research article effects of active components of fuzi and...

Research ArticleEffects of Active Components of Fuzi and Gancao Compatibilityon Bax Bcl-2 and Caspase-3 in Chronic Heart Failure Rats

Liqin Wang Yu He Yuyan Zhang Huifen Zhou Li Yu Jiehong Yang and Haitong Wan

Zhejiang Chinese Medical University 548 Binwen Road Hangzhou Zhejiang 310053 China

Correspondence should be addressed to Jiehong Yang yannoo7376126com and Haitong Wan whtong126com

Received 4 July 2016 Revised 2 October 2016 Accepted 9 October 2016

Academic Editor Giuseppe Caminiti

Copyright copy 2016 Liqin Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Hypaconitine (HA) and glycyrrhetinic acid (GA) are active components of Fuzi (Aconitum carmichaelii) and Gancao (Glycyrrhizauralensis Fisch) they have been used in compatibility for chronic heart failure (CHF) fromancient timesThe purpose of the presentresearch was to explore whether apoptosis pathways were related with the protective effects of HA + GA against CHF rats or notThe rats were progressed with transverse-aortic constriction (TAC) operation for 4 weeks to build the CHF state and then theDigoxin (1mgkg) HA (207mgkg) GA (25mgkg) and HA (207mgkg) + GA (25mgkg) were orally administrated to rats for 1week The levels of BNP and cTnI in the plasma were decreased in the HA + GA group and the heartbody weight ratio (HB) andleft ventricular (LV) parameters of transthoracic echocardiography were also declined moreover the expressions of Bax Bcl-2 andcaspase-3 were all improved in the HA + GA group than other groups in the immunohistochemistry and western blot methods Ingeneral the data suggested that Fuzi and Gancao compatibility could protect the CHF rats from apoptosis which provided a strongevidence for further searching for mechanisms of them

1 Introduction

Fuzi is the processed lateral root of Aconitum carmichaeliiDebeaux it has been used in traditional Chinese medicine(TCM) for thousands of years Fuzi has widespread biologicalactivities besides the effects on cardiovascular system thereare also including many other systems effects such as theimmune system the kidney system and the metabolismsystem The common effects reveal as anti-inflammationand analgesic actions antitumor activity and antiaging Ithas been extensively used for chronic heart failure (CHF)low blood pressure coronary heart disease and so on It issaid that 122 chemical constituents have been isolated andidentified from Fuzi among which C

19-diterpenoid alkaloids

and C20-diterpenoid alkaloids are the predominant ingredi-

ents [1] Aconite alkaloids have been proved to be primarytherapeutic as well as toxic in Fuzi The aconitines of Fuzisuch as aconitine hypaconitine (HA) and mesaconitinewere detected through gut sac model and in situ single-pass intestinal perfusion model in the Fuzi-Gancao herb-pair precipitation The results indicated that 3 diester diter-penoid alkaloids in Fuzi-Gancao could be decomposed in

gastrointestinal tract and then absorbed in blood after oraladministration and the permeability of HA appeared bestin ileum [2] The chromatographic fingerprinting of HA wasachieved [3] and the structure of HA is shown in Figure 1(a)It has been found that theT (max) of HAdelayed significantlyinDahuangFuzi Decoction group compared with the value ofFuzi group that is to say HA has a longer metabolite time invivo maybe it has a better effect time with combination herbsthan other aconitines [4]

In TCM Gancao (Glycyrrhiza uralensis Fisch) is alwaysused as a concerted application of herbs and the acid sub-stance is one of its major activity ingredients Glycyrrhetinicacid (GA) is the metabolite of glycyrrhizinic acid (GL) invivo [5] the fingerprinting of GA had been obtained [6] andthe structure of GA is shown in Figure 1(b) There have beenmany researches about GA such as the metabolic processescaused by mesaconitine-treated rats that could be mitigatedby prophylaxis with GA which meant that pretreatmentwith GA might reduce the toxicity of mesaconitine in themetabolic level [7] GA has also been proved to be a typicalactive component of Shaoyao-Gancao Decoction for painrelief and be the major active constituent of Kushen-Gancao

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 7686045 12 pageshttpdxdoiorg10115520167686045

2 Evidence-Based Complementary and Alternative Medicine

OH

109

8

765

112

4

1

NMe

1918

OMe

OAc

OC14

O

OHH

1712

13 16OMe

OMe

15

3

1998400

16998400

1998400998400

2998400998400 3998400998400

4998400998400

5998400998400

6998400998400

6998400

18998400H3CO

(a)

COOH

HO

O

(b)

Figure 1 (a) Chemical structure of hypaconitine (HA) molecular formula C33H45NO10 (b) chemical structure of Glycyrrhetinic acid (GA)

molecular formula C30H46O4

Decoction after oral administration in rat by HPLC-MSMS[8 9] In another study about the comparative pharmacoki-netic profiles of GA GL and Gancao-Fuzi-Tang after orallytaking GL and Gancao-Fuzi-Tang the results demonstratedan increased effect ofGAafter oral administration ofGancao-Fuzi-Tang in comparison with GL which implied the GAwasone of the crucial elements in Gancao-Fuzi-Tang [10]

As we all know TCMconsists ofmany kinds of herbs andit is a complex system Every Chinese herb performs one ormore specific functions when prescribed alone Neverthelessthey will play much more complicated effects when usedin combination Because TCM includes lots of chemicalcomponents they influence each other even sometimes theherb compatibility produces new substances and results inmultiply therapeutic effects These actions are always causedby multicompounds For example the Fuzi-Gancao herb-pair generates the reducing toxicities and enhancing effectsmay be related on the interaction of acid and base whichbrings about some other new chemical compounds [11] Inconclusions to study the effects of Fuzi-Gancao herb pairwe chose HA and GA on behalf of this herb pair for furtherinvestigations

2 Materials and Methods

21 Material HA (purity ge98) and GA (purity ge98)were purchased from Baoji Chenguang Biotechnology CoLtd (Xirsquoan China) Digoxin pills were obtained from Shang-hai sym pharma Co Ltd (Shanghai China) penicillin wasbought from REYOUNG Co Ltd (Shandong China) isofl-urane was purchased from Shenzhen Huabao Medical Sup-plies Industry amp Trade Development Co Ltd (ShenzhenChina) B-type natriuretic peptide (BNP) and cardiac tro-ponin I (cTnI) ELISA kits were bought from Shanghai yua-nye biological agent company (Shanghai China) anti-Baxanti-Bcl-2 anti-caspase-3 and anti-120573-actin antibodies were

obtained from Santa Cruz Biotechnology Co (CA USA)immunohistochemical detection kit was purchased fromBeijing Zhongshan Biotechnology Co Ltd (Beijing China)(import packing)

22 Animal Healthy male SPF rats (body weight 160ndash170 g)were provided by Shanghai Slack Laboratory Animal CoLtd (animal license number SCXK (Shanghai) 2012-0002animal use certificate number SYXK (Zhejiang) 2013-184)The rats were watered ad libitum and fed a standard dietThe animals were maintained in the 12 h dark and 12 h lightcycle at the temperature of 20 plusmn 2∘C and the humidity was50 plusmn 2 All animal experimental protocols were approvedby the Animal Care and Use Committee of Zhejiang ChineseMedical University and complied with laboratory animalmanagement and use regulations

23 Animal Model The CHF model was induced bytransverse-aortic constriction (TAC) operation which wasmodified from deAlmeidarsquos methods [12] In this study it wasperformed after the rats were allowed to acclimate for 1 weekThe rats were anesthetized by inhalation of 20 isofluraneand then they were endotracheally intubated with positive-pressure ventilation inputting 100 oxygen gas with a tidalvolume of 250120583L at a velocity of 120 breaths per minuteby a Small Animal Ventilator (Hallowell Inc USA) Afteropening the chest wall via an upper sternotomy the aortawas carefully picked out A self-regulating ldquoLrdquo needle with theexternal diameter of approximately 09mmwas placed on theaorta on which a 3-0 prolene ligation was placed betweenthe right and left common carotid arteries The needle wasremoved cautiously and the aortic constriction was then cre-ated After the procedure the wound was closed respectivelyand the animal was allowed to recover from anesthesia withobservation Penicillin was injected intramuscular once a day

Evidence-Based Complementary and Alternative Medicine 3

for 3 days The rats in Sham group were processed as Modelgroup but without TAC operation

We chose the TAC operation to create chronic pressureoverload responses in the cardiac myocyte in order to induceCHF and after 4 weeksrsquo operation the major situations of ratswere in the cardiac hypertrophy period [13]

24 Experimental Protocol RatswithTACoperationwere fedand watered normally for 4 weeks and then the surviving ratswere randomly divided into six groups Sham group (119899 = 6)Model group (119899 = 6) Digoxin group (119899 = 6) HA group(119899 = 8) GA group (119899 = 7) and HA + GA group (119899 = 7)According to our previous work and professor Chen [14] thedosage of GA orally administered to rats was 25mgkg perday And based on the preliminary tests [15] and the dosageof HA required to kill half of a tested population (LD50 valueper day) the dosage of HA was chosen as 207mgkg perday Digoxin (1mgkg) HA (207mgkg) GA (25mgkg)and HA (207mgkg) + GA (25mgkg) were administered byintragastric once a day for 1 week and an equal volume ofnormal saline was fed to the rats in Sham and Model groupsAt the end of oral administration in the HA group two ratsdied and the other groups all survived

25 Transthoracic Echocardiography An atraumatic transt-horacic echocardiography method [16] was used to evaluatethe morphology and function of the heart Rats were anes-thetized by inhalation of 20 isoflurane before transtho-racic echocardiography by Vevo2100 imaging system (VisualSonics Inc Toronto Canada) with a 30MHz probe Stablepictures and measurements were obtained from B-mode andM-mode

26 Sample Preparation After 1 weekrsquos treatment the ratswere weighed and anesthetized The blood was drew fromthe abdominal aortic and immediately the plasma wasobtained from supernatant of centrifugation of the bloodThe heart were gained and weighedThen the left ventriculartissues were separated into double parts one fixed in 4paraformaldehyde and embedded in paraffin for histologicalanalysis and the other preserved in minus80∘C for assessing ofprotein expressions

27 Enzyme-Linked Immunosorbent Assay (ELISA) Theplasma samples for ELISA analysis were prepared followingthe instructions and levels of BNP and cTnI were detected byELISA kits according to the manufacturerrsquos instructions Theoptical density (OD) values from the samples were measuredwith a microplate reader in the 490 nm wavelength

28 Immunohistochemistry Staining Immunohistochemicalstaining was handled on formalin-fixed paraffin-embedded3120583m sections and the sections were rehydrated Theywere incubated with anti-Bax anti-Bcl-2 and anti-caspase-3 antibody (1 200) 60min at 37∘C rewashed with PBSand then used horseradish peroxidase coupled goat sec-ondary antibodies incubated 40min at 37∘CThe tissues werethen washed by PBS and incubated in Diaminobenzidine(DAB) substrate The sections were finally counterstained

Table 1 The effects of HA + GA on CHF rats in HB and LVdysfunction (119909 plusmn 119904)

Group HB (mgg) EF () FS ()Sham 030 plusmn 001 7244 plusmn 063 4335 plusmn 120

Model 051 plusmn 002 9229 plusmn 069 5708 plusmn 130

Digoxin 033 plusmn 001lowastlowast 7740 plusmn 172lowast 4841 plusmn 091lowast

HA 048 plusmn 002 8959 plusmn 148 5471 plusmn 017

GA 043 plusmn 001 8669 plusmn 105lowast 5268 plusmn 058

HA + GA 036 plusmn 001lowast 8056 plusmn 039lowastlowast 4990 plusmn 073lowast

Note HA hypaconitine GA glycyrrhetinic acid HB the heartbodyweight ratio EF ejection fraction FS fractional shortening Sham group(119899 = 6) Model group (119899 = 6) Digoxin group (119899 = 6) HA group (119899 = 6)GA group (119899 = 7) and HA + GA group (119899 = 7) All data were presentedas mean plusmn SD 119875 lt 001 versus Sham group lowast119875 lt 005 and lowastlowast119875 lt 001versus Model group

with hematoxylin and xylene before being rewashed inethanol Images were collected by DAB light microscope(Leica Microsystems Ltd Wetzlar Germany) Integratedoptical density (IOD) was obtained to represent the relativeamount of positive staining and 5 fields (magnification 200x)were randomly chosen to analyze All immunohistochemicalresults were repeated at least three times and the representa-tive images were presented

29 Western Blot Analysis The cryopreserved heart tissueswere homogenized and then lysed in an ice-cold proteinlysis buffer and centrifuged at 14000 rpm for 5 minutes at4∘C to collect proteins [17] The protein concentrations weredetermined by BCA methods using a BCA protein assay kit(Beyotime Biotechnology Hangzhou Zhejiang China) Therelevant proteins were separated using 15 SDS-PAGE andtransferred to polyvinylidene difluoride (PVDF)membranesand then theywere probedwith rabbit anti-rat polyclone anti-Bax anti-Bcl and anti-caspase-3 at 4∘C overnight Themem-branes were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1 2000) for 2 h at room temper-ature The blots were visualized with ECL-Plus reagent (GEHealthcare Pisca-taway NJ) In these experiments the blotswere reprobed with an anti-120573-actin antibody to control theprotein loading Image J (NIH image Bethesda MD) wasapplied to analyze the gel images

210 Statistical Analysis All experimental data are displayedas the mean plusmn the standard deviation (SD) single factoranalysis of variance (ANOVA)was performed using SPSS 190statistical software followed by Tukeyrsquos test In this study thevalue at 119875 lt 005 was considered statistically significant

3 Results

31 Effects of HA + GA on Left Ventricular (LV) CardiacDysfunction and Remodeling The heartbody weight ratio(HB) and transthoracic echocardiography results about LVcardiac dysfunction are presented in Table 1 As has beenstated above the rats were in cardiac hypertrophy periodsso the ejection fraction (EF) and fractional shortening (FS)


Sham Model Digoxin

HA GA HA + GA

Figure 2 Typical transthoracic echocardiography images fromeffects of HA + GA on CHF rats HA hypaconitine GA gly-cyrrhetinic acid Sham group (119899 = 6) Model group (119899 = 6) Digoxingroup (119899 = 6) HA group (119899 = 6) GA group (119899 = 7) and HA + GAgroup (119899 = 7)

measurements were higher than the CHF standards Butafter the treatments they changed respectively Comparedwith the Sham group the HB EF and FS were elevateddramatically in the Model group (119875 lt 001) Comparedwith the Model group the HB EF and FS of the HA grouphad no significant difference (119875 gt 005) but the EF of GAgroup and all of the measurements in the Digoxin and HA +GA group were decreased (119875 lt 005) especially the HB inDigoxin group and the EF in HA +GA group (119875 lt 001)Theresults revealed that HA + GA could protect the CHF rat onregulating LV dysfunctions particularly on the EF

32 Effects ofHA+GAonLVCardiac Remodeling Theresultsof LV cardiac remodeling in LV end-diastolic dimension(LVIDd) LV end-systolic dimension (LVIDs) the LV end-diastolic posterior wall (LVPWd) and the LV end-systolicposterior wall (LVPWs) were showed in Figure 2 and Table 2Comparing to the Sham group the values were all ascendedin the Model group (119875 lt 001) When compared with theModel group in the measurement of LVIDd except the HAgroup (119875 gt 005) the other groups all declined distinctly(119875 lt 001) in the measurement of LVIDs the Digoxin andHA + GA groups descended apparently (119875 lt 001) the HAandGAgroups had no significant difference (119875 gt 005) in themeasurement of LVPWd the GA group receded it (119875 lt 005)and the other groups were all reduced dramatically (119875 lt001) in the levels of LVPWs the HA + GA group was obvi-ously declined (119875 lt 001) and the Digoxin and GA groups(119875 lt 005) and the HA group had no significance difference(119875 gt 005) The results demonstrated that the parameters ofLV remodeling were significantly modified by the HA + GAtreatment much better than the HA and GA groups

33 Effects of HA + GA on CHF Rats in the Levels of BNP andcTnI Cardiac functions were determined by BNP and cTnIELISA assays as revealed in Figures 3(a) and 3(b) In contrastwith the Sham group theModel group extremely elevated thereleases of BNP and cTnI levels in the plasmaof CHF rats (119875 lt001) When comparing with the Model group the DigoxinHAGA andHA+GAgroups all attenuated the levels of BNPand cTnI (119875 lt 001) and from the amounts of the releaseswe could conclude the HA + GA group ameliorated the CHFstate better than HA and GA groups

34 Effects of HA + GA on Bax Bcl-2 and Caspase-3Protein Expressions by Immunohistochemistry The immuno-histochemistry staining was used to detect the expressions ofBax Bcl-2 and caspase-3 these proteins were related withapoptosis In this method the positive parts were stainedto brown as displayed in Figures 4(a) 5(a) and 6(a) Thequantitative image analyses were performed based on theIODof positive immunostaining as exhibited in Figures 4(b)5(b) and 6(b) besides the ratio of Bcl-2Bax in IOD wasshowed in Figure 5(c) Comparing to the Sham group theIOD of Bax and caspase-3 appeared to be uptrend (119875 lt 001)and the IOD of Bcl-2 and ratio of Bcl-2Bax performed tobe in downtrend (119875 lt 001) When compared to the Modelgroup the IODof Bax and caspase-3 inDigoxin HAGA andHA +GA groups emerged to be dropped (119875 lt 001) with theHA + GA group holding the least quantity the IOD of Bcl-2and ratio of Bcl-2Bax in Digoxin GA and HA + GA groupsrevealed to be raised dramatically (119875 lt 001) and the IOD ofBcl-2 in the HA group also increased (119875 lt 005) but the ratioof Bcl-2Bax showed no significant difference (119875 gt 005) Wecould summarize that the HA+GA group improved the anti-apoptosis effects on the CHF rats

35 Effects of HA + GA on Expressions of Bax Bcl-2 andCaspase-3 byWestern Blot Method Thewestern blot methodis a frequently used for further study of protein expres-sions in this research the representative photographs weremanifested in Figure 7(a) and the quantitative analysis ofprotein expressions of Bax Bcl-2 and caspase-3 was showedin Figure 7(b) By comparison with the Sham group theModel group elevated the expressions of Bax and caspase-3 proteins (119875 lt 001) in the meantime the Model grouphad lower levels of the expression of Bcl-2 protein and ratioof Bcl-2Bax (119875 lt 001) While in contrast with the Modelgroup except for the expression of caspase-3 in the HA grouphad no significant difference (119875 gt 005) all the other groupsameliorated the expressions of Bax and caspase-3 greatly(119875 lt 001) with the HA + GA group being much less thanthe HA and GA groups at the same time all the groupsimproved the expression of Bcl-2 protein and ratio of Bcl-2Bax tremendously (119875 lt 001) with the HA + GA groupin a much higher degree than the HA and GA groups So wedrew the conclusion that the HA+GA could protect the CHFrats from apoptosis better than the HA and GA groups

4 Discussion

CHFhas been a worldwide health problem with a prevalenceof over 23 million rising year by year Its prevalence greatlyincreases with advancing age while the percentage is 07in 45 to 54-year-old people compared with 84 in peopleaged more than 75 years [18] In clinic the CHF also has highrates of outpatient visits hospitalizations and readmissionsCHF is not a simple single disease but the final stage of manyheart diseases with a series of clinical syndromes that mayhave been caused by myocardial hypertrophy hypertensionischemic heart disease and so on [19 20] Despite themanagement and therapymethods advanced themortality of


Sham Model Digoxin HA GA HA + GA

lowastlowast

lowastlowastlowastlowast

lowastlowast

BNP

(pg

mL)

1200

800

400

0

(a)


lowastlowast

lowastlowast

lowastlowast lowastlowast

cTnI

(ng

mL)

24

16

8

0

(b)

Figure 3 Effects of HA + GA on CHF rats in levels of BNP and cTnI by ELISA kits (a) The levels of BNP in the plasma (b) the levels ofcTnI in the plasma HA hypaconitine GA glycyrrhetinic acid Sham group (119899 = 6) Model group (119899 = 6) Digoxin group (119899 = 6) HA group(119899 = 6) GA group (119899 = 7) and HA + GA group (119899 = 7) All data were presented as mean plusmn SD 119875 lt 001 versus Sham group lowastlowast119875 lt 001versusModel group

Table 2 The effects of HA + GA on CHF rats in LV remodeling (119909 plusmn 119904)

Group LVIDd (mm) LVIDs (mm) LVPWd (mm) LVPWs (mm)Sham 375 plusmn 020 224 plusmn 017 216 plusmn 011 253 plusmn 014

Model 804 plusmn 028 473 plusmn 015 456 plusmn 003 494 plusmn 021

Digoxin 482 plusmn 086lowastlowast 253 plusmn 008lowastlowast 238 plusmn 010lowastlowast 301 plusmn 007lowast

HA 754 plusmn 007 404 plusmn 002 310 plusmn 005lowastlowast 452 plusmn 011

GA 698 plusmn 017lowastlowast 328 plusmn 027 280 plusmn 018lowast 378 plusmn 017lowast

HA + GA 642 plusmn 015lowastlowast 275 plusmn 013lowastlowast 251 plusmn 003lowastlowast 328 plusmn 019lowastlowast

Note HA hypaconitine GA glycyrrhetinic acid LV left ventricular LVIDd LV end-diastolic dimension LVIDs LV end-systolic dimension LVPWd LV end-diastolic posteriorwall LVPWs LV end-systolic posteriorwall Sham group (119899 = 6) Model group (119899 = 6) Digoxin group (119899 = 6) HAgroup (119899 = 6) GA group(119899 = 7) and HA + GA group (119899 = 7) All data were presented as mean plusmn SD 119875 lt 001 versus Sham group lowast119875 lt 005 and lowastlowast119875 lt 001 versus Model group

CHF still increased along with years Based on the Framing-ham Heart Study 30-day mortality of CHF is approximately10 1-year mortality is about 20ndash30 and 5-year mortalityis between 45 and 60 [21]Themore effective treatments forCHF patients themore surviving rates of older patients couldbe increased [22] In clinic almost of the CHF patients takeChinese herbs or patent medicines combined with westernmedicines because TCM can both improve the cardiacfunctions and reduce the side effects of western medicines

It is generally believed that CHF is based on a complicatedpathological mechanism but without a clear pathogenesis yet[23] Apoptosis is related with CHF and great advances havebeen made in certifying the role of apoptosis in various heartdiseases and also in elucidating themechanisms Beyond thatthe importance of apoptosis in the pathogenesis of CHF andthe relationship between cardiac hypertrophic and apoptosishas been investigated [24] So in this study we selected therelevant apoptotic proteins such as Bax Bcl-2 and caspase-3to testify the pharmacologic effects and relevant mechanismsof HA + GA on CHF rats

Cardiac hypertrophy is classified as pathological whenassociated with cardiac insufficiency The pathological hyper-trophy is induced by lots of factors such as prolonged

and abnormal hemodynamic stress activation of proinflam-matory cytokines and cellular dysfunction and all thesecomplicated responses will cause cardiac remodeling changesand be the potential process to develop into CHF [25 26]Thus the inhibition of cardiac hypertrophy is a promisingtherapeutic option for CHF That is why in this research wegive medicines in the cardiac hypertrophy period of CHFAnd the results of BNP cTnI and EF FS LVIDd LVIDsLVPWd and LVPWs are consistent with these theories

From the perspective of TCM the fundamental problemin heart failure is the deficiency of heart yang and qi whichmakes the heart become tooweak to transport fluid andmoveblood and gradually leads to CHF day after day Fuzi hasthe functions of warming yang dispelling cold removingdampness and so on As for yang the heart yang is the mostcrucial yang There have been lots of studies about Fuzi as themajor medicine in TCM patent medicines such as Shenfudecoction (SFD) which is a water extract of Renshen andFuzi It has been used for CHF for many years it can protectmyocardial structures enhance the heart contractility andeven ameliorate the ischemic myocardial metabolism [27]Though Fuzi has powerful effects in clinic at the sametime it was confined by its toxic effects And Gancao has


(a)

60

40

20

0Sham Model Digoxin HA GA HA + GA

lowastlowast

lowastlowast

lowastlowast

lowastlowast

IOD

of B

ax

(b)

Figure 4 (a) Immunostainingmicrophotographs of Bax (magnification 200x) (b)Quantitative image analyses of Bax proteinwere performedbased on the IOD of positive immunostaining (brown) in the heart tissues All data were presented as mean plusmn SD 119875 lt 001 versus Shamgroup lowastlowast119875 lt 001 versus Model group A Sham group (119899 = 6) B Model group (119899 = 6) C Digoxin group (119899 = 6) D HA group (119899 = 6) EGA group (119899 = 7) and F HA + GA group (119899 = 7) HA hypaconitine GA glycyrrhetinic acid

the functions of tonifying qi and detoxifying In ancienttimes TCM doctors used them together to warming heartyang tonifying heart qi and particularly detoxifying But themechanisms of their functions are still not so well revealedso in the present study we chose the main active componentsof Fuzi and Gancao to explore the effects and possible relatedmechanisms on CHF

As mentioned above the cardiac hypertrophy is theprocess of CHF and we can observe the hypertrophy degreethrough the analysis of HB which is an ordinary measure-ment index reflecting the cardiac remodeling changes [28]In this paper HA + GA group reduced the HB and showed

a better effect than other groups on CHF rats We acquiredthat the combination of HA + GA could ameliorate thecardiac remodeling better than the single activity ingredientAnd in 2016 ESC guidelines for CHF it also points out thattransthoracic echocardiography is a crucial diagnosismethodin CHF [29] In the meantime transthoracic echocardiogra-phy is also often used in diagnosis of cardiovascular diseasesand prognosis of curements For example it can measure thethickness of ventricularwall and carotid intima-media whichare risk factors of cardiovascular diseases And we knowthat heart failure is a state of most cardiovascular diseases[30 31] So in this research we selected the transthoracic


(a)

60

40

20


lowastlowast

lowastlowast

lowastlowast

lowast

IOD

of B

cl-2

(b)


lowastlowast

lowastlowast

lowastlowast

IOD

of B

cl-2

Bax

24

16

08

00

(c)Figure 5 (a) Immunostaining microphotographs of Bcl-2 (magnification 200x) (b) Quantitative image analyses of Bcl-2 protein wereperformed based on the IOD of positive immunostaining (brown) in the heart tissues (c) The ratio of Bcl-2Bax in IOD All data werepresented as mean plusmn SD 119875 lt 001 versus Sham group lowast119875 lt 005 and lowastlowast119875 lt 001 versus Model group A Sham group (119899 = 6) B Modelgroup (119899 = 6) C Digoxin group (119899 = 6) D HA group (119899 = 6) E GA group (119899 = 7) and F HA + GA group (119899 = 7) HA hypaconitine GAglycyrrhetinic acid

echocardiography for evaluations of cardiac functions In theresults we confirmed that HA + GA compatibility coulddescend the parameters of LV remodeling measurementswhich are crucial in improving the cardiac functions in ratswith CHF

The correlation between levels in cardiac troponin anddegrees in cardiac functions has been well established In aresearch the level of cTnIwas significantly higher in heart fail-ure patients and exhibited increasing precise concentrationswith rising NewYorkHeart Association classification of heartfailure severity which meant cTnI is an accurate indicatorfor the diagnosis of heart failure [32] There are also other

studies demonstrated that the release of cTnI is associatedwith increased 1 year mortality and heart failure relatedreadmission and the cTnI level can act as an independentpredicator and complete in prognostic utility of CHF patients[33] The level of plasma BNP is a cost-effective method forevaluating the LV dysfunction And some studies presentedthat BNP-guided therapies showed a decrease in death andhospital stay of CHF patients [34] That means BNP ispositively associated with all-cause mortality of CHF andresearchers also found that the standard of BNP was usefulin estimating prognosis in stable CHF patients [35] In ourstudy we received that the HA + GA group could decrease


(a)

IOD

of c

aspa

se-3


lowastlowast

lowastlowast


27

24

21

18

15

12

9

6

3

0

(b)Figure 6 (a) Immunostaining microphotographs of caspase-3 (magnification 200x) (b) Quantitative image analyses of caspase-3 proteinwere performed based on the IOD of positive immunostaining (brown) in the heart tissues All data were presented as mean plusmn SD 119875 lt 001versus Sham group lowastlowast119875 lt 001 versusModel group A Sham group (119899 = 6) B Model group (119899 = 6) C Digoxin group (119899 = 6) D HA group(119899 = 6) E GA group (119899 = 7) and F HA + GA group (119899 = 7) HA hypaconitine GA glycyrrhetinic acid

the levels of cTnI and BNP in plasma of CHF rats and thecardiac protection was much better than the HA group andGA group and even nearly equaled to the Digoxin groupwhich is still one of the major treatments for CHF in 2016ESC guidelines [29]

As for the relationship between apoptosis and CHF weused immunohistochemistry assays and western blot meth-odswhich belonged to quantitative and qualitative analysis todetect the levels of apoptotic and antiapoptotic proteins suchas Bax Bcl-2 and caspase-3 they are on the downstream ofapoptotic signal pathway and theymediate the final morpho-logical and biochemical alterationswhich are characteristic ofapoptosis [24] The apoptosis process is vital to the survival

of the organism which can cause degenerative diseases if toomuch andmay lead to the onset of proliferative diseases if toolittle that is to say the disturbance in the balance betweencell growth and cell death is very crucial in incentives ofmany diseases so it is of great significance to explore theprotein expressions of apoptosis in diseases [36] It is wellknown that Bcl-2 is a protein that inhibits cell apoptosis andleads to cells survival while Bax is a protein that promotescell apoptosis and leads to cells death There were studiesrevealing that the inhabitation of Bcl-2 and other relatedantiapoptotic proteins could make proliferation pathologicchanges become better [37ndash39] Furthermore a protectiveeffect of ischemic preconditioningwas certified to be involved



Bax

Bcl-2

Caspase-3

120573-Actin

(a)

Bax120573

-act

in

Bcl-2

120573-a

ctin

Bcl-2

Bax

Casp

ase-3

120573-a

ctin

012

008

004

000

009

006

003

000

4

3

2

1

0

0004

0002

0000

(A) (B)

(C) (D)

Sham Model Digoxin HA GA HA + GA Sham Model Digoxin HA GA HA + GA

Sham Model Digoxin HA GA HA + GASham Model Digoxin HA GA HA + GA


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

Figure 7 (a)The representative photographs of western blot in expressions of Bax Bcl-2 and caspase-3 proteins in heart tissues of CHF rats(b) The quantitative analysis of Bax Bcl-2 and caspase-3 protein expressions (A) Expressions of Bax (B) expressions of Bcl-2 (C) the ratioof Bcl-2Bax of protein expressions (D) expressions of caspase-3 HA hypaconitine GA glycyrrhetinic acid Sham group (119899 = 6) Modelgroup (119899 = 6) Digoxin group (119899 = 6) HA group (119899 = 6) GA group (119899 = 7) and HA + GA group (119899 = 7) All data were presented as mean plusmnSD 119875 lt 001 versus Sham group lowastlowast119875 lt 001 versus Model group


with upregulation of Bcl-2 [40] Besides the ratio betweenantiapoptotic and proapoptotic proteins is considered to bea key index for estimating homeostasis states in survival ofcells [41] for example the change of Bcl-2Bax ratio wassuggested to promote cell apoptosis in an apoptotic stimulus[42] while other studies demonstrated that the decrease ofthe Bcl-2Bax ratio might lead to an increase rate of apoptosisin cardiomyocytes [43]

In the activity of apoptosis pathway we know thatcaspase-3 is a fatal factor and it is one of the major caspasesfamilies which are involved in the final execution step ofapoptosis process Caspase-3 is responsible for the cleavageof numerous other apoptosis proteins In addition caspase-3 can cause a decrease in the amount of Bcl-2 protein andin contrast Bcl-2 protein also inhibits caspsae-3 protein [42]There were studies as well showing that the regulation ofcaspase-3 could reflect the anti-apoptosis effects in medicineson CHF patients [44] What is more the vital role of caspasesin apoptosis process makes them a potential target for anti-apoptotic therapies For example the over expression of p35in a pacing-induced CHF model resulting in the decrease ofcaspase-3 activity and turned to be related to the improvedcardiac functions [45] In present research we obtainedthat the HA + GA group upregulated the expression ofBcl-2 protein and the ratio of Bcl-2Bax while it reverselydownregulated the expressions of Bax and caspase-3 proteinsThat means the combination of HA and GA could protectthe cardiomyocytes from apoptosis and the effects mightbe associated with apoptotic signal pathways belonging tothe mitochondrial pathway such as PI3KAkt pathway orothers Although our findings can provide some strategiesfor modulating cardiac apoptosis further investigation isrequired to clarify the exact pathway about the anti-apoptosiseffect on CHF rats by Fuzi and Gancao

5 Conclusions

In conclusion HA + GA could significantly inhibit the devel-opment of cardiac hypertrophy remodeling and dysfunctionin the CHF processing and the underlying mechanismmaybe attributed to the attenuation of cardiac apoptosisAlthough the exact mechanism of the effects of HA + GAremains to be further explored our data point out thedirection for the upcoming mechanism research for Fuzi andGancao compatibility on CHF patients

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

Liqin Wang and Yu He contributed equally to this paper

Acknowledgments

This study was supported by grants from theNational NaturalScience Foundation of China (nos 81473412 81274176 and

81573868) The Zhejiang Provincial Natural Science Foun-dation of China (no LZ14H270001 LR13H270001) ZhejiangProvincial Program for the Cultivation of High-level Inno-vative Health talents The first author sincerely thanks otherauthors of all experimental work for preparations of thismanuscript

References

[1] G Zhou L Tang X Zhou T Wang Z Kou and Z WangldquoA review on phytochemistry and pharmacological activities ofthe processed lateral root of Aconitum carmichaelii DebeauxrdquoJournal of Ethnopharmacology vol 160 pp 173ndash193 2015

[2] J-M Zhang W Liao Y-X He Y He D Yan and C-MFu ldquoStudy on intestinal absorption and pharmacokinetic char-acterization of diester diterpenoid alkaloids in precipitationderived from Fuzi-Gancao herb-pair decoction for its potentialinteractionmechanism investigationrdquo Journal of Ethnopharma-cology vol 147 no 1 pp 128ndash135 2013

[3] N Zhang Y Song Q Song et al ldquoQualitative and quantitativeassessments of Aconiti lateralis radix praeparata using high-performance liquid chromatography coupled with diode arraydetection and hybrid ion trap-time-of-flight mass spectrome-tryrdquo Journal of Chromatographic Science vol 54 no 6 pp 888ndash901 2016

[4] X Liu H Li X Song et al ldquoComparative pharmacokineticsstudies of benzoylhypaconine benzoylmesaconine benzoyla-conine and hypaconitine in rats by LC-MS method after admi-nistration of Radix Aconiti Lateralis Praeparata extract andDahuang Fuzi Decoctionrdquo Biomedical Chromatography vol 28no 7 pp 966ndash973 2014

[5] R Fan N Li H Xu J Xiang L Wang and Y Gao ldquoThemechanism of hydrothermal hydrolysis for glycyrrhizic acidinto glycyrrhetinic acid and glycyrrhetinic acid 3-O-mono-120573-d-glucuronide in subcritical waterrdquo Food Chemistry vol 190 pp912ndash921 2016

[6] F Rauchensteiner Y Matsumura Y Yamamoto S Yamajiand T Tani ldquoAnalysis and comparison of Radix Glycyrrhizae(licorice) fromEurope and China by capillary-zone electropho-resis (CZE)rdquo Journal of Pharmaceutical and Biomedical Analysisvol 38 no 4 pp 594ndash600 2005

[7] B Sun M Zhang Q Zhang et al ldquoMetabonomics studyof the effects of pretreatment with glycyrrhetinic acid onmesaconitine-induced toxicity in ratsrdquo Journal of Ethnopharma-cology vol 154 no 3 pp 839ndash846 2014

[8] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats Competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[9] Q Wang L Shi X Tang Q Wang X Dang and Y ZhangldquoPharmacokinetic study of multiple active constituents fromKushen-Gancao Decoction after oral administration in ratby HPLC-MSMSrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 965 pp 19ndash26 2014

[10] Q-T Gao X-H Chen and K-S Bi ldquoComparative pharma-cokinetic behavior of glycyrrhetic acid after oral administrationof glycyrrhizic acid and Gancao-Fuzi-Tangrdquo Biological andPharmaceutical Bulletin vol 27 no 2 pp 226ndash228 2004

[11] G Zhu G Zhang M Wang J Wang W Zeng and X GaoldquoSimultaneous determination of nine active compounds of the


traditional Chinese medicinal prescription Shaoyao-Gancao-Tang and analysis of the relationship between therapeuticaleffect and compatibility of medicinesrdquo Evidence-Based Comple-mentary and Alternative Medicine vol 2014 Article ID 52103810 pages 2014

[12] A C deAlmeida R J van Oort and X H T WehrensldquoTransverse aortic constriction in micerdquo Journal of VisualizedExperiments JoVE no 38 article 1729 2010

[13] T J LaRocca D Jeong E Kohlbrenner et al ldquoCXCR4 genetransfer prevents pressure overload induced heart failurerdquoJournal of Molecular and Cellular Cardiology vol 53 no 2 pp223ndash232 2012

[14] P-P Xing W-HWu P Du F-MHan and Y Chen ldquoEffects ofbrucine combined with glycyrrhetinic acid or liquiritin on rathepatic cytochromeP450 activities in vivordquoActa PharmaceuticaSinica vol 46 no 5 pp 573ndash580 2011

[15] W Peng M J Yi J J Wang et al ldquoEffects of hypaconitinecombined with liquiritin on the mRNA expression of ion chan-nel genes related with cardiac muscle excitation-contractioncouplingrdquo China Journal of Traditional Chinese Medicine andPharmacy vol 28 no 1 pp 117ndash120 2013 (Chinese)

[16] Y Z Zou L Lin Y Ye et al ldquoQiliqiangxin inhibits thedevelopment of cardiac hypertrophy remodeling and dysfunc-tion during 4 weeks of pressure overload in micerdquo Journal ofCardiovascular Pharmacology vol 59 no 3 pp 268ndash280 2012

[17] H-J Lin Y-S Chang L-H Lin C-F Haung C-Y Wu andK-L Ou ldquoAn immunomodulatory protein (Ling Zhi-8) from aGanoderma lucidum induced acceleration of wound healing inrat liver tissues aftermonopolar electrosurgeryrdquoEvidence-BasedComplementary and Alternative Medicine vol 2014 Article ID916531 12 pages 2014

[18] M M Redfield S J Jacobsen J C Burnett Jr D W MahoneyK R Bailey and R J Rodeheffer ldquoBurden of systolic and dias-tolic ventricular dysfunction in the community appreciatingthe scope of the heart failure epidemicrdquo Journal of the AmericanMedical Association vol 289 no 2 pp 194ndash202 2003

[19] A L Bui T BHorwich andG C Fonarow ldquoEpidemiology andrisk profile of heart failurerdquo Nature Reviews Cardiology vol 8no 1 pp 30ndash41 2011

[20] D Yach C Hawkes C L Gould and K J Hofman ldquoTheglobal burden of chronic diseases overcoming impediments toprevention and controlrdquo The Journal of the American MedicalAssociation vol 291 no 21 pp 2616ndash2622 2004

[21] D Levy S Kenchaiah M G Larson et al ldquoLong-term trendsin the incidence of and survival with heart failurerdquo The NewEngland Journal ofMedicine vol 347 no 18 pp 1397ndash1402 2002

[22] L Bonneux J J Barendregt K Meeter G J Bonsel and P JVan der Maas ldquoEstimating clinical morbidity due to ischemicheart disease and congestive heart failure the future rise of heartfailurerdquoAmerican Journal of Public Health vol 84 no 1 pp 20ndash28 1994

[23] A Yu J Zhang H Liu B Liu and L Meng ldquoIdentificationof nondiabetic heart failure-associated genes by bioinformaticsapproaches in patients with dilated ischemic cardiomyopathyrdquoExperimental andTherapeutic Medicine vol 11 no 6 pp 2602ndash2608 2016

[24] PM Kang and S Izumo ldquoApoptosis in heart basicmechanismsand implications in cardiovascular diseasesrdquo Trends in Molecu-lar Medicine vol 9 no 4 pp 177ndash182 2003

[25] I Shimizu and T Minamino ldquoPhysiological and pathologicalcardiac hypertrophyrdquo Journal of Molecular and Cellular Cardi-ology vol 97 pp 245ndash262 2016

[26] Z G Ma J Dai W Y Wei et al ldquoAsiatic acid protects againstcardiac hypertrophy through activating AMPK120572 signallingpathwayrdquo International Journal of Biological Sciences vol 12 no7 pp 861ndash871 2016

[27] HWei HWuW Yu X Yan and X Zhang ldquoShenfu decoctionas adjuvant therapy for improving quality of life and hepaticdysfunction in patientswith symptomatic chronic heart failurerdquoJournal of Ethnopharmacology vol 169 pp 347ndash355 2015

[28] A E Khodir H A Ghoneim M A Rahim and G SuddekldquoMontelukast attenuates lipopolysaccharide-induced cardiacinjury in ratsrdquo Human amp Experimental Toxicology vol 35 no4 pp 388ndash397 2016

[29] P Ponikowski A A Voors S D Anker et al ldquo2016 ESCGuidelines for the diagnosis and treatment of acute and chronicheart failure the Task Force for the diagnosis and treatmentof acute and chronic heart failure of the European Society ofCardiology (ESC) developed with the special contribution ofthe Heart Failure Association (HFA) of the ESCrdquo EuropeanHeart Journal vol 37 no 27 pp 2129ndash2200 2016

[30] R Laszlo T Baumann H Konz et al ldquoRight ventricularfunction assessed by tissue Doppler echocardiography in oldersubjects without evidence for structural cardiac diseaserdquo AgingClinical and Experimental Research 2016

[31] M Evran G Akkus I Berk Bozdogan et al ldquoCarotidintima-media thickness as the cardiometabolic risk indicatorin patients with nonfunctional adrenal mass and metabolicsyndrome screeningrdquoMedical Science Monitor vol 22 pp 991ndash997 2016

[32] P Jarolim P P Patel M J Conrad L Chang V Melenovskyand D H Wilson ldquoFully automated ultrasensitive digitalimmunoassay for cardiac troponin i based on single moleculearray technologyrdquo Clinical Chemistry vol 61 no 10 pp 1283ndash1291 2015

[33] H Cheng W Z Fan S C Wang et al ldquoPrognostic utilityof combination of NT-proBNP with high sensitive cTn I inpatientswith heart failure results from retrospective study in anemergency departmentrdquo Scandinavian Journal of Clinical andLaboratory Investigation vol 76 no 5 pp 361ndash367 2016

[34] A Joharimoghadam M Tajdini and A Bozorgi ldquoSalivaryB-type natriuretic peptide a new method for heart failurediagnosis and follow uprdquo Kardiologia Polska 2016

[35] M Oremus A Don-Wauchope R McKelvie et al ldquoBNP andNT-proBNP as prognostic markers in persons with chronicstable heart failurerdquoHeart Failure Reviews vol 19 no 4 pp 471ndash505 2014

[36] R S Wong ldquoApoptosis in cancer from pathogenesis to treat-mentrdquo Journal of Experimental and Clinical Cancer Researchvol 30 no 1 article 87 2011

[37] M S Davids and A Letai ldquoTargeting the B-cell lym-phomaleukemia 2 family in cancerrdquo Journal of Clinical Oncol-ogy vol 30 no 25 pp 3127ndash3135 2012

[38] M Tomek T Akiyama andC R Dass ldquoRole of Bcl-2 in tumourcell survival and implications for pharmacotherapyrdquoThe Journalof Pharmacy and Pharmacology vol 64 no 12 pp 1695ndash17022012

[39] B Leibowitz and J Yu ldquoMitochondrial signaling in cell deathvia the Bcl-2 familyrdquo Cancer Biology ampTherapy vol 9 no 6 pp417ndash422 2010

[40] N Maulik R M Engelman J A Rousou J E Flack III DDeaton andD K Das ldquoIschemic preconditioning reduces apo-ptosis by upregulating anti-death gene Bcl-2rdquo Circulation vol100 no 19 pp II369ndashII375 1999


[41] R Malla S Gopinath K Alapati et al ldquoDownregulation ofuPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3KAkt pathway in gliomasrdquoPLoS ONE vol 5 no 10 Article ID e13731 2010

[42] J Zhao J Li W Li et al ldquoEffects of spironolactone on atrialstructural remodelling in a canine model of atrial fibrillationproduced by prolonged atrial pacingrdquo British Journal of Phar-macology vol 159 no 8 pp 1584ndash1594 2010

[43] B Bartling H Milting H Schumann et al ldquoMyocardial geneexpression of regulators of myocyte apoptosis and myocytecalcium homeostasis during hemodynamic unloading by ven-tricular assist devices in patients with end-stage heart failurerdquoCirculation vol 100 no 19 pp II216ndashII223 1999

[44] R A de Boer D J van Veldhuisen J van der Wijk et alldquoAdditional use of immunostaining for active caspase 3 andcleaved actin and PARP fragments to detect apoptosis inpatients with chronic heart failurerdquo Journal of Cardiac Failurevol 6 no 4 pp 330ndash337 2000

[45] K-L Laugwitz A Schomig M Ungerer et al ldquoBlocking cas-pase-activated apoptosis improves contractility in failing myo-cardiumrdquo Human Gene Therapy vol 12 no 17 pp 2051ndash20632001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


OH

109

8

765

112

4

1

NMe

1918

OMe

OAc

OC14

O

OHH

1712

13 16OMe

OMe

15

3

1998400

16998400

1998400998400

2998400998400 3998400998400

4998400998400

5998400998400

6998400998400

6998400

18998400H3CO

(a)

COOH

HO

O

(b)

Figure 1 (a) Chemical structure of hypaconitine (HA) molecular formula C33H45NO10 (b) chemical structure of Glycyrrhetinic acid (GA)

molecular formula C30H46O4

Decoction after oral administration in rat by HPLC-MSMS[8 9] In another study about the comparative pharmacoki-netic profiles of GA GL and Gancao-Fuzi-Tang after orallytaking GL and Gancao-Fuzi-Tang the results demonstratedan increased effect ofGAafter oral administration ofGancao-Fuzi-Tang in comparison with GL which implied the GAwasone of the crucial elements in Gancao-Fuzi-Tang [10]

As we all know TCMconsists ofmany kinds of herbs andit is a complex system Every Chinese herb performs one ormore specific functions when prescribed alone Neverthelessthey will play much more complicated effects when usedin combination Because TCM includes lots of chemicalcomponents they influence each other even sometimes theherb compatibility produces new substances and results inmultiply therapeutic effects These actions are always causedby multicompounds For example the Fuzi-Gancao herb-pair generates the reducing toxicities and enhancing effectsmay be related on the interaction of acid and base whichbrings about some other new chemical compounds [11] Inconclusions to study the effects of Fuzi-Gancao herb pairwe chose HA and GA on behalf of this herb pair for furtherinvestigations

2 Materials and Methods

21 Material HA (purity ge98) and GA (purity ge98)were purchased from Baoji Chenguang Biotechnology CoLtd (Xirsquoan China) Digoxin pills were obtained from Shang-hai sym pharma Co Ltd (Shanghai China) penicillin wasbought from REYOUNG Co Ltd (Shandong China) isofl-urane was purchased from Shenzhen Huabao Medical Sup-plies Industry amp Trade Development Co Ltd (ShenzhenChina) B-type natriuretic peptide (BNP) and cardiac tro-ponin I (cTnI) ELISA kits were bought from Shanghai yua-nye biological agent company (Shanghai China) anti-Baxanti-Bcl-2 anti-caspase-3 and anti-120573-actin antibodies were

obtained from Santa Cruz Biotechnology Co (CA USA)immunohistochemical detection kit was purchased fromBeijing Zhongshan Biotechnology Co Ltd (Beijing China)(import packing)

22 Animal Healthy male SPF rats (body weight 160ndash170 g)were provided by Shanghai Slack Laboratory Animal CoLtd (animal license number SCXK (Shanghai) 2012-0002animal use certificate number SYXK (Zhejiang) 2013-184)The rats were watered ad libitum and fed a standard dietThe animals were maintained in the 12 h dark and 12 h lightcycle at the temperature of 20 plusmn 2∘C and the humidity was50 plusmn 2 All animal experimental protocols were approvedby the Animal Care and Use Committee of Zhejiang ChineseMedical University and complied with laboratory animalmanagement and use regulations

23 Animal Model The CHF model was induced bytransverse-aortic constriction (TAC) operation which wasmodified from deAlmeidarsquos methods [12] In this study it wasperformed after the rats were allowed to acclimate for 1 weekThe rats were anesthetized by inhalation of 20 isofluraneand then they were endotracheally intubated with positive-pressure ventilation inputting 100 oxygen gas with a tidalvolume of 250120583L at a velocity of 120 breaths per minuteby a Small Animal Ventilator (Hallowell Inc USA) Afteropening the chest wall via an upper sternotomy the aortawas carefully picked out A self-regulating ldquoLrdquo needle with theexternal diameter of approximately 09mmwas placed on theaorta on which a 3-0 prolene ligation was placed betweenthe right and left common carotid arteries The needle wasremoved cautiously and the aortic constriction was then cre-ated After the procedure the wound was closed respectivelyand the animal was allowed to recover from anesthesia withobservation Penicillin was injected intramuscular once a day




















3 Results



Sham Model Digoxin

HA GA HA + GA







4 Discussion




lowastlowast


lowastlowast

BNP

(pg

mL)

1200

800

400

0

(a)


lowastlowast

lowastlowast


cTnI

(ng

mL)

24

16

8

0

(b)
















(a)

60

40

20


lowastlowast

lowastlowast

lowastlowast

lowastlowast

IOD

of B

ax

(b)






(a)

60

40

20


lowastlowast

lowastlowast

lowastlowast

lowast

IOD

of B

cl-2

(b)


lowastlowast

lowastlowast

lowastlowast

IOD

of B

cl-2

Bax

24

16

08

00






(a)

IOD

of c

aspa

se-3


lowastlowast

lowastlowast


27

24

21

18

15

12

9

6

3

0







Bax

Bcl-2

Caspase-3

120573-Actin

(a)

Bax120573

-act

in

Bcl-2

120573-a

ctin

Bcl-2

Bax

Casp

ase-3

120573-a

ctin

012

008

004

000

009

006

003

000

4

3

2

1

0

0004

0002

0000

(A) (B)

(C) (D)




lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)





5 Conclusions


Competing Interests




Acknowledgments



References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































3 Results



Sham Model Digoxin

HA GA HA + GA







4 Discussion




lowastlowast


lowastlowast

BNP

(pg

mL)

1200

800

400

0

(a)


lowastlowast

lowastlowast


cTnI

(ng

mL)

24

16

8

0

(b)
















(a)

60

40

20


lowastlowast

lowastlowast

lowastlowast

lowastlowast

IOD

of B

ax

(b)






(a)

60

40

20


lowastlowast

lowastlowast

lowastlowast

lowast

IOD

of B

cl-2

(b)


lowastlowast

lowastlowast

lowastlowast

IOD

of B

cl-2

Bax

24

16

08

00






(a)

IOD

of c

aspa

se-3


lowastlowast

lowastlowast


27

24

21

18

15

12

9

6

3

0







Bax

Bcl-2

Caspase-3

120573-Actin

(a)

Bax120573

-act

in

Bcl-2

120573-a

ctin

Bcl-2

Bax

Casp

ase-3

120573-a

ctin

012

008

004

000

009

006

003

000

4

3

2

1

0

0004

0002

0000

(A) (B)

(C) (D)




lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)





5 Conclusions


Competing Interests




Acknowledgments



References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sham Model Digoxin

HA GA HA + GA







4 Discussion




lowastlowast


lowastlowast

BNP

(pg

mL)

1200

800

400

0

(a)


lowastlowast

lowastlowast


cTnI

(ng

mL)

24

16

8

0

(b)
















(a)

60

40

20


lowastlowast

lowastlowast

lowastlowast

lowastlowast

IOD

of B

ax

(b)






(a)

60

40

20


lowastlowast

lowastlowast

lowastlowast

lowast

IOD

of B

cl-2

(b)


lowastlowast

lowastlowast

lowastlowast

IOD

of B

cl-2

Bax

24

16

08

00






(a)

IOD

of c

aspa

se-3


lowastlowast

lowastlowast


27

24

21

18

15

12

9

6

3

0







Bax

Bcl-2

Caspase-3

120573-Actin

(a)

Bax120573

-act

in

Bcl-2

120573-a

ctin

Bcl-2

Bax

Casp

ase-3

120573-a

ctin

012

008

004

000

009

006

003

000

4

3

2

1

0

0004

0002

0000

(A) (B)

(C) (D)




lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)





5 Conclusions


Competing Interests




Acknowledgments



References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















lowastlowast


lowastlowast

BNP

(pg

mL)

1200

800

400

0

(a)


lowastlowast

lowastlowast


cTnI

(ng

mL)

24

16

8

0

(b)
















(a)

60

40

20


lowastlowast

lowastlowast

lowastlowast

lowastlowast

IOD

of B

ax

(b)






(a)

60

40

20


lowastlowast

lowastlowast

lowastlowast

lowast

IOD

of B

cl-2

(b)


lowastlowast

lowastlowast

lowastlowast

IOD

of B

cl-2

Bax

24

16

08

00






(a)

IOD

of c

aspa

se-3


lowastlowast

lowastlowast


27

24

21

18

15

12

9

6

3

0







Bax

Bcl-2

Caspase-3

120573-Actin

(a)

Bax120573

-act

in

Bcl-2

120573-a

ctin

Bcl-2

Bax

Casp

ase-3

120573-a

ctin

012

008

004

000

009

006

003

000

4

3

2

1

0

0004

0002

0000

(A) (B)

(C) (D)




lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)





5 Conclusions


Competing Interests




Acknowledgments



References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

60

40

20


lowastlowast

lowastlowast

lowastlowast

lowastlowast

IOD

of B

ax

(b)






(a)

60

40

20


lowastlowast

lowastlowast

lowastlowast

lowast

IOD

of B

cl-2

(b)


lowastlowast

lowastlowast

lowastlowast

IOD

of B

cl-2

Bax

24

16

08

00






(a)

IOD

of c

aspa

se-3


lowastlowast

lowastlowast


27

24

21

18

15

12

9

6

3

0







Bax

Bcl-2

Caspase-3

120573-Actin

(a)

Bax120573

-act

in

Bcl-2

120573-a

ctin

Bcl-2

Bax

Casp

ase-3

120573-a

ctin

012

008

004

000

009

006

003

000

4

3

2

1

0

0004

0002

0000

(A) (B)

(C) (D)




lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)





5 Conclusions


Competing Interests




Acknowledgments



References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

60

40

20


lowastlowast

lowastlowast

lowastlowast

lowast

IOD

of B

cl-2

(b)


lowastlowast

lowastlowast

lowastlowast

IOD

of B

cl-2

Bax

24

16

08

00






(a)

IOD

of c

aspa

se-3


lowastlowast

lowastlowast


27

24

21

18

15

12

9

6

3

0







Bax

Bcl-2

Caspase-3

120573-Actin

(a)

Bax120573

-act

in

Bcl-2

120573-a

ctin

Bcl-2

Bax

Casp

ase-3

120573-a

ctin

012

008

004

000

009

006

003

000

4

3

2

1

0

0004

0002

0000

(A) (B)

(C) (D)




lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)





5 Conclusions


Competing Interests




Acknowledgments



References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

IOD

of c

aspa

se-3


lowastlowast

lowastlowast


27

24

21

18

15

12

9

6

3

0







Bax

Bcl-2

Caspase-3

120573-Actin

(a)

Bax120573

-act

in

Bcl-2

120573-a

ctin

Bcl-2

Bax

Casp

ase-3

120573-a

ctin

012

008

004

000

009

006

003

000

4

3

2

1

0

0004

0002

0000

(A) (B)

(C) (D)




lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)





5 Conclusions


Competing Interests




Acknowledgments



References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Bax

Bcl-2

Caspase-3

120573-Actin

(a)

Bax120573

-act

in

Bcl-2

120573-a

ctin

Bcl-2

Bax

Casp

ase-3

120573-a

ctin

012

008

004

000

009

006

003

000

4

3

2

1

0

0004

0002

0000

(A) (B)

(C) (D)




lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)





5 Conclusions


Competing Interests




Acknowledgments



References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusions


Competing Interests




Acknowledgments



References






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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