia Mondal The facilities ofDST-FIST and UGC-CAS programs in the Department ofZoology and BHU-DBT ISLS facilities in Faculty of Scienceare also acknowledged The authors thank Professor HemantSingh Lumen Research Foundation Chennai for providingthe ethanolic extract of Bacopa monnieri (CDRI-08)

References

[1] V Felipo ldquoHyperammonemiardquo inHandbook of Neurochemistryand Molecular Neurobiology Brain and Spinal Cord Trauma Alaztha N Banik and S K Ray Eds pp 43ndash69 Springer BerlinGermany 3rd edition 2009

[2] S Singh and S K Trigun ldquoLow grade cirrhosis inducescognitive impairment and motor dysfunction in rats couldbe a model for minimal hepatic encephalopathyrdquo NeuroscienceLetters vol 559 pp 136ndash140 2014

[3] A T Blei J Cordoba and The Practice Parameters Com-mittee of the American College of Gastroenterology ldquoHepaticencephalopathyrdquo American Journal of Gastroenterology vol 96pp 635ndash643 2001

[4] J S Bajaj J B Wade and A J Sanyal ldquoSpectrum of neurocog-nitive impairment in cirrhosis implications for the assessmentof hepatic encephalopathyrdquoHepatology vol 50 no 6 pp 2014ndash2021 2009

[5] F F Poordad ldquoReview article the burden of hepaticencephalopathyrdquo Alimentary Pharmacology and Therapeuticsvol 25 no 1 pp 3ndash9 2007

[6] J S Bajaj ldquoMinimal hepatic encephalopathy matters in dailyliferdquoWorld Journal of Gastroenterology vol 14 no 23 pp 3609ndash3615 2008

[7] S Mechtcheriakov I W Graziadei A Kugener et al ldquoMotordysfunction in patients with liver cirrhosis impairment ofhandwritingrdquo Journal of Neurology vol 253 no 3 pp 349ndash3562006

[8] S J Gilberstadt H Gilberstadt L Zieve B Buegel R O CollierJr and C J McClain ldquoPsychomotor performance defects incirrhotic patients without overt encephalopathyrdquo Archives ofInternal Medicine vol 140 no 4 pp 519ndash521 1980

[9] V Felipo and R F Butterworth ldquoNeurobiology of ammoniardquoProgress in Neurobiology vol 67 no 4 pp 259ndash279 2002

[10] C C Gandhi R M Kelly R G Wiley and T J WalshldquoImpaired acquisition of a Morris water maze task followingselective destruction of cerebellar purkinje cells with OX7-saporinrdquo Behavioural Brain Research vol 109 no 1 pp 37ndash472000

[11] M G Leggio P Neri A Graziano L Mandolesi M Molinariand L Petrosini ldquoCerebellar contribution to spatial event pro-cessing characterization of procedural learningrdquo ExperimentalBrain Research vol 127 no 1 pp 1ndash11 1999

[12] S Singh R K Koiri and S K Trigun ldquoAcute and chronichyperammonemiamodulate antioxidant enzymes differently incerebral cortex and cerebellumrdquo Neurochemical Research vol33 no 1 pp 103ndash113 2008

[13] S Singh and S K Trigun ldquoActivation of neuronal nitric oxidesynthase in cerebellum of chronic hepatic encephalopathyrats is associated with up-regulation of NADPH-producingpathwayrdquo Cerebellum vol 9 no 3 pp 384ndash397 2010

[14] P Mondal and S K Trigun ldquoPannexin1 as a novel cerebraltarget in pathogenesis of hepatic encephalopathyrdquo MetabolicBrain Disease vol 29 no 4 pp 1007ndash1015 2014

[15] S Cull-Candy S Brickley and M Farrant ldquoNMDA receptorsubunits diversity development and diseaserdquo Current Opinionin Neurobiology vol 11 no 3 pp 327ndash335 2001

[16] Y Liu PW TakM Aarts et al ldquoNMDA receptor subunits havedifferential roles in mediating excitotoxic neuronal death bothin vitro and in vivordquoThe Journal of Neuroscience vol 27 no 11pp 2846ndash2857 2007

8 Evidence-Based Complementary and Alternative Medicine

[17] O A Shipton and O Paulsen ldquoGluN2A and GluN2B subunit-containing NMDA receptors in hippocampal plasticityrdquo Philo-sophical Transactions of the Royal Society B Biological Sciencesvol 369 no 1633 2014

[18] C Montoliu R Rodrigo P Monfort et al ldquoCyclic GMP path-ways in hepatic encephalopathy neurological and therapeuticimplicationsrdquoMetabolic Brain Disease vol 25 no 1 pp 39ndash482010

[19] M V Doucet A Harkin and K K Dev ldquoThe PSD-95nNOScomplex new drugs for depressionrdquo Pharmacology amp Thera-peutics vol 133 no 2 pp 218ndash229 2012

[20] H Prast andA Philippu ldquoNitric oxide asmodulator of neuronalfunctionrdquo Progress in Neurobiology vol 64 no 1 pp 51ndash682001

[21] F X Guix I Uribesalgo M Coma and F J Munoz ldquoThephysiology and pathophysiology of nitric oxide in the brainrdquoProgress in Neurobiology vol 76 no 2 pp 126ndash152 2005

[22] E A Waxman and D R Lynch ldquoN-methyl-D-aspartate recep-tor subtypes multiple roles in excitotoxicity and neurologicaldiseaserdquo Neuroscientist vol 11 no 1 pp 37ndash49 2005

[23] V Felipo ldquoContribution of altered signal transduction asso-ciated to glutamate receptors in brain to the neurologicalalterations of hepatic encephalopathyrdquo World Journal of Gas-troenterology vol 12 no 48 pp 7737ndash7743 2006

[24] M Llansola R Rodrigo P Monfort et al ldquoNMDA receptorsin hyperammonemia and hepatic encephalopathyrdquo MetabolicBrain Disease vol 22 no 3-4 pp 321ndash335 2007

[25] S A Lipton and P A Rosenberg ldquoMechanisms of disease exci-tatory amino acids as a final common pathway for neurologicdisordersrdquoTheNew England Journal of Medicine vol 330 no 9pp 613ndash622 1994

[26] D R Lynch and R P Guttmann ldquoExcitotoxicity perspectivesbased on N-methyl-D-aspartate receptor subtypesrdquo Journal ofPharmacology and Experimental Therapeutics vol 300 no 3pp 717ndash723 2002

[27] C Ikonomidou and L Turski ldquoWhy did NMDA receptorantagonists fail clinical trials for stroke and traumatic braininjuryrdquoThe Lancet Neurology vol 1 no 6 pp 383ndash386 2002

[28] K J Gohil and J J Patel ldquoA review onBacopamonniera currentresearch and future prospectsrdquo International Journal of GreenPharmacy vol 4 no 1 pp 1ndash9 2010

[29] A Krishnakumar P M Abraham J Paul and C S PauloseldquoDown-regulation of cerebellar 5-HT2C receptors inpilocarpine-induced epilepsy in rats therapeutic role ofBacopa monnieri extractrdquo Journal of the Neurological Sciencesvol 284 no 1-2 pp 124ndash128 2009

[30] M Singh V Murthy and C Ramassamy ldquoNeuroprotectivemechanisms of the standardized extract of Bacopa monnierain a paraquatdiquat-mediated acute toxicityrdquo NeurochemistryInternational vol 62 no 5 pp 530ndash539 2013

[31] N Uabundit J Wattanathorn S Mucimapura and K Ingkan-inan ldquoCognitive enhancement and neuroprotective effects ofBacopa monnieri in Alzheimerrsquos disease modelrdquo Journal ofEthnopharmacology vol 127 no 1 pp 26ndash31 2010

[32] R Khan A Krishnakumar and C S Paulose ldquoDecreasedglutamate receptor binding and NMDA R1 gene expression inhippocampus of pilocarpine-induced epileptic rats neuropro-tective role of Bacopa monnieri extractrdquo Epilepsy and Behaviorvol 12 no 1 pp 54ndash60 2008

[33] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[34] K V H Sastry R P Moudgal J Mohan J S Tyagi and G SRao ldquoSpectrophotometric determination of serum nitrite andnitrate by copper-cadmium alloyrdquo Analytical Biochemistry vol306 no 1 pp 79ndash82 2002

[35] D R Lynch and R P Guttmann ldquoNMDA receptor pharma-cology perspectives from molecular biologyrdquo Current DrugTargets vol 2 no 3 pp 215ndash231 2001

[36] L V Kalia S K Kalia and M W Salter ldquoNMDA receptorsin clinical neurology excitatory times aheadrdquo The LancetNeurology vol 7 no 8 pp 742ndash755 2008

[37] A Lau and M Tymianski ldquoGlutamate receptors neurotoxicityand neurodegenerationrdquo Pflugers Archiv European Journal ofPhysiology vol 460 no 2 pp 525ndash542 2010

[38] M M Zeron O Hansson N Chen et al ldquoIncreased sensitivityto N-methyl-D-aspartate receptor-mediated excitotoxicity in amousemodel ofHuntingtonrsquos diseaserdquoNeuron vol 33 no 6 pp849ndash860 2002

[39] A J Williams J R Dave X M Lu G Ling and F C TortellaldquoSelective NR2B NMDA receptor antagonists are protectiveagainst staurosporine-induced apoptosisrdquo European Journal ofPharmacology vol 452 no 1 pp 135ndash136 2002

[40] M Chen T-J Lu X-J Chen et al ldquoDifferential roles of NMDAreceptor subtypes in ischemic neuronal cell death and ischemictolerancerdquo Stroke vol 39 no 11 pp 3042ndash3048 2008

[41] K Erreger S M Dravid T G Banke D J A Wyllie and SF Traynelis ldquoSubunit-specific gating controls rat NR1NR2AandNR1NR2BNMDAchannel kinetics and synaptic signallingprofilesrdquoThe Journal of Physiology vol 563 no 2 pp 345ndash3582005

[42] C S Paulose F Chathu S Reas Khan and A KrishnakumarldquoNeuroprotective role of Bacopa monnieri extract in epilepsyand effect of glucose supplementation during hypoxia gluta-mate receptor gene expressionrdquoNeurochemical Research vol 33no 9 pp 1663ndash1671 2008

[43] V L R Rao R M Audet and R F Butterworth ldquoIncreasednitric oxide synthase activities and L-[3H]arginine uptake inbrain following portacaval anastomosisrdquo Journal of Neurochem-istry vol 65 no 2 pp 677ndash681 1995

[44] P Monfort M-D Munoz A ElAyadi E Kosenko and VFelipo ldquoEffects of hyperammonemia and liver failure on glu-tamatergic neurotransmissionrdquoMetabolic Brain Disease vol 17no 4 pp 237ndash250 2002

[45] C M Snyder E H Shroff J Liu and N S Chandel ldquoNitricoxide induces cell death by regulating anti-apoptotic Bcl2 familymembersrdquo PLoS ONE vol 4 no 9 Article ID e7059 2009

[46] R K Koiri and S K Trigun ldquoDimethyl sulfoxide acti-vates tumor necrosis factor 120572-p53 mediated apoptosis anddown regulates D-fructose-6-phosphate-2-kinase and lactatedehydrogenase-5 in Daltonrsquos Lymphoma in vivordquo LeukemiaResearch vol 35 no 7 pp 950ndash956 2011

[47] R B Thomas S Joy M S Ajayan and C S Paulose ldquoNeuro-protective potential of bacopa monnieri and bacoside a againstdopamine receptor dysfunction in the cerebral cortex of neona-tal hypoglycaemic ratsrdquo Cellular and Molecular Neurobiologyvol 33 no 8 pp 1065ndash1074 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

Bcl2

C CLF CLF + BM

120573-actin

(a)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (B

cl2120573

-act

in)

C CLF CLF + BM

lowast

(b)

Bax

C CLF CLF + BM

120573-actin

(c)

000

020

040

060

080

100

120

140

160

Den

sitom

etric

val

ue (B

ax120573

-act

in)

C CLF CLF + BM

lowastlowast

(d)

000

020

040

060

080

100

120

140

Den

sitom

etric

val

ue (B

cl2B

ax)

C CLF CLF + BM

lowastlowast

(e)

Figure 3 Expression profile of Bcl2 ((a) and (b)) and Bax ((c) and (d)) in cerebellum of the CLF and BM extract treated CLF rats Westernblot analysis of Bcl2 and Bax was performed as described in the text of methods The level of 120573-actin was probed as the loading control Inpanels (b) (d) and (e) normalized densitometric values of Bcl2120573-actin Bax120573-actin and Bcl2Bax ratio respectively have been presentedas mean plusmn SD from three western blot repeats lowast119875 lt 005 lowastlowast119875 lt 001 (control versus CLF rats) 119875 lt 005 119875 lt 001 (CLF versus CLF +BM rats) C control CLF chronic liver failure CLF + BM chronic liver failure + Bacopa monnieri extract

an epilepsy model it has been demonstrated that BM couldalter NR1 gene expression [32] thus suggesting BM as amod-ulator of NMDAR subunit expression A similar finding onmodulation of NR1 by BM was reported in a hypoxia modelalso [42] Importantly such a change in NR1 expression wasfound accountable for modulating glutamatergic signaling incerebellum of those rats [32] Thus it is argued that shiftingfrom a neurodegenerative NR2B overexpression towards

the neuroprotective NR2A level due to the treatment withBME (Figure 1) could also account for preventing down-stream undesirable neurochemical changes in cerebellum ofthe CLF rats

In a number of excitotoxic models including CLF ledHE nNOS activation is considered as the most commonevent after NMDAR activation [13 43] It is initiated by influxof Ca2+ which overactivates this enzyme to produce excess

Evidence-Based Complementary and Alternative Medicine 7

NO in the postsynaptic neurons [44] NO is a moleculeof pleiotropic effects however when produced in excessin the brain cells it uses more than one mechanism toinduce neuronal dysfunction [20 21] A milder increase inNO level is likely to initiate mitochondrial dysfunction ledneuronal apoptosis Ratio of Bcl2 versus Bax is consideredas the most effective regulators of mitochondrial dysfunctionled apoptosis [45] and hence relative levels of both theseproteins are considered as a reliable tool to assay whether acell is likely to undergo internal apoptotic process [46] Incerebellum of the CLF rats a significantly increased level ofnNOS coincides well with a similar increment in NO level(Figure 2) This is consistent with a significant decline in theBcl2Bax ratio (Figure 3) Moreover all these factors werefound to be reversed to regain their normal levels in thecerebellumwhen thoseCLF rats were treatedwith BMextract(Figures 1ndash3) Recent studies have also shown that BM extractdownregulates Bax and upregulates Bcl2 and thus providesneuroprotection in several neurological disease models [47]Thus the findings of Figures 1ndash3 together advocate forconcordant modulation of NMDA receptor constitution andnNOS led apoptotic activation by ethanolic extract of BM(CDRI-08) in cerebellum of the CLF rats

5 Conclusion

Opposing roles of the two main glutamate binding subunitsNR2A and NR2B of NMDA receptor advocate for modu-lation of NMDAR constitution as an effective mechanismto normalize its excitotoxic effects without blocking its ion-channel activity During recent past studies on efficacy ofBacopa monnieri extract have been shown to prevent neu-rodegenerative diseases by modulating neurochemistry ofthe brain cells but with little information about modulationof NMDAR overactivation led excitotoxicity The presentfindings demonstrate that BM extract is able to modulateNMDA receptor signaling by bringing reciprocal changes inthe expression of its two glutamate binding subunits NR2Aand NR2B and thus provide a novel approach to normalizeNMDAR overactivation effects without blocking this ionchannel

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The work was financially supported by DBT Grant (BTPR14049Med303442010) to Surendra Kumar Trigun andthe award of CSIR SRF to Papia Mondal The facilities ofDST-FIST and UGC-CAS programs in the Department ofZoology and BHU-DBT ISLS facilities in Faculty of Scienceare also acknowledged The authors thank Professor HemantSingh Lumen Research Foundation Chennai for providingthe ethanolic extract of Bacopa monnieri (CDRI-08)

References

[1] V Felipo ldquoHyperammonemiardquo inHandbook of Neurochemistryand Molecular Neurobiology Brain and Spinal Cord Trauma Alaztha N Banik and S K Ray Eds pp 43ndash69 Springer BerlinGermany 3rd edition 2009

[2] S Singh and S K Trigun ldquoLow grade cirrhosis inducescognitive impairment and motor dysfunction in rats couldbe a model for minimal hepatic encephalopathyrdquo NeuroscienceLetters vol 559 pp 136ndash140 2014

[3] A T Blei J Cordoba and The Practice Parameters Com-mittee of the American College of Gastroenterology ldquoHepaticencephalopathyrdquo American Journal of Gastroenterology vol 96pp 635ndash643 2001

[4] J S Bajaj J B Wade and A J Sanyal ldquoSpectrum of neurocog-nitive impairment in cirrhosis implications for the assessmentof hepatic encephalopathyrdquoHepatology vol 50 no 6 pp 2014ndash2021 2009

[5] F F Poordad ldquoReview article the burden of hepaticencephalopathyrdquo Alimentary Pharmacology and Therapeuticsvol 25 no 1 pp 3ndash9 2007

[6] J S Bajaj ldquoMinimal hepatic encephalopathy matters in dailyliferdquoWorld Journal of Gastroenterology vol 14 no 23 pp 3609ndash3615 2008

[7] S Mechtcheriakov I W Graziadei A Kugener et al ldquoMotordysfunction in patients with liver cirrhosis impairment ofhandwritingrdquo Journal of Neurology vol 253 no 3 pp 349ndash3562006

[8] S J Gilberstadt H Gilberstadt L Zieve B Buegel R O CollierJr and C J McClain ldquoPsychomotor performance defects incirrhotic patients without overt encephalopathyrdquo Archives ofInternal Medicine vol 140 no 4 pp 519ndash521 1980

[9] V Felipo and R F Butterworth ldquoNeurobiology of ammoniardquoProgress in Neurobiology vol 67 no 4 pp 259ndash279 2002

[10] C C Gandhi R M Kelly R G Wiley and T J WalshldquoImpaired acquisition of a Morris water maze task followingselective destruction of cerebellar purkinje cells with OX7-saporinrdquo Behavioural Brain Research vol 109 no 1 pp 37ndash472000

[11] M G Leggio P Neri A Graziano L Mandolesi M Molinariand L Petrosini ldquoCerebellar contribution to spatial event pro-cessing characterization of procedural learningrdquo ExperimentalBrain Research vol 127 no 1 pp 1ndash11 1999

[12] S Singh R K Koiri and S K Trigun ldquoAcute and chronichyperammonemiamodulate antioxidant enzymes differently incerebral cortex and cerebellumrdquo Neurochemical Research vol33 no 1 pp 103ndash113 2008

[13] S Singh and S K Trigun ldquoActivation of neuronal nitric oxidesynthase in cerebellum of chronic hepatic encephalopathyrats is associated with up-regulation of NADPH-producingpathwayrdquo Cerebellum vol 9 no 3 pp 384ndash397 2010

[14] P Mondal and S K Trigun ldquoPannexin1 as a novel cerebraltarget in pathogenesis of hepatic encephalopathyrdquo MetabolicBrain Disease vol 29 no 4 pp 1007ndash1015 2014

[15] S Cull-Candy S Brickley and M Farrant ldquoNMDA receptorsubunits diversity development and diseaserdquo Current Opinionin Neurobiology vol 11 no 3 pp 327ndash335 2001

[16] Y Liu PW TakM Aarts et al ldquoNMDA receptor subunits havedifferential roles in mediating excitotoxic neuronal death bothin vitro and in vivordquoThe Journal of Neuroscience vol 27 no 11pp 2846ndash2857 2007

8 Evidence-Based Complementary and Alternative Medicine

[17] O A Shipton and O Paulsen ldquoGluN2A and GluN2B subunit-containing NMDA receptors in hippocampal plasticityrdquo Philo-sophical Transactions of the Royal Society B Biological Sciencesvol 369 no 1633 2014

[18] C Montoliu R Rodrigo P Monfort et al ldquoCyclic GMP path-ways in hepatic encephalopathy neurological and therapeuticimplicationsrdquoMetabolic Brain Disease vol 25 no 1 pp 39ndash482010

[19] M V Doucet A Harkin and K K Dev ldquoThe PSD-95nNOScomplex new drugs for depressionrdquo Pharmacology amp Thera-peutics vol 133 no 2 pp 218ndash229 2012

[20] H Prast andA Philippu ldquoNitric oxide asmodulator of neuronalfunctionrdquo Progress in Neurobiology vol 64 no 1 pp 51ndash682001

[21] F X Guix I Uribesalgo M Coma and F J Munoz ldquoThephysiology and pathophysiology of nitric oxide in the brainrdquoProgress in Neurobiology vol 76 no 2 pp 126ndash152 2005

[22] E A Waxman and D R Lynch ldquoN-methyl-D-aspartate recep-tor subtypes multiple roles in excitotoxicity and neurologicaldiseaserdquo Neuroscientist vol 11 no 1 pp 37ndash49 2005

[23] V Felipo ldquoContribution of altered signal transduction asso-ciated to glutamate receptors in brain to the neurologicalalterations of hepatic encephalopathyrdquo World Journal of Gas-troenterology vol 12 no 48 pp 7737ndash7743 2006

[24] M Llansola R Rodrigo P Monfort et al ldquoNMDA receptorsin hyperammonemia and hepatic encephalopathyrdquo MetabolicBrain Disease vol 22 no 3-4 pp 321ndash335 2007

[25] S A Lipton and P A Rosenberg ldquoMechanisms of disease exci-tatory amino acids as a final common pathway for neurologicdisordersrdquoTheNew England Journal of Medicine vol 330 no 9pp 613ndash622 1994

[26] D R Lynch and R P Guttmann ldquoExcitotoxicity perspectivesbased on N-methyl-D-aspartate receptor subtypesrdquo Journal ofPharmacology and Experimental Therapeutics vol 300 no 3pp 717ndash723 2002

[27] C Ikonomidou and L Turski ldquoWhy did NMDA receptorantagonists fail clinical trials for stroke and traumatic braininjuryrdquoThe Lancet Neurology vol 1 no 6 pp 383ndash386 2002

[28] K J Gohil and J J Patel ldquoA review onBacopamonniera currentresearch and future prospectsrdquo International Journal of GreenPharmacy vol 4 no 1 pp 1ndash9 2010

[29] A Krishnakumar P M Abraham J Paul and C S PauloseldquoDown-regulation of cerebellar 5-HT2C receptors inpilocarpine-induced epilepsy in rats therapeutic role ofBacopa monnieri extractrdquo Journal of the Neurological Sciencesvol 284 no 1-2 pp 124ndash128 2009

[30] M Singh V Murthy and C Ramassamy ldquoNeuroprotectivemechanisms of the standardized extract of Bacopa monnierain a paraquatdiquat-mediated acute toxicityrdquo NeurochemistryInternational vol 62 no 5 pp 530ndash539 2013

[31] N Uabundit J Wattanathorn S Mucimapura and K Ingkan-inan ldquoCognitive enhancement and neuroprotective effects ofBacopa monnieri in Alzheimerrsquos disease modelrdquo Journal ofEthnopharmacology vol 127 no 1 pp 26ndash31 2010

[32] R Khan A Krishnakumar and C S Paulose ldquoDecreasedglutamate receptor binding and NMDA R1 gene expression inhippocampus of pilocarpine-induced epileptic rats neuropro-tective role of Bacopa monnieri extractrdquo Epilepsy and Behaviorvol 12 no 1 pp 54ndash60 2008

[33] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[34] K V H Sastry R P Moudgal J Mohan J S Tyagi and G SRao ldquoSpectrophotometric determination of serum nitrite andnitrate by copper-cadmium alloyrdquo Analytical Biochemistry vol306 no 1 pp 79ndash82 2002

[35] D R Lynch and R P Guttmann ldquoNMDA receptor pharma-cology perspectives from molecular biologyrdquo Current DrugTargets vol 2 no 3 pp 215ndash231 2001

[36] L V Kalia S K Kalia and M W Salter ldquoNMDA receptorsin clinical neurology excitatory times aheadrdquo The LancetNeurology vol 7 no 8 pp 742ndash755 2008

[37] A Lau and M Tymianski ldquoGlutamate receptors neurotoxicityand neurodegenerationrdquo Pflugers Archiv European Journal ofPhysiology vol 460 no 2 pp 525ndash542 2010

[38] M M Zeron O Hansson N Chen et al ldquoIncreased sensitivityto N-methyl-D-aspartate receptor-mediated excitotoxicity in amousemodel ofHuntingtonrsquos diseaserdquoNeuron vol 33 no 6 pp849ndash860 2002

[39] A J Williams J R Dave X M Lu G Ling and F C TortellaldquoSelective NR2B NMDA receptor antagonists are protectiveagainst staurosporine-induced apoptosisrdquo European Journal ofPharmacology vol 452 no 1 pp 135ndash136 2002

[40] M Chen T-J Lu X-J Chen et al ldquoDifferential roles of NMDAreceptor subtypes in ischemic neuronal cell death and ischemictolerancerdquo Stroke vol 39 no 11 pp 3042ndash3048 2008

[41] K Erreger S M Dravid T G Banke D J A Wyllie and SF Traynelis ldquoSubunit-specific gating controls rat NR1NR2AandNR1NR2BNMDAchannel kinetics and synaptic signallingprofilesrdquoThe Journal of Physiology vol 563 no 2 pp 345ndash3582005

[42] C S Paulose F Chathu S Reas Khan and A KrishnakumarldquoNeuroprotective role of Bacopa monnieri extract in epilepsyand effect of glucose supplementation during hypoxia gluta-mate receptor gene expressionrdquoNeurochemical Research vol 33no 9 pp 1663ndash1671 2008

[43] V L R Rao R M Audet and R F Butterworth ldquoIncreasednitric oxide synthase activities and L-[3H]arginine uptake inbrain following portacaval anastomosisrdquo Journal of Neurochem-istry vol 65 no 2 pp 677ndash681 1995

[44] P Monfort M-D Munoz A ElAyadi E Kosenko and VFelipo ldquoEffects of hyperammonemia and liver failure on glu-tamatergic neurotransmissionrdquoMetabolic Brain Disease vol 17no 4 pp 237ndash250 2002

[45] C M Snyder E H Shroff J Liu and N S Chandel ldquoNitricoxide induces cell death by regulating anti-apoptotic Bcl2 familymembersrdquo PLoS ONE vol 4 no 9 Article ID e7059 2009

[46] R K Koiri and S K Trigun ldquoDimethyl sulfoxide acti-vates tumor necrosis factor 120572-p53 mediated apoptosis anddown regulates D-fructose-6-phosphate-2-kinase and lactatedehydrogenase-5 in Daltonrsquos Lymphoma in vivordquo LeukemiaResearch vol 35 no 7 pp 950ndash956 2011

[47] R B Thomas S Joy M S Ajayan and C S Paulose ldquoNeuro-protective potential of bacopa monnieri and bacoside a againstdopamine receptor dysfunction in the cerebral cortex of neona-tal hypoglycaemic ratsrdquo Cellular and Molecular Neurobiologyvol 33 no 8 pp 1065ndash1074 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

NO in the postsynaptic neurons [44] NO is a moleculeof pleiotropic effects however when produced in excessin the brain cells it uses more than one mechanism toinduce neuronal dysfunction [20 21] A milder increase inNO level is likely to initiate mitochondrial dysfunction ledneuronal apoptosis Ratio of Bcl2 versus Bax is consideredas the most effective regulators of mitochondrial dysfunctionled apoptosis [45] and hence relative levels of both theseproteins are considered as a reliable tool to assay whether acell is likely to undergo internal apoptotic process [46] Incerebellum of the CLF rats a significantly increased level ofnNOS coincides well with a similar increment in NO level(Figure 2) This is consistent with a significant decline in theBcl2Bax ratio (Figure 3) Moreover all these factors werefound to be reversed to regain their normal levels in thecerebellumwhen thoseCLF rats were treatedwith BMextract(Figures 1ndash3) Recent studies have also shown that BM extractdownregulates Bax and upregulates Bcl2 and thus providesneuroprotection in several neurological disease models [47]Thus the findings of Figures 1ndash3 together advocate forconcordant modulation of NMDA receptor constitution andnNOS led apoptotic activation by ethanolic extract of BM(CDRI-08) in cerebellum of the CLF rats

5 Conclusion

Opposing roles of the two main glutamate binding subunitsNR2A and NR2B of NMDA receptor advocate for modu-lation of NMDAR constitution as an effective mechanismto normalize its excitotoxic effects without blocking its ion-channel activity During recent past studies on efficacy ofBacopa monnieri extract have been shown to prevent neu-rodegenerative diseases by modulating neurochemistry ofthe brain cells but with little information about modulationof NMDAR overactivation led excitotoxicity The presentfindings demonstrate that BM extract is able to modulateNMDA receptor signaling by bringing reciprocal changes inthe expression of its two glutamate binding subunits NR2Aand NR2B and thus provide a novel approach to normalizeNMDAR overactivation effects without blocking this ionchannel

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The work was financially supported by DBT Grant (BTPR14049Med303442010) to Surendra Kumar Trigun andthe award of CSIR SRF to Papia Mondal The facilities ofDST-FIST and UGC-CAS programs in the Department ofZoology and BHU-DBT ISLS facilities in Faculty of Scienceare also acknowledged The authors thank Professor HemantSingh Lumen Research Foundation Chennai for providingthe ethanolic extract of Bacopa monnieri (CDRI-08)

References

[1] V Felipo ldquoHyperammonemiardquo inHandbook of Neurochemistryand Molecular Neurobiology Brain and Spinal Cord Trauma Alaztha N Banik and S K Ray Eds pp 43ndash69 Springer BerlinGermany 3rd edition 2009

[2] S Singh and S K Trigun ldquoLow grade cirrhosis inducescognitive impairment and motor dysfunction in rats couldbe a model for minimal hepatic encephalopathyrdquo NeuroscienceLetters vol 559 pp 136ndash140 2014

[3] A T Blei J Cordoba and The Practice Parameters Com-mittee of the American College of Gastroenterology ldquoHepaticencephalopathyrdquo American Journal of Gastroenterology vol 96pp 635ndash643 2001

[4] J S Bajaj J B Wade and A J Sanyal ldquoSpectrum of neurocog-nitive impairment in cirrhosis implications for the assessmentof hepatic encephalopathyrdquoHepatology vol 50 no 6 pp 2014ndash2021 2009

[5] F F Poordad ldquoReview article the burden of hepaticencephalopathyrdquo Alimentary Pharmacology and Therapeuticsvol 25 no 1 pp 3ndash9 2007

[6] J S Bajaj ldquoMinimal hepatic encephalopathy matters in dailyliferdquoWorld Journal of Gastroenterology vol 14 no 23 pp 3609ndash3615 2008

[7] S Mechtcheriakov I W Graziadei A Kugener et al ldquoMotordysfunction in patients with liver cirrhosis impairment ofhandwritingrdquo Journal of Neurology vol 253 no 3 pp 349ndash3562006

[8] S J Gilberstadt H Gilberstadt L Zieve B Buegel R O CollierJr and C J McClain ldquoPsychomotor performance defects incirrhotic patients without overt encephalopathyrdquo Archives ofInternal Medicine vol 140 no 4 pp 519ndash521 1980

[9] V Felipo and R F Butterworth ldquoNeurobiology of ammoniardquoProgress in Neurobiology vol 67 no 4 pp 259ndash279 2002

[10] C C Gandhi R M Kelly R G Wiley and T J WalshldquoImpaired acquisition of a Morris water maze task followingselective destruction of cerebellar purkinje cells with OX7-saporinrdquo Behavioural Brain Research vol 109 no 1 pp 37ndash472000

[11] M G Leggio P Neri A Graziano L Mandolesi M Molinariand L Petrosini ldquoCerebellar contribution to spatial event pro-cessing characterization of procedural learningrdquo ExperimentalBrain Research vol 127 no 1 pp 1ndash11 1999

[12] S Singh R K Koiri and S K Trigun ldquoAcute and chronichyperammonemiamodulate antioxidant enzymes differently incerebral cortex and cerebellumrdquo Neurochemical Research vol33 no 1 pp 103ndash113 2008

[13] S Singh and S K Trigun ldquoActivation of neuronal nitric oxidesynthase in cerebellum of chronic hepatic encephalopathyrats is associated with up-regulation of NADPH-producingpathwayrdquo Cerebellum vol 9 no 3 pp 384ndash397 2010

[14] P Mondal and S K Trigun ldquoPannexin1 as a novel cerebraltarget in pathogenesis of hepatic encephalopathyrdquo MetabolicBrain Disease vol 29 no 4 pp 1007ndash1015 2014

[15] S Cull-Candy S Brickley and M Farrant ldquoNMDA receptorsubunits diversity development and diseaserdquo Current Opinionin Neurobiology vol 11 no 3 pp 327ndash335 2001

[16] Y Liu PW TakM Aarts et al ldquoNMDA receptor subunits havedifferential roles in mediating excitotoxic neuronal death bothin vitro and in vivordquoThe Journal of Neuroscience vol 27 no 11pp 2846ndash2857 2007

8 Evidence-Based Complementary and Alternative Medicine

[17] O A Shipton and O Paulsen ldquoGluN2A and GluN2B subunit-containing NMDA receptors in hippocampal plasticityrdquo Philo-sophical Transactions of the Royal Society B Biological Sciencesvol 369 no 1633 2014

[18] C Montoliu R Rodrigo P Monfort et al ldquoCyclic GMP path-ways in hepatic encephalopathy neurological and therapeuticimplicationsrdquoMetabolic Brain Disease vol 25 no 1 pp 39ndash482010

[19] M V Doucet A Harkin and K K Dev ldquoThe PSD-95nNOScomplex new drugs for depressionrdquo Pharmacology amp Thera-peutics vol 133 no 2 pp 218ndash229 2012

[20] H Prast andA Philippu ldquoNitric oxide asmodulator of neuronalfunctionrdquo Progress in Neurobiology vol 64 no 1 pp 51ndash682001

[21] F X Guix I Uribesalgo M Coma and F J Munoz ldquoThephysiology and pathophysiology of nitric oxide in the brainrdquoProgress in Neurobiology vol 76 no 2 pp 126ndash152 2005

[22] E A Waxman and D R Lynch ldquoN-methyl-D-aspartate recep-tor subtypes multiple roles in excitotoxicity and neurologicaldiseaserdquo Neuroscientist vol 11 no 1 pp 37ndash49 2005

[23] V Felipo ldquoContribution of altered signal transduction asso-ciated to glutamate receptors in brain to the neurologicalalterations of hepatic encephalopathyrdquo World Journal of Gas-troenterology vol 12 no 48 pp 7737ndash7743 2006

[24] M Llansola R Rodrigo P Monfort et al ldquoNMDA receptorsin hyperammonemia and hepatic encephalopathyrdquo MetabolicBrain Disease vol 22 no 3-4 pp 321ndash335 2007

[25] S A Lipton and P A Rosenberg ldquoMechanisms of disease exci-tatory amino acids as a final common pathway for neurologicdisordersrdquoTheNew England Journal of Medicine vol 330 no 9pp 613ndash622 1994

[26] D R Lynch and R P Guttmann ldquoExcitotoxicity perspectivesbased on N-methyl-D-aspartate receptor subtypesrdquo Journal ofPharmacology and Experimental Therapeutics vol 300 no 3pp 717ndash723 2002

[27] C Ikonomidou and L Turski ldquoWhy did NMDA receptorantagonists fail clinical trials for stroke and traumatic braininjuryrdquoThe Lancet Neurology vol 1 no 6 pp 383ndash386 2002

[28] K J Gohil and J J Patel ldquoA review onBacopamonniera currentresearch and future prospectsrdquo International Journal of GreenPharmacy vol 4 no 1 pp 1ndash9 2010

[29] A Krishnakumar P M Abraham J Paul and C S PauloseldquoDown-regulation of cerebellar 5-HT2C receptors inpilocarpine-induced epilepsy in rats therapeutic role ofBacopa monnieri extractrdquo Journal of the Neurological Sciencesvol 284 no 1-2 pp 124ndash128 2009

[30] M Singh V Murthy and C Ramassamy ldquoNeuroprotectivemechanisms of the standardized extract of Bacopa monnierain a paraquatdiquat-mediated acute toxicityrdquo NeurochemistryInternational vol 62 no 5 pp 530ndash539 2013

[31] N Uabundit J Wattanathorn S Mucimapura and K Ingkan-inan ldquoCognitive enhancement and neuroprotective effects ofBacopa monnieri in Alzheimerrsquos disease modelrdquo Journal ofEthnopharmacology vol 127 no 1 pp 26ndash31 2010

[32] R Khan A Krishnakumar and C S Paulose ldquoDecreasedglutamate receptor binding and NMDA R1 gene expression inhippocampus of pilocarpine-induced epileptic rats neuropro-tective role of Bacopa monnieri extractrdquo Epilepsy and Behaviorvol 12 no 1 pp 54ndash60 2008

[33] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[34] K V H Sastry R P Moudgal J Mohan J S Tyagi and G SRao ldquoSpectrophotometric determination of serum nitrite andnitrate by copper-cadmium alloyrdquo Analytical Biochemistry vol306 no 1 pp 79ndash82 2002

[35] D R Lynch and R P Guttmann ldquoNMDA receptor pharma-cology perspectives from molecular biologyrdquo Current DrugTargets vol 2 no 3 pp 215ndash231 2001

[36] L V Kalia S K Kalia and M W Salter ldquoNMDA receptorsin clinical neurology excitatory times aheadrdquo The LancetNeurology vol 7 no 8 pp 742ndash755 2008

[37] A Lau and M Tymianski ldquoGlutamate receptors neurotoxicityand neurodegenerationrdquo Pflugers Archiv European Journal ofPhysiology vol 460 no 2 pp 525ndash542 2010

[38] M M Zeron O Hansson N Chen et al ldquoIncreased sensitivityto N-methyl-D-aspartate receptor-mediated excitotoxicity in amousemodel ofHuntingtonrsquos diseaserdquoNeuron vol 33 no 6 pp849ndash860 2002

[39] A J Williams J R Dave X M Lu G Ling and F C TortellaldquoSelective NR2B NMDA receptor antagonists are protectiveagainst staurosporine-induced apoptosisrdquo European Journal ofPharmacology vol 452 no 1 pp 135ndash136 2002

[40] M Chen T-J Lu X-J Chen et al ldquoDifferential roles of NMDAreceptor subtypes in ischemic neuronal cell death and ischemictolerancerdquo Stroke vol 39 no 11 pp 3042ndash3048 2008

[41] K Erreger S M Dravid T G Banke D J A Wyllie and SF Traynelis ldquoSubunit-specific gating controls rat NR1NR2AandNR1NR2BNMDAchannel kinetics and synaptic signallingprofilesrdquoThe Journal of Physiology vol 563 no 2 pp 345ndash3582005

[42] C S Paulose F Chathu S Reas Khan and A KrishnakumarldquoNeuroprotective role of Bacopa monnieri extract in epilepsyand effect of glucose supplementation during hypoxia gluta-mate receptor gene expressionrdquoNeurochemical Research vol 33no 9 pp 1663ndash1671 2008

[43] V L R Rao R M Audet and R F Butterworth ldquoIncreasednitric oxide synthase activities and L-[3H]arginine uptake inbrain following portacaval anastomosisrdquo Journal of Neurochem-istry vol 65 no 2 pp 677ndash681 1995

[44] P Monfort M-D Munoz A ElAyadi E Kosenko and VFelipo ldquoEffects of hyperammonemia and liver failure on glu-tamatergic neurotransmissionrdquoMetabolic Brain Disease vol 17no 4 pp 237ndash250 2002

[45] C M Snyder E H Shroff J Liu and N S Chandel ldquoNitricoxide induces cell death by regulating anti-apoptotic Bcl2 familymembersrdquo PLoS ONE vol 4 no 9 Article ID e7059 2009

[46] R K Koiri and S K Trigun ldquoDimethyl sulfoxide acti-vates tumor necrosis factor 120572-p53 mediated apoptosis anddown regulates D-fructose-6-phosphate-2-kinase and lactatedehydrogenase-5 in Daltonrsquos Lymphoma in vivordquo LeukemiaResearch vol 35 no 7 pp 950ndash956 2011

[47] R B Thomas S Joy M S Ajayan and C S Paulose ldquoNeuro-protective potential of bacopa monnieri and bacoside a againstdopamine receptor dysfunction in the cerebral cortex of neona-tal hypoglycaemic ratsrdquo Cellular and Molecular Neurobiologyvol 33 no 8 pp 1065ndash1074 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

[17] O A Shipton and O Paulsen ldquoGluN2A and GluN2B subunit-containing NMDA receptors in hippocampal plasticityrdquo Philo-sophical Transactions of the Royal Society B Biological Sciencesvol 369 no 1633 2014

[18] C Montoliu R Rodrigo P Monfort et al ldquoCyclic GMP path-ways in hepatic encephalopathy neurological and therapeuticimplicationsrdquoMetabolic Brain Disease vol 25 no 1 pp 39ndash482010

[19] M V Doucet A Harkin and K K Dev ldquoThe PSD-95nNOScomplex new drugs for depressionrdquo Pharmacology amp Thera-peutics vol 133 no 2 pp 218ndash229 2012

[20] H Prast andA Philippu ldquoNitric oxide asmodulator of neuronalfunctionrdquo Progress in Neurobiology vol 64 no 1 pp 51ndash682001

[21] F X Guix I Uribesalgo M Coma and F J Munoz ldquoThephysiology and pathophysiology of nitric oxide in the brainrdquoProgress in Neurobiology vol 76 no 2 pp 126ndash152 2005

[22] E A Waxman and D R Lynch ldquoN-methyl-D-aspartate recep-tor subtypes multiple roles in excitotoxicity and neurologicaldiseaserdquo Neuroscientist vol 11 no 1 pp 37ndash49 2005

[23] V Felipo ldquoContribution of altered signal transduction asso-ciated to glutamate receptors in brain to the neurologicalalterations of hepatic encephalopathyrdquo World Journal of Gas-troenterology vol 12 no 48 pp 7737ndash7743 2006

[24] M Llansola R Rodrigo P Monfort et al ldquoNMDA receptorsin hyperammonemia and hepatic encephalopathyrdquo MetabolicBrain Disease vol 22 no 3-4 pp 321ndash335 2007

[25] S A Lipton and P A Rosenberg ldquoMechanisms of disease exci-tatory amino acids as a final common pathway for neurologicdisordersrdquoTheNew England Journal of Medicine vol 330 no 9pp 613ndash622 1994

[26] D R Lynch and R P Guttmann ldquoExcitotoxicity perspectivesbased on N-methyl-D-aspartate receptor subtypesrdquo Journal ofPharmacology and Experimental Therapeutics vol 300 no 3pp 717ndash723 2002

[27] C Ikonomidou and L Turski ldquoWhy did NMDA receptorantagonists fail clinical trials for stroke and traumatic braininjuryrdquoThe Lancet Neurology vol 1 no 6 pp 383ndash386 2002

[28] K J Gohil and J J Patel ldquoA review onBacopamonniera currentresearch and future prospectsrdquo International Journal of GreenPharmacy vol 4 no 1 pp 1ndash9 2010

[29] A Krishnakumar P M Abraham J Paul and C S PauloseldquoDown-regulation of cerebellar 5-HT2C receptors inpilocarpine-induced epilepsy in rats therapeutic role ofBacopa monnieri extractrdquo Journal of the Neurological Sciencesvol 284 no 1-2 pp 124ndash128 2009

[30] M Singh V Murthy and C Ramassamy ldquoNeuroprotectivemechanisms of the standardized extract of Bacopa monnierain a paraquatdiquat-mediated acute toxicityrdquo NeurochemistryInternational vol 62 no 5 pp 530ndash539 2013

[31] N Uabundit J Wattanathorn S Mucimapura and K Ingkan-inan ldquoCognitive enhancement and neuroprotective effects ofBacopa monnieri in Alzheimerrsquos disease modelrdquo Journal ofEthnopharmacology vol 127 no 1 pp 26ndash31 2010

[32] R Khan A Krishnakumar and C S Paulose ldquoDecreasedglutamate receptor binding and NMDA R1 gene expression inhippocampus of pilocarpine-induced epileptic rats neuropro-tective role of Bacopa monnieri extractrdquo Epilepsy and Behaviorvol 12 no 1 pp 54ndash60 2008

[33] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[34] K V H Sastry R P Moudgal J Mohan J S Tyagi and G SRao ldquoSpectrophotometric determination of serum nitrite andnitrate by copper-cadmium alloyrdquo Analytical Biochemistry vol306 no 1 pp 79ndash82 2002

[35] D R Lynch and R P Guttmann ldquoNMDA receptor pharma-cology perspectives from molecular biologyrdquo Current DrugTargets vol 2 no 3 pp 215ndash231 2001

[36] L V Kalia S K Kalia and M W Salter ldquoNMDA receptorsin clinical neurology excitatory times aheadrdquo The LancetNeurology vol 7 no 8 pp 742ndash755 2008

[37] A Lau and M Tymianski ldquoGlutamate receptors neurotoxicityand neurodegenerationrdquo Pflugers Archiv European Journal ofPhysiology vol 460 no 2 pp 525ndash542 2010

[38] M M Zeron O Hansson N Chen et al ldquoIncreased sensitivityto N-methyl-D-aspartate receptor-mediated excitotoxicity in amousemodel ofHuntingtonrsquos diseaserdquoNeuron vol 33 no 6 pp849ndash860 2002

[39] A J Williams J R Dave X M Lu G Ling and F C TortellaldquoSelective NR2B NMDA receptor antagonists are protectiveagainst staurosporine-induced apoptosisrdquo European Journal ofPharmacology vol 452 no 1 pp 135ndash136 2002

[40] M Chen T-J Lu X-J Chen et al ldquoDifferential roles of NMDAreceptor subtypes in ischemic neuronal cell death and ischemictolerancerdquo Stroke vol 39 no 11 pp 3042ndash3048 2008

[41] K Erreger S M Dravid T G Banke D J A Wyllie and SF Traynelis ldquoSubunit-specific gating controls rat NR1NR2AandNR1NR2BNMDAchannel kinetics and synaptic signallingprofilesrdquoThe Journal of Physiology vol 563 no 2 pp 345ndash3582005

[42] C S Paulose F Chathu S Reas Khan and A KrishnakumarldquoNeuroprotective role of Bacopa monnieri extract in epilepsyand effect of glucose supplementation during hypoxia gluta-mate receptor gene expressionrdquoNeurochemical Research vol 33no 9 pp 1663ndash1671 2008

[43] V L R Rao R M Audet and R F Butterworth ldquoIncreasednitric oxide synthase activities and L-[3H]arginine uptake inbrain following portacaval anastomosisrdquo Journal of Neurochem-istry vol 65 no 2 pp 677ndash681 1995

[44] P Monfort M-D Munoz A ElAyadi E Kosenko and VFelipo ldquoEffects of hyperammonemia and liver failure on glu-tamatergic neurotransmissionrdquoMetabolic Brain Disease vol 17no 4 pp 237ndash250 2002

[45] C M Snyder E H Shroff J Liu and N S Chandel ldquoNitricoxide induces cell death by regulating anti-apoptotic Bcl2 familymembersrdquo PLoS ONE vol 4 no 9 Article ID e7059 2009

[46] R K Koiri and S K Trigun ldquoDimethyl sulfoxide acti-vates tumor necrosis factor 120572-p53 mediated apoptosis anddown regulates D-fructose-6-phosphate-2-kinase and lactatedehydrogenase-5 in Daltonrsquos Lymphoma in vivordquo LeukemiaResearch vol 35 no 7 pp 950ndash956 2011

[47] R B Thomas S Joy M S Ajayan and C S Paulose ldquoNeuro-protective potential of bacopa monnieri and bacoside a againstdopamine receptor dysfunction in the cerebral cortex of neona-tal hypoglycaemic ratsrdquo Cellular and Molecular Neurobiologyvol 33 no 8 pp 1065ndash1074 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom