research article salidroside stimulates mitochondrial...

Research ArticleSalidroside Stimulates Mitochondrial Biogenesis and Protectsagainst H2O2-Induced Endothelial Dysfunction

Shasha Xing12 Xiaoyan Yang12 Wenjing Li12 Fang Bian12

Dan Wu12 Jiangyang Chi12 Gao Xu12 Yonghui Zhang12 and Si Jin12

1 Department of Pharmacology Tongji Medical College Huazhong University of Science and Technology Wuhan 430030 China2The Key Laboratory of Drug Target Research and Pharmacodynamic Evaluation of Hubei Province Wuhan 430030 China

Correspondence should be addressed to Si Jin jinsimailhusteducn

Received 13 February 2014 Accepted 19 March 2014 Published 24 April 2014

Academic Editor Shiwei Deng

Copyright copy 2014 Shasha Xing 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

Salidroside (SAL) is an active component of Rhodiola rosea with documented antioxidative properties The purpose of this studyis to explore the mechanism of the protective effect of SAL on hydrogen peroxide- (H

2O2-) induced endothelial dysfunction

Pretreatment of the human umbilical vein endothelial cells (HUVECs) with SAL significantly reduced the cytotoxicity brought byH2O2 Functional studies on the rat aortas found that SAL rescued the endothelium-dependent relaxation and reduced superoxide

anion (O2

∙minus) production induced by H2O2 Meanwhile SAL pretreatment inhibited H

2O2-induced nitric oxide (NO) production

The underlying mechanisms involve the inhibition of H2O2-induced activation of endothelial nitric oxide synthase (eNOS)

adenosinemonophosphate-activated protein kinase (AMPK) and Akt as well as the redox sensitive transcription factor NF-kappaB (NF-120581B) SAL also increasedmitochondrial mass and upregulated the mitochondrial biogenesis factors peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1120572) and mitochondrial transcription factor A (TFAM) in the endothelialcells H

2O2-induced mitochondrial dysfunction as demonstrated by reduced mitochondrial membrane potential (Δ120595m) and ATP

production was rescued by SAL pretreatment Taken together these findings implicate that SAL could protect endothelium againstH2O2-induced injury via promoting mitochondrial biogenesis and function thus preventing the overactivation of oxidative stress-

related downstream signaling pathways

1 Introduction

The role of oxidative stress in the development of theendothelial dysfunction has been studied extensively [1ndash4]Excessive reactive oxygen species (ROS) not only reducesbioavailable nitric oxide (NO) through direct reaction toform peroxynitrite but also leads to eNOS uncoupling andfurther induces more ROS production [5]

As a major cellular source of ROS the contributions ofmitochondria to the detrimental effects of cardiovascularrisk factors have recently received increased attention [6ndash8] Excessive mitochondrial ROS (mtROS) act to inspirepathologic cell-signaling cascades under conditions of com-prehensive excessive oxidative stress [9ndash11] NF-kappa B (NF-120581B) activation occurs secondary to excessive mitochondrialROS production in the endothelium participating in a range

of proinflammatory and prothrombotic alterations in theendothelial cells [12 13]

Endothelial mitochondria have been found to have cru-cial roles in vascular path-physiology [14ndash16] and increasingevidences have indicated the importance of mitochondrialdysfunction in various vascular diseases such as atheroscle-rosis heart failure and cardiac ischemiareperfusion injury[17ndash19] Previous studies have shown that dysregulation ofmitochondrial biogenesis represents an early manifestationof endothelial dysfunction shifting cell metabolism towardmetabolic hypoxia in animals with impairedNObioavailabil-ity [14] Impairment ofmitochondrial biogenesis is frequentlyobserved in atherosclerosis and is thus likely to contribute tocellular energetic imbalance oxidative stress and endothelialdysfunction in these pathological conditions [20] Sinceincreased mitochondrial production of ROS due to impaired

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 904834 13 pageshttpdxdoiorg1011552014904834

2 Oxidative Medicine and Cellular Longevity

mitochondrial biogenesis also appears to be a key event in thedevelopment of aging-related vascular pathologies [13 21 22]identification of mechanisms that promote mitochondrialbiogenesis in the endothelial cells may provide new clues onthe pathogenesis of vascular disease

The health of mitochondria is in part regulated by theirbiogenesis and peroxisome proliferator-activated receptorgamma-coactivator-alpha (PGC-1120572) is regarded as the keyregulator [23] In endothelial cells PGC-1120572 also orchestratescellular defenses against oxidative stress [24] Mitochondrialtranscription factor A (TFAM) is responsible for the tran-scriptional control of mtDNA and its translocation to themitochondria is important to initiate mtDNA transcriptionand replication [25]

As described above mitochondria are highly dynamicorganelles and their biogenesis is likely to be involved in theregulation of endothelial cell metabolism redox regulationand signal transduction [6 16 26] Pathways that regulatemitochondrial biogenesis are potential therapeutic targetsfor the amelioration of endothelial dysfunction and vasculardiseases [27]

Salidroside (SAL) is an active ingredient of the rootof Rhodiola rosea a well-known herb used to relieve highaltitude sickness [28] SALhas also been used to enhance boththe physical and mental performance SAL upregulates thelevels of antioxidative enzymes glutathione peroxidase-1 andthioredoxin-1 to counteract oxidative stress [29] Previousstudies have shown that SAL promotes DNA repair enzymeParp-1 to counteract oxidative stress [30] Meanwhile arecent study reported that SAL attenuated homocysteine-induced endothelial dysfunction by reducing oxidative stress[31] As noted above there is tightly relationship betweenreduction of mitochondrial biogenesis and endothelial dys-function the present study was conducted to determinewhether SAL recovers the endothelial dysfunction inducedby H2O2through stimulating mitochondrial biogenesis and

counteracting the oxidative stress-related eNOS and NF-120581Bsignaling pathways

2 Materials and Methods

21 Animals Animals were treated in accordance with theguide for the Care and Use of Laboratory Animals publishedby the US National Institutes of Health and approved bythe Local Animal Care Committee Healthy Wistar rats(200ndash250 g) were purchased from the Center of Experimen-tal Animals (Tongji Medical College Huazhong Universityof Science and Technology China) and maintained in acontrolled environment with a lightdark cycle of 12 h atemperature of 20 plusmn 2∘C and humidity of 50 plusmn 2

22 Cell Culture The collection of human umbilical cordswas approved by the Ethics Committee of Tongji MedicalCollege Huazhong University of Science and Technology(Wuhan China) and conducted in accordance with theDeclaration of [32] Primary cultured human umbilical veinendothelial cells (HUVECs) were prepared as described in[33] In brief the umbilical cord was washed with cold

PBS and then infused with 025 trypsin After digestionstopped the cells were collected by centrifuging for 10minat 1 000 rpm The cells were resuspended and then culturedin endothelial cell medium (ECM Sciencell Carlsbad CA)at 37∘C in an incubator with a humidified atmosphere of 5CO2 In all experiments cells were used at passages 2ndash7

23 Cell Viability Assays To study the effect of SAL onH2O2-

induced cytotoxicity HUVECs were inoculated at a densityof 2 times 104 per well in 96-well plates and cultured overnightthe cell viability was evaluated using the Cell CountingKit-8 (Dojindo Laboratories Kumamoto) [34] Briefly theHUVECs treated with H

2O2(Sigma 4 h) or SAL (National

Institute for Food and Drug Control purity gt 98 24 h) atthe indicated concentration in OPTI-MEM (Gibco) beforecell viability was measured Moreover to study the effect ofSAL on H

2O2-induced inhibition of cell viability the cells

were cultured as described above after treatment with SALfor 20 h the H

2O2(100 120583M) was added to the medium for

another 4 hours At the end of the time period the culturemedium was removedThe cells were washed twice with PBSand incubated with CCK-8 solution at 37∘C for 30min Theabsorbance was measured using a microplate reader with atest wavelength of 450 nm

24 Free RadicalMeasurement in Cell Free System Theeffectsof SAL on scavenging hydroxyl radical (OH∙) superoxideradical (O

2

minus) and H2O2were measured with commercially

available kits (Nanjing Jiancheng Bioengineering InstituteChina) according to the manufacturerrsquos instructions In briefthe OH∙ was generated by the Fenton reaction and thentreated with a chromogenic substrate nitrotetrazolium bluechloride (NBT) to yield a stable colored substance which wasmeasured using a microplate reader with a test wavelengthof 450 nm O

2

minus was generated by the xanthinexanthineoxidase system O

2

minus was detected by nitrite method and theabsorbance at 550 nm was measured The reaction productof H2O2and molybdic acid can be detected at 405 nm

Deionized water and ascorbic acid (Vc) were used as theblank and positive controls respectivelyThe inhibition rate =(optical density of blank control groupsmdashoptical density oftreatment groups)optical density of blank control groups

25 Vascular Function Measurement The thoracic segmentsof the rat aorta were dissected and the surrounding con-nective tissues were cleaned off Each aorta was cut intoring segments of 2sim3mm in length The aortic rings weremounted in organ chambers filledwithKrebs-Henseleit (KH)solution at 37∘C with constant bubbling of 95 oxygen5carbon dioxide KH solution contained (mM) 133 NaCl 475KCl 15 CaCl

2 125 MgCl

2 25 NaHCO

3 and 11 D-glucose

Isometric tension was recorded with a force transducer(RM6240C Chengdu Instrument Factory) A basal tensionof 20mN was applied to each vascular ring After beingplaced in organ baths for 90min 05120583M phenylephrine(PE Sigma) was first administered to the rings to test theircontractility and then 1 120583M acetylcholine (ACh Sigma) wasadministered to assess the integrity of the endothelial layer

Oxidative Medicine and Cellular Longevity 3

Rings with less than 80 relaxation response to ACh werediscarded The aortic rings were pretreated with or without10 120583M SAL for 30min before H

2O2100 120583M was added to

the bath After precontracting with PE (01 120583M) ACh (1 times10minus8 sim1times 10minus4M)was added cumulatively to the bath to evokethe endothelium-dependent relaxationThe relaxation rate isthe ratio between the tension relaxed by ACh and the tensioncontracted by PE

26 Measurement of NO and Superoxide Anion (O2

∙minus) Tomeasure intracellular NO and O

2

∙minus levels the NO andO2

∙minus-specific fluorescent dye 45-diaminofluorescein diac-etate (DAF-FM-DA Beyotime Institute of Biotechnology)[35] and dihydroethidium (DHE Beyotime Institute ofBiotechnology) [36] were used to measure intracellular NOand O

2

∙minus levels respectively Briefly confluent HUVECs in96-well plates after SAL (10 120583M) or PBS treatment for 24 hwere washed twice with PBS followed by staining with 25120583MDAF-FM-DAor 5 120583MDHE for 30min at 37∘C After washingtwice with PBS the fluorescence intensities were measured asbasal and then 100120583MH

2O2was addedUsing a fluorescence

spectrophotometer (TECAN INFINITE F200PRO) the flu-orescence intensities of DAF-FM-DA were measured at anexcitation wavelength of 485 nm and an emission wavelengthof 535 nm respectively and DHE were measured at anexcitation wavelength of 535 nm and an emission wavelengthof 610 nm respectively

27 NF-120581B Activity Assay An ELISA-based assay was used tomeasure the NF-120581B activity as described previously [37ndash39]In brief after stimulation cells were rinsed twice with coldPBS and then RIPA containing a protease inhibitor cocktail(Roche Basel Switzerland) was added After incubationon ice for 30min the lysate was centrifuged for 15min at14 000 rpm and the supernatant was collected After beingquantified with BCA reagent (Pierce Rockford IL) thecell extracts were incubated in a 96-well plate coated withthe oligonucleotide containing theNF-120581B consensus-bindingsite (51015840-GGGACTTTCC-31015840) Activated transcription factorsfrom extracts specifically bound to the respective immobi-lized oligonucleotide NF-120581B activity was then detected withthe primary antibody to NF-120581B p65 (1 1000 ProteintechChina) and secondary antibody conjugated to horseradishperoxidase (1 10 1000 Abbkine CA) Tetramethylbenzidine(100 120583L Sigma) was added in each microwell at 37∘C beforeadding 100 120583Lof stopping solution (2MH

2SO4)NF-120581Bactiv-

ity was finally determined as absorbance values measuredwith a microplate reader at a wavelength of 450 nm

28 Measurement of Mitochondrial Mass Mitochondriamass was determined by using MitoTracker green amitochondrial-selective membrane potential-independentdye [40] The cells grown on cover slips coated with 2gelatin were incubated with SAL (1 10 120583M) for 24 h At theend of the incubation suspensions were removed and thecells were incubated with 200 nM MitoTracker green (Bey-otime Institute of Biotechnology China) in 37∘C for 30minThe images were captured with a fluorescence microscope

(Olympus FV500) using 40times magnification objective [41]The integrated fluorescence intensities were measured usingthe Image-Pro Plus software and normalized to the numberof cells

29 Measurement of Intracellular ATP Levels After treat-ment the HUVECs from each well of a 6-well dish (5 times105well) were washed twice with cold PBS and lysed with05M perchloric acid and briefly sonicated (5 to 10 timesof a 1 sec burst) until cells were clearly disrupted Sampleswere then neutralized with 2M KOH and then centrifugedto remove the precipitate ATP content was analyzed byHPLC (Agilent Palo Alto CA) with an LC-18T reverse-phasecolumn (Agilent Palo Alto CA) at a flow rate of 03mLminand the absorbance at 254 nmwas recordedThe elution peakwas compared with ATP standards (National Institute forFood and Drug Control Beijing) to confirm its identity

210 Assessment ofMitochondrialMembrane Potential (Δ120595m)JC-1 is a positively charged fluorescent compound whichis taken up by mitochondria proportionally to the innermitochondrial membrane potential [42] The ratio of red(J-aggregate)green (monomeric JC-1) emission is directlyproportional to the Δ120595m HUVECs were grown on 96-well plates treated with SAL (10 120583M) for 20 h and thenH2O2(100 120583M) was added for another 4 h Cells were rinsed

with PBS and incubated in 100 120583L JC-1 staining solutionat 37∘C for 20min Cells were then rinsed twice with JC-1 washing solution and analyzed with a fluorescence spec-trophotometer J-aggregates were recorded with an excitationwavelength of 535 nm and an emission wavelength of 610 nmrespectively and monomeric JC-1 was recorded with anexcitation wavelength of 485 nm and an emission wavelengthof 535 nm respectively

211 Western Blot Analysis Cells were homogenized in ice-cold RIPA lysis buffer containing protease inhibitor cocktailand phosSTOP (Roche Basel Switzerland) Equal amountsof protein (60120583g) were mixed with the loading buffer(Beyotime Institute of Biotechnology) boiled for 10min andseparated by SDS-PAGE After electrophoresis proteins weretransferred to polyvinylidene difluoride membranes (PVDFMillipore Temecula CA) The membranes were blockedfor 1 h in 5 milk The membranes were then incubatedovernight at 4∘C with one of the following specific pri-mary antibodies rabbit anti-eNOS ser1177 anti-eNOS anti-AMPK120572 thr172 anti-AMPK120572 anti-120573-actin (1 1 000 Cell Sig-naling Technology Beverly MA) anti-Akt ser473 (1 1 000EPITOMICS CA) anti-Akt (1 600 Proteintech) anti-TFAM(1 300 Proteintech) and anti-PGC-1120572 (1 200 Santa CruzCA) After washing the membranes were incubated for 2 hat room temperature with secondary antibodies (Goat anti-rabbit IgG goat anti-mouse IgG 1 10 000 Abbkine CA) andthenwashed Finally the blots were developedwith enhancedchemiluminescence detection reagents (Thermo ScientificWaltham MA) Membranes were scanned using the Micro-Chemi bioimage analyzer (NDR Israel) and quantified usingImage J program and normalized against 120573-actin


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dagger dagger

H2O2

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minusSAL (120583M)+ + + +

1

(c)

Figure 1 Protective effects of SAL on H2O2-induced cytotoxicity in HUVECs (a) HUVECs were exposed to various concentrations of H

2O2

for 4 h (b) Cells were exposed to various concentrations of SAL for 24 h (c) HUVECs were pretreated with SAL or vehicle for 20 h and thenexposed to H

2O2(100120583M) for 4 h Cell viability was detected by CCK-8 Cell viability in untreated cells was assigned the value of 1 lowast119875 lt 005

lowastlowast

119875 lt 001 versus control dagger119875 lt 005 versus H2O2 119899 = 3ndash5

212 Statistical Analysis All data in this study are expressedas the mean plusmn SEM from at least three separate experimentsSPSS 130 was used for statistical analysis Individual groupstatistical comparisons were analysed by unpaired Studentrsquos119905-test with Bonferroni correction and multiple groups com-parisons were evaluated by one-way ANOVA with post hoctesting A probability value of 119875 lt 005 was consideredstatistically significant

3 Results

31 SAL Alleviates the Cytotoxicity Induced by H2O2in

HUVECs In this study H2O2was used to induce oxidative

stress in HUVECs After exposure to H2O2(100ndash1000120583M)

for 4 h HUVECs viability was reduced (Figure 1(a)) SALat the concentration below 10 120583M had no obvious effect oncell viability compared to control (Figure 1(b)) Pretreatmentwith SAL could reduce cell death induced by H

2O2in a

concentration-dependent manner (Figure 1(c))

32 Effect of SAL on ROS in Cell Free System SAL has theeffect of scavenging OH∙ but not O

2

minus or H2O2at indicated

concentration (Figures 2(a) 2(b) and 2(c))

33 SAL Recovers H2O2-Induced Impairment of Endothelium-

Dependent Relaxation in Rat Aortas Treatment with H2O2

(100 120583M) for 30min markedly attenuated ACh-inducedendothelium-dependent relaxation (EDR) in rat aortasExposure to SAL (10 120583M 30min) prior to the addition ofH2O2partially rescued the impaired EDR (Figure 3(a))

Meanwhile pretreatment with SAL (10 120583M 24 h) inhib-ited NO and O

2

∙minus production induced by H2O2(100 120583M)

(Figures 3(b) and 3(c))

34 SAL Decreased eNOS Activation Induced by H2O2in

HUVECs Compared with control H2O2(100 120583M 4 h) or

SAL (10 120583M 24 h) treatment significantly increased eNOSphosphorylation at ser1177 and Akt phosphorylation atSer473 in HUVECs Pretreatment with SAL (10 120583M) for 24 h


70

50

30

10

minus1001 1 10 100 1000

Vc

lowastlowast

lowastlowast

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(120583M)

Inhi

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te (

)

SAL

(a)

Vc

01 1 10 100 1000

(120583M)

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60

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minus20

Inhi

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)

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(b)

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Vc(120583M)

6

2

minus2

minus6

minus10

minus14

Inhi

bitio

n ra

te (

)

SAL

(c)

Figure 2 Effect of SAL on cell free ROS detection using different assay systems (a) OH∙ was generated by Fenton reaction (b) O2

minus wasgenerated by xanthinexanthine oxidase and (c) H

2O2was exogenously added lowast119875 lt 005 lowastlowast119875 lt 001 versus blank control 119899 = 3

attenuated the activation of eNOS AMPK120572 and Akt inducedby 100 120583MH

2O2(Figures 4(a) 4(b) 4(c) and 4(d))

35 SAL Inhibited the Activation of Transcription Factor NF-120581B Induced by H

2O2in HUVECs Activation of the tran-

scription factor NF-120581B has been associated with endothelialcells dysfunction and vascular inflammation in atherogenesis[43] In line with previous studies exposure to H

2O2could

result in transient activation of NF-120581B (Figure 5(a)) whileexposure to SAL (10 120583M) for indicated time significantlydecreased the activity of transcription factorNF-120581B in a time-dependent manner (Figure 5(b)) Pretreatment with SALinhibited the activation of the NF-120581B induced by 01120583MH2O2(Figure 5(c)) To our surprise we found that expo-

sure to H2O2(01 120583M) for 30min could result in transient

activation of NF-120581B whereas H2O2at the concentration of

100 120583M slightly reduced rather than increased the NF-120581Bactivity SAL pretreatment can further reduce the activity of

NF-120581B (Figure 5(c)) To explain this result we used TNF-120572 aclassical NF-120581B inducer to treat HUVECs to detect the directeffect of H

2O2on DNA binding activity The result indicated

that H2O2inhibited the DNA binding activity of activated

NF-120581B in a dose-dependent manner (Figure 5(d))

36 SAL Induced Mitochondrial Biogenesis in EndothelialCells Our results showed that SAL (10120583M) increased the flu-orescent intensity of MitoTracker green suggesting that SALincreased the mitochondrial mass in HUVECs (Figure 6(a))Consistent with this the expression of PCG-1120572 and TFAMthe key regulators of mitochondrial biogenesis were signif-icantly increased in HUVECs incubated with SAL (Figures6(b) and 6(c))

37 SAL Restores H2O2-Induced Mitochondrial Dysfunction

We used independent parameters to evaluate mitochondrialfunction ATP production and ΔΨm As shown in Figure 7


minus20

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20

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minus8 minus7 minus6 minus5 minus4

Rela

xatio

n (

)

CtrH2O2

SAL + H2O2

lowastlowast

lowastlowast

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daggerdaggerdagger

daggerdagger

daggerdagger

daggerdagger

daggerdagger

ACh (lgM)

(a)

CtrH2O2

SAL + H2O2

0 3 6 9 12 15 18 21 24 27 30

(min)

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lowastlowast lowast

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ized

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E flu

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(b)

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CtrH2O2

SAL + H2O2

0 3 6 9 12 15 18 21 24 27 30

(min)

Nor

mal

ized

DA

F-FM

-DA

fluor

esce

nce (

)

lowast

lowast

lowast lowast

lowastlowast

lowastlowast

daggerdaggerdagger

dagger

(c)

Figure 3 Protective effects of SAL onH2O2-induced impairment of endothelium-dependent relaxation (a) Rat thoracic aorta was pretreated

with or without SAL for 30min and then H2O2(100120583M) was added to incubate for another 30min After precontracting with 1 120583MPE ACh

was added accumulatively Complete relaxation of aorta induced by ACh was considered as 100 lowast119875 lt 005 lowastlowast119875 lt 001 versus controldagger119875 lt 005 daggerdagger119875 lt 001 versusH

2O2 119899 = 8ndash16 (b)HUVECswere pretreatedwith orwithout 10 120583MSAL for 20 h and then loadedwithDHEdye

After acquisition of basal data H2O2(100120583M) was added and the fluorescence was measured every 5min within 30min The fluorescence

of basal data was assigned the value of 100 lowast119875 lt 005 lowastlowast119875 lt 001 versus control dagger119875 lt 005 versus H2O2 119899 = 4 (c) HUVECs werepretreated with or without SAL for 20 h and then loaded with DAF-FM-DA dye After acquisition of basal data H

2O2(100120583M) was added

and the fluorescence was measured every 5min within 30min The fluorescence of basal data was assigned the value of 100 lowast119875 lt 005versus control dagger119875 lt 005 versus H

2O2 119899 = 4

H2O2(100 120583M) inducedΔΨmcollapse (a) and decreasedATP

production (b) after 4 h of treatment in cultured HUVECsPretreatment with SAL (10120583M) for 24 h rescued mitochon-drial function

4 Discussion

There is increasing attention on the relationship betweenoxidative stress and endothelial cell injury [44ndash46] Thepresent study employed H

2O2-induced oxidative stress in

HUVECs as a cellular model to study the protective effect of

SAL In our experiments pretreatment of SAL significantlyprevents the impaired viability of HUVECs caused by H

2O2

exposureThese results are in accordance with previous stud-ies [47] and further confirmed the protective effects of SALagainst injury induced by H

2O2 Moreover the antioxidative

mechanism of SAL is not due to the direct reaction betweenSAL and H

2O2(Figure 2)

Overproduced ROS are known to harm the normalvascular function by limiting the beneficial effects of endothe-lium derived NO [48] The enhanced production and releaseof ROS andor the diminished bioavailability of NO withinvascular wall lead to endothelial dysfunction that is widely


p-eNOS (ser1177)

eNOS

p-AMPK120572 (thr172)

AMPK120572

p-Akt (ser473)

Akt

H2O2

10 10 10

01 01 100 100

minus minus minus

minus minusSAL (120583M)

120573-Actin

(a)

3

25

2

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1

05

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p-eNOSeNOS

lowastlowast

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H2O2

10 10 10

01 01 100 100

minus minus minusminus minus

SAL (120583M)

Expr

essio

n ra

tio (f

old)

(b)

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p-AMPK120572AMPK120572

H2O2

10 10 10

01 01 100 100


SAL (120583M)

lowastlowast

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Expr

essio

n ra

tio (f

old)

(c)

H2O2

10 10 10

01 01 100 100

minus minus minus

minus minus

SAL (120583M)

2

15

1

05

0

p-AktAkt

lowast

daggerlowastlowast

Expr

essio

n ra

tio (f

old)

(d)

Figure 4 Effects of SAL onH2O2-induced activation of eNOS AMPK and Akt After pretreatment with SAL (10120583M) for 20 h H

2O2(01 120583M

or 100120583M) was added for another 4 h and the lysates were analyzed by western blot (a) Representative immunoblots (b c d)The histogramshows quantitation of eNOS-ser1177 eNOS AMPKa-thr172 AMPK Akt-ser473 and Akt expression The expressions were normalized tothat obtained in untreated cells lowast119875 lt 005 lowastlowast119875 lt 001 versus control dagger119875 lt 005 versus H2O2 119899 = 4ndash6

believed to be the early key event in the pathogenesis ofvarious vascular complications [4 49] AlthoughH

2O2elicits

relaxation in rat mouse and rabbit aortas [50ndash53] andstimulates eNOS resulting in higher NO levels [54] the effectof prolonged elevation of H

2O2is to impair endothelium-

dependent relaxation [55] Previous study showed that SALprevented homocysteine-induced endothelial dysfunctionthrough curtailing oxidative stress [31] In our study pretreat-ment with H

2O2induced significant impairment of endothe-

lial dependent relaxation while SAL had the capacity torescue this impairment As expected incubation of HUVECswith H

2O2strikingly increased intracellular O

2

∙minus and thiscan be suppressed by pretreatment with SAL These resultsstrongly suggest that SAL inhibits H

2O2-induced ROS pro-

duction contributing to the restoration of the endothelium-dependent vasorelaxation

NO derived from the action of eNOS in endothelial cellsis one of the most important mediators in the regulationof endothelial functions [56] Phosphorylation of eNOSat ser1177 activates eNOS while increased oxidative fluxdirectly scavenges NO to lower NO bioavailability [57] andsubsequently impairs endothelium-dependent vasodilatation[58] Interestingly as shown in many previous studies H

2O2

directly upregulated the levels of NO in endothelial cellssuggesting that overproduction of NO by endothelial cellsin response to H

2O2stimulation was intended to protect

the cells rather than damage cells [59 60] An intriguingquestion that arises from this study is why SAL tends toinhibit H

2O2-induced eNOS activation and NO production

while it per se appears to upregulate eNOS expression andactivation (Figures 4 and 3(c)) We speculated here thatthe upregulation of eNOS activity and NO production by


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daggerdaggerdaggerdagger daggerdagger

TNF-120572

(d)

Figure 5 Effects of SAL on H2O2-induced activation of transcription factor NF-120581B Effects of H

2O2(01 120583M) (a) and SAL (10 120583M) (b) on

transcription factor NF-120581B activity H2O2(01 120583M) or SAL (10120583M) was added for the indicated periods of time (0ndash4 h) The transcription

activity in untreated cells was assigned the value of 1 lowast119875 lt 005 lowastlowast119875 lt 001 versus control 119899 = 4 (c) Effects of SAL on H2O2-induced

NF-120581B activity HUVECs were pretreated with SAL (1 10120583M) for 10min and then incubated with H2O2(01 100120583M) for 30min lowast119875 lt 005

lowastlowast

119875 lt 001 versus control dagger119875 lt 005 daggerdagger119875 lt 001 versus H2O2 119899 = 4ndash6 (d) HUVECs were treated with TNF-120572 (30 ngmL) for 30min andthen total protein was extracted The proteins were incubated with increasing concentrations of H

2O2(01 1 10 and 100120583M) for 30min and

then the activities of NF-120581B were detected as described in Section 2 The transcription activity was normalized to that obtained in untreatedcells lowastlowast119875 lt 001 versus control daggerdagger119875 lt 001 versus TNF-120572 119899 = 3

long-term SAL treatment may be initiated by a transientmild mitochondrial depolarization and reduced oxygendemand which in turn increase the tolerance of affected cellsto subsequent oxidative insult that is greater in severity Butthis needs to be further proved

There are evidences that NO can repress the activationof NF-120581B through degradation of I120581B120572 [61] or inhibitionof NF-120581B DNA binding [62] We also analyzed the effectof SAL on the activation of NF-120581B induced by H

2O2 Our

date showed that SAL decreased the basal activity of NF-120581B meanwhile blocking the activation of NF-120581B inducedby H2O2(Figure 5) H

2O2at the concentration of 100 120583M

slightly reduced rather than increased the NF-120581B activitymeanwhile pretreatment with SAL further reduced theactivity The possible explanation is that H

2O2per se induces

the formation of active dimmers of P65 and P50 subunits

and leads to their translocation to the nucleus Howeverwithin the nucleus the Cys 62 residue on the P50 subunitis oxidized to sulfenic acid and is further followed by S-glutathionylation which inhibits the binding of NF-120581B tothe DNA [63ndash65] To strengthen this hypothesis we detectedthe direct effect of H

2O2on DNA binding activity We

used TNF-120572 a classical NF-120581B inducer to treat HUVECsAfter cell lysis and total protein extraction we incubated theproteins with increasing concentrations of H

2O2(01 1 10

and 100 120583M) for 30min and then the activities of NF-120581Bwere detected as described in Section 2The results indicatedthat H

2O2dose dependently inhibited the DNA binding

activity of activated NF-120581B Since NF-120581B is a redox sensitivetranscription factor its activation participates in inflam-mation and mitochondrial biogenesis impairment [66] Wespeculated that the protective effect of SAL on HUVECs


Ctr SAL (1120583M)

SAL (10120583M)2

16

12

08

04

0

Rela

tive

fluor

esce

nce i

nten

sity

Ctr SAL (1120583M) SAL (10120583M)

lowast

(a)

Ctr SAL (1120583M) SAL (10120583M)

PGC-1120572

TFAM

120573-Actin

(b)

Ctr SAL (1120583M) SAL (10120583M)

2

16

12

08

04

0

lowast

lowast

PGC-1120572TFAM

Expr

essio

n ra

tio (f

old)

(c)

Figure 6 Effects of SAL on mitochondrial biogenesis (a) Mitochondria mass was quantified using Mito Tracker Green Scale bars = 50120583mThe fluorescence in untreated cells was assigned the value of 1 lowast119875 lt 005 versus control 119899 = 4 HUVECs were treated with SAL (1 10120583M) for24 h and the total protein was extracted the lysates were analyzed by western blot (b) Representative immunoblots (c) Summary histogramsof the relative density of PGC-1120572 and TFAM normalized to 120573-actin The expression in untreated cells was assigned the value of 1 lowast119875 lt 005versus control 119899 = 5

might be related to an interference with the NF-120581B signalingpathway

Moderate increases in NO stimulate mitochondrial bio-genesis mainly through cGMP-dependent gene expressionand activation of regulatory factors including PGC-1120572 andTFAM [67 68] In cardiomyocytes coimmunoprecipitationexperiments demonstrated that the p65 subunit of NF-120581B isconstitutively bound to PGC-1120572 coactivator and blocks itsactivation of gene transcription and that NF-120581B activationincreases this binding [69] Moreover PGC-1120572 overexpres-sion inhibits NF-120581B activation in human aortic smooth

muscular and endothelial cells [70] So it is reasonable thatthe agents stimulating PGC-1120572 expression andmitochondrialbiogenesis in the endothelial cells are beneficial to preventthe development of cardiovascular disease Here we reportedfor the first time that SAL increased mitochondrial mass inHUVECs (Figure 6(a)) Inducible mitochondrial biogenesisis very important in vascular health [27 71] Moreover mito-chondrial proliferation reduces the flow of electron per unitmitochondria SAL-induced mitochondrial biogenesis maycontribute to the reduction ofmitochondrial ROS productionin HUVECs


12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

JC-1

(RG

) (fo

ld)

(a)

12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

Cel

lula

r ATP

leve

l (fo

ld)

(b)

Figure 7 Effects of SAL on H2O2-induced mitochondrial dysfunction HUVECs were pretreated with SAL (10 120583M) for 20 h and then

H2O2(100120583M)was added to incubate for another 4 h (a)Mitochondrial membrane potential and (b) ATP content were detected as described

in Section 2 ΔΨm and ATP content in untreated cells were assigned the value of 1 lowast119875 lt 005 versus control dagger119875 lt 005 versus H2O2 119899 = 4

eNOS

NO

OO

ActivationInhibition

Mitochondrialbiogenesis

Nucleus

NF-120581B

HO

HO

HOOH

Salidroside

OH

TFAM

Akt

p65

p50

PGC-1120572

Figure 8 Schematic diagram of the potential mechanisms of SAL to induce mitochondrial biogenesis SAL inhibited transcription factorsNF-120581B activity enhanced eNOS activity and NO production which induced mitochondrial biogenesis subsequently

To determine whether the effect of SAL onmitochondrialbiogenesis is a consequence of the activation ofmitochondrialbiogenesis regulatory factors we examined the expressionof PGC-1120572 and TFAM We found that SAL increasedthe expression of PGC-1120572 and TFAM (Figures 6(b) and6(c))

In addition to stimulating mitochondrial biogenesisPGC-1120572 also contributes to the induction of ROS detoxify-ing enzymes including catalase superoxide dismutase andheme oxygenase [72ndash75] These findings imply that PGC-1120572-mediated mitochondrial biogenesis in oxidative injured cellsseems to offer a good source of ldquohealthymitochondriardquo which


detoxify mtROS by a large antioxidant defense system con-taining numerous redox enzymes of the electron-transportchain ultimately decreasing net ROS production Theseeffects of SAL on mitochondrial biogenesis may partiallyexplain its antioxidant properties

Many evidences have revealed thatH2O2caused endothe-

lial cell injury by inducing mitochondrial dysfunction [7677] Due to localization to the inner mitochondrial mem-brane lack of histone-like coverage and a less efficient DNArepair system compared with nuclear DNA mtDNA is proneto oxidative stress [78] Furthermore the mutation is morelikely to affect gene integrity because of the absence of intronin the mitochondrial genome [79] Damaged mitochondriaproduce less ATP but more greater amounts of ROS potenti-ating the signal and entering a vicious circle which aggravatecardiovascular diseases [80] As shown in Figure 7 SALpretreatment abrogated the H

2O2-induced collapse of ΔΨm

and rescued themitochondrial function proved by increasedATP production These effects of SAL decrease the potentialfor mtDNA damaged by H

2O2 moreover SAL enhanced

mitochondrial biogenesis which provides healthy mitochon-dria to replace the mitochondrial components damagedby ROS and maintain normal mitochondrial function Theprotective effect of SAL on mitochondria is of paramountimportance to maintain the endothelial homeostasis

In summary the present study demonstrated a novelmechanism of SAL to protect the endothelial cells fromoxidative damage By stimulating mitochondrial biogenesis(Figure 8) and counteracting the reactive oxygen speciesburst within mitochondria SAL administration preventedthe overactivation of several signaling pathways evoked bydamaging oxidative stimuli and preserved the viability ofendothelial cells as well as the endothelial dependent vesselfunctions Novel compounds with mitochondria biogenesisstimulating activities may become potential drug candidatesfor the prevention or treatment of such disorders associatedwith oxidative stress as metabolic syndrome or cancer and soforth

Conflict of Interests

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

Authorsrsquo Contribution

Shasha Xing and Xiaoyan Yang contributed equally to thiswork

Acknowledgments

This work was supported by Grants from the NationalNatural Science Foundation of China (81373413 81072634and 81000080) the Ministry of Education of China (NCET-10-0409 and 2013YGYL008) and the National Scienceand Technology Major Projects (2013zx09103-001-020 and2011zx09102-004-001)

References

[1] V V Makarenko P V Usatyuk G Yuan et al ldquoIntermit-tent hypoxia-induced Endothelial barrier Dysfunction requiresROS-dependent MAP kinase activationrdquo American Journal ofPhysiology Cell Physiology 2014

[2] N Hermida and J L Balligand ldquoLow-density lipoprotein-cholesterol-induced endothelial dysfunction and oxidativestress the role of statinsrdquo Antioxidants amp Redox Signaling vol20 no 8 pp 1216ndash1237 2013

[3] S Wang M Zhang B Liang et al ldquoAMPK1205722 deletion causesaberrant expression and activation of NAD(P)H oxidase andconsequent endothelial dysfunction in vivo role of 26S protea-somesrdquo Circulation Research vol 106 no 6 pp 1117ndash1128 2010

[4] C M Sena A M Pereira and R Seica ldquoEndothelialdysfunctionmdasha major mediator of diabetic vascular diseaserdquoBiochimica et Biophysica Acta vol 1832 no 12 pp 2216ndash22312013

[5] A L Moens R Kietadisorn J Y Lin and D Kass ldquoTarget-ing endothelial and myocardial dysfunction with tetrahydro-biopterinrdquo Journal of Molecular and Cellular Cardiology vol 51no 4 pp 559ndash563 2011

[6] P Dromparis and E D Michelakis ldquoMitochondria in vascularhealth and diseaserdquo Annual Review of Physiology vol 75 pp95ndash126 2013

[7] E Marzetti A Csiszar D Dutta G Balagopal R Calvaniand C Leeuwenburgh ldquoRole of mitochondrial dysfunction andaltered autophagy in cardiovascular aging and disease frommechanisms to therapeuticsrdquo American Journal of PhysiologymdashHeart and Circulatory Physiology vol 305 no 4 pp H459ndashH476 2013

[8] J R Mercer ldquoMitochondrial bioenergetics and therapeuticintervention in cardiovascular diseaserdquo Pharmacology amp Ther-apeutics vol 141 no 1 pp 13ndash20 2014

[9] R O Poyton K A Ball and P R Castello ldquoMitochondrialgeneration of free radicals and hypoxic signalingrdquo Trends inEndocrinology andMetabolism vol 20 no 7 pp 332ndash340 2009

[10] S R Thomas P K Witting and G R Drummond ldquoRedoxcontrol of endothelial function and dysfunction molecularmechanisms and therapeutic opportunitiesrdquo Antioxidants ampRedox Signaling vol 10 no 10 pp 1713ndash1765 2008

[11] M T Sorbara and S E Girardin ldquoMitochondrial ROS fuel theinflammasomerdquo Cell Research vol 21 no 4 pp 558ndash560 2011

[12] C Banfi M Brioschi S S Barbieri et al ldquoMitochondrialreactive oxygen species a common pathway for PAR1- andPAR2-mediated tissue factor induction in human endothelialcellsrdquo Journal of Thrombosis and Haemostasis vol 7 no 1 pp206ndash216 2009

[13] Z Ungvari Z Orosz N Labinskyy et al ldquoIncreased mitochon-drial H

2O2production promotes endothelial NF-120581B activation

in aged rat arteriesrdquoAmerican Journal of PhysiologymdashHeart andCirculatory Physiology vol 293 no 1 pp H37ndashH47 2007

[14] F Addabbo B Ratliff H-C Park et al ldquoThe krebs cycle andmitochondrial mass are early victims of endothelial dysfunc-tion proteomic approachrdquo American Journal of Pathology vol174 no 1 pp 34ndash43 2009

[15] S M Davidson and M R Duchen ldquoEndothelial mitochondriacontributing to vascular function and diseaserdquo CirculationResearch vol 100 no 8 pp 1128ndash1141 2007

[16] M A Kluge J L Fetterman and J A Vita ldquoMitochondria andendothelial functionrdquo Circulation Research vol 112 no 8 pp1171ndash1188 2013


[17] K J Krishnan A K Reeve D C Samuels et al ldquoWhat causesmitochondrial DNAdeletions in human cellsrdquoNatureGeneticsvol 40 no 3 pp 275ndash279 2008

[18] M G Rosca and C L Hoppel ldquoMitochondria in heart failurerdquoCardiovascular Research vol 88 no 1 pp 40ndash50 2010

[19] A PHalestrap ldquoCalciummitochondria and reperfusion injurya pore way to dierdquoBiochemical Society Transactions vol 34 part2 pp 232ndash237 2006

[20] J Gutierrez SW Ballinger VMDarley-Usmar andA LandarldquoFree radicals mitochondria and oxidized lipids the emerg-ing role in signal transduction in vascular cellsrdquo CirculationResearch vol 99 no 9 pp 924ndash932 2006

[21] Z Ungvari N Labinskyy S Gupte P N Chander J GEdwards and A Csiszar ldquoDysregulation of mitochondrial bio-genesis in vascular endothelial and smooth muscle cells of agedratsrdquo American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 294 no 5 pp H2121ndashH2128 2008

[22] R Blake and I A Trounce ldquoMitochondrial dysfunction andcomplications associated with diabetesrdquo Biochimica et Biophys-ica Acta vol 1840 no 4 pp 1404ndash1412 2014

[23] R C Scarpulla ldquoMetabolic control of mitochondrial biogenesisthrough the PGC-1 family regulatory networkrdquo Biochimica etBiophysica Acta vol 1813 no 7 pp 1269ndash1278 2011

[24] I S Patten and Z Arany ldquoPGC-1 coactivators in the cardiovas-cular systemrdquo Trends in Endocrinology and Metabolism vol 23no 2 pp 90ndash97 2012

[25] R C Scarpulla ldquoNuclear control of respiratory chain expressionby nuclear respiratory factors and PGC-1-related coactivatorrdquoAnnals of the New York Academy of Sciences vol 1147 pp 321ndash334 2008

[26] J M Santos S Tewari A F X Goldberg and R A KowluruldquoMitochondrial biogenesis and the development of diabeticretinopathyrdquo Free Radical Biology and Medicine vol 51 no 10pp 1849ndash1860 2011

[27] Q Xu P Xia X Li W Wang Z Liu and X Gao ldquoTetram-ethylpyrazine ameliorates high glucose-induced endothelialdysfunction by increasing mitochondrial biogenesisrdquo PLoSONE vol 9 no 2 Article ID e88243 2014

[28] A Panossian and HWagner ldquoStimulating effect of adaptogensan overview with particular reference to their efficacy followingsingle dose administrationrdquo Phytotherapy Research vol 19 no10 pp 819ndash838 2005

[29] EWQian D T Ge and S-K Kong ldquoSalidroside promotes ery-thropoiesis and protects erythroblasts against oxidative stress byup-regulating glutathione peroxidase and thioredoxinrdquo Journalof Ethnopharmacology vol 133 no 2 pp 308ndash314 2011

[30] X Li J SippleQ Pang andWDu ldquoSalidroside stimulatesDNArepair enzyme Parp-1 activity in mouse HSC maintenancerdquoBlood vol 119 no 18 pp 4162ndash4173 2012

[31] S B LeungH Zhang CW Lau YHuang andZ Lin ldquoSalidro-side improves homocysteine-induced endothelial dysfunctionby reducing oxidative stressrdquo Evidence-Based Complementaryand Alternative Medicine vol 2013 Article ID 679635 8 pages2013

[32] J R Williams ldquoThe declaration of Helsinki and public healthrdquoBulletin of the World Health Organization vol 86 pp 650ndash6522008

[33] E A Jaffe R L Nachman C G Becker and C R MinickldquoCulture of human endothelial cells derived from umbilicalveins Identification bymorphologic and immunologic criteriardquoThe Journal of Clinical Investigation vol 52 no 11 pp 2745ndash2756 1973

[34] Y Bai A K Suzuki andM Sagai ldquoThe cytotoxic effects of dieselexhaust particles on human pulmonary artery endothelial cellsin vitro role of active oxygen speciesrdquo Free Radical Biology andMedicine vol 30 no 5 pp 555ndash562 2001

[35] S Song F Zhou W R Chen and D Xing ldquoPDT-inducedHSP70 externalization up-regulates NO production via TLR2signal pathway in macrophagesrdquo FEBS Letters vol 587 no 2pp 128ndash135 2013

[36] K Dasuri P Ebenezer S O Fernandez-Kim et al ldquoRole ofphysiological levels of 4-hydroxynonenal on adipocyte biologyimplications for obesity and metabolic syndromerdquo Free RadicalResearch vol 47 no 1 pp 8ndash19 2013

[37] S Jin D Lu S Ye et al ldquoA simplified probe preparation forELISA-based NF-120581B activity assayrdquo Journal of Biochemical andBiophysical Methods vol 65 no 1 pp 20ndash29 2005

[38] Y Zhang X Yang F Bian et al ldquoTNF-120572 promotes earlyatherosclerosis by increasing transcytosis of LDL acrossendothelial cells crosstalk between NF-120581B and PPAR-120574rdquo Jour-nal of Molecular and Cellular Cardiology vol 72 pp 85ndash942014

[39] LWangH ZhenWYao et al ldquoLipid raft-dependent activationof dual oxidase 1H

2O2NF-120581B pathway in bronchial epithelial

cellsrdquo American Journal of PhysiologymdashCell Physiology vol 301no 1 pp C171ndashC180 2011

[40] R Zhou A S Yazdi P Menu and J Tschopp ldquoA role formitochondria inNLRP3 inflammasome activationrdquoNature vol469 no 7329 pp 221ndash226 2011

[41] V Parra V Eisner M Chiong et al ldquoChanges in mitochondrialdynamics during ceramide-induced cardiomyocyte early apop-tosisrdquo Cardiovascular Research vol 77 no 2 pp 387ndash397 2008

[42] M Reers T W Smith and L B Chen ldquoJ-aggregate formationof a carbocyanine as a quantitative fluorescent indicator ofmembrane potentialrdquo Biochemistry vol 30 no 18 pp 4480ndash4486 1991

[43] T Collins ldquoBiology of disease endothelial nuclear factor-120581Band the initiation of the atherosclerotic lesionrdquo LaboratoryInvestigation vol 68 no 5 pp 499ndash508 1993

[44] E Schulz T Gori and T Munzel ldquoOxidative stress andendothelial dysfunction in hypertensionrdquo HypertensionResearch vol 34 no 6 pp 665ndash673 2011

[45] F Giacco and M Brownlee ldquoOxidative stress and diabeticcomplicationsrdquo Circulation Research vol 107 no 9 pp 1058ndash1070 2010

[46] A Briasoulis D Tousoulis E S Androulakis N PapageorgiouG Latsios and C Stefanadis ldquoEndothelial dysfunction andatherosclerosis focus on novel therapeutic approachesrdquo RecentPatents onCardiovascular DrugDiscovery vol 7 no 1 pp 21ndash322012

[47] M C Xu H M Shi H Wang and X F Gao ldquoSalidrosideprotects against hydrogen peroxide-induced injury inHUVECsvia the regulation of REDD1 and mTOR activationrdquo MolecularMedicine Reports vol 8 no 1 pp 147ndash153 2013

[48] L M Yung F P Leung X Yao Z-Y Chen and Y HuangldquoReactive oxygen species in vascular wallrdquo Cardiovascular andHematological DisordersmdashDrug Targets vol 6 no 1 pp 1ndash192006

[49] M Feletou and P M Vanhoutte ldquoEndothelial dysfunction amultifaceted disorderrdquo American Journal of PhysiologymdashHeartand Circulatory Physiology vol 291 no 3 pp H985ndashH10022006


[50] S Fujimoto T AsanoM Sakai et al ldquoMechanisms of hydrogenperoxide-induced relaxation in rabbit mesenteric small arteryrdquoEuropean Journal of Pharmacology vol 412 no 3 pp 291ndash3002001

[51] Z-W Yang A Zhang B T Altura and BM Altura ldquoHydrogenperoxide-induced endothelium-dependent relaxation of rataorta involvement of Ca2+ and other cellular metabolitesrdquoGeneral Pharmacology vol 33 no 4 pp 325ndash336 1999

[52] A Ellis M Pannirselvam T J Anderson and C R TriggleldquoCatalase has negligible inhibitory effects on endothelium-dependent relaxations inmouse isolated aorta and smallmesen-teric arteryrdquo British Journal of Pharmacology vol 140 no 7 pp1193ndash1200 2003

[53] A Zembowicz R J Hatchett A M Jakubowski and R JGryglewski ldquoInvolvement of nitric oxide in the endothelium-dependent relaxation induced by hydrogen peroxide in therabbit aortardquo British Journal of Pharmacology vol 110 no 1 pp151ndash158 1993

[54] H Cai Z Li S Dikalov et al ldquoNAD(P)H oxidase-derivedhydrogen peroxide mediates endothelial nitric oxide produc-tion in response to angiotensin IIrdquo The Journal of BiologicalChemistry vol 277 no 50 pp 48311ndash48317 2002

[55] C M Wong L M Yung F P Leung et al ldquoRaloxifene protectsendothelial cell function against oxidative stressrdquoBritish Journalof Pharmacology vol 155 no 3 pp 326ndash334 2008

[56] S Dimmeler and A M Zeiher ldquoNitric oxidemdashan endothelialcell survival factorrdquo Cell Death and Differentiation vol 6 no10 pp 964ndash968 1999

[57] W T Wong S L Wong X Y Tian and Y Huang ldquoEndothelialdysfunction the common consequence in diabetes and hyper-tensionrdquo Journal of Cardiovascular Pharmacology vol 55 no 4pp 300ndash307 2010

[58] X-F Niu C W Smith and P Kubes ldquoIntracellular oxidativestress induced by nitric oxide synthesis inhibition increasesendothelial cell adhesion to neutrophilsrdquo Circulation Researchvol 74 no 6 pp 1133ndash1140 1994

[59] H Cai Z Li M E Davis W Kanner D G Harrison andS C Dudley Jr ldquoAkt-dependent phosphorylation of serine1179 and mitogen-activated protein kinase kinaseextracellularsignal-regulated kinase 12 cooperatively mediate activation ofthe endothelial nitric-oxide synthase by hydrogen peroxiderdquoMolecular Pharmacology vol 63 no 2 pp 325ndash331 2003

[60] Z XiaM Liu YWu et al ldquoN-acetylcysteine attenuates TNF-120572-induced human vascular endothelial cell apoptosis and restoreseNOS expressionrdquo European Journal of Pharmacology vol 550no 1ndash3 pp 134ndash142 2006

[61] S Mohan M Hamuro G P Sorescu et al ldquoI120581B120572-dependentregulation of low-shear flow-induced NF-120581B activity role ofnitric oxiderdquo American Journal of PhysiologymdashCell Physiologyvol 284 no 4 pp C1039ndashC1047 2003

[62] J RMatthews C H BottingM Panico H RMorris and R THay ldquoInhibition ofNF-120581BDNAbinding by nitric oxiderdquoNucleicAcids Research vol 24 no 12 pp 2236ndash2242 1996

[63] E Pineda-Molina P Klatt J Vazquez et al ldquoGlutathionylationof the p50 subunit of NF-120581B a mechanism for redox-inducedinhibition of DNA bindingrdquo Biochemistry vol 40 no 47 pp14134ndash14142 2001

[64] L Jornot H Petersen andA F Junod ldquoModulation of theDNAbinding activity of transcription factors CREP NF120581B and HSFby H2O2and TNF120572 Differences between in vivo and in vitro

effectsrdquo FEBS Letters vol 416 no 3 pp 381ndash386 1997[65] V Oliveira-Marques H S Marinho L Cyrne and F Antunes

ldquoRole of hydrogen peroxide in NF-120581B activation from inducer

to modulatorrdquo Antioxidants amp Redox Signaling vol 11 no 9 pp2223ndash2243 2009

[66] Y Hasegawa T Saito T Ogihara et al ldquoBlockade of thenuclear factor-120581B pathway in the endothelium prevents insulinresistance and prolongs life spansrdquo Circulation vol 125 no 9pp 1122ndash1133 2012

[67] E Nisoli and M O Carruba ldquoNitric oxide and mitochondrialbiogenesisrdquo Journal of Cell Science vol 119 part 14 pp 2855ndash2862 2006

[68] E Nisoli E Clementi C Paolucci et al ldquoMitochondrialbiogenesis in mammals the role of endogenous nitric oxiderdquoScience vol 299 no 5608 pp 896ndash899 2003

[69] D Alvarez-Guardia X Palomer T Coll et al ldquoThe p65 subunitof NF-B binds to PGC-1 linking inflammation and metabolicdisturbances in cardiac cellsrdquo Cardiovascular Research vol 87no 3 pp 449ndash458 2010

[70] H-J Kim K-G Park E-K Yoo et al ldquoEffects of PGC-1120572on TNF-120572-induced MCP-1 and VCAM-1 expression and NF-120581B activation in human aortic smooth muscle and endothelialcellsrdquo Antioxidants amp Redox Signaling vol 9 no 3 pp 301ndash3072007

[71] A Csiszar N Labinskyy J T Pinto et al ldquoResveratrol inducesmitochondrial biogenesis in endothelial cellsrdquo American Jour-nal of PhysiologymdashHeart and Circulatory Physiology vol 297no 1 pp H13ndashH20 2009

[72] J Schilling L Lai N Sambandam C E Dey T C Leone andD P Kelly ldquoToll-like receptor-mediated inflammatory signalingreprograms cardiac energy metabolism by repressing peroxi-some proliferator-activated receptor 120574 coactivator-1 signalingrdquoCirculation Heart Failure vol 4 no 4 pp 474ndash482 2011

[73] J C Won J-Y Park Y M Kim et al ldquoPeroxisome proliferator-activated receptor-120574 coactivator 1-120572 overexpression preventsendothelial apoptosis by increasing ATPADP translocaseactivityrdquo Arteriosclerosis Thrombosis and Vascular Biology vol30 no 2 pp 290ndash297 2010

[74] I Valle A Alvarez-Barrientos E Arza S Lamas and MMonsalve ldquoPGC-1120572 regulates the mitochondrial antioxidantdefense system in vascular endothelial cellsrdquo CardiovascularResearch vol 66 no 3 pp 562ndash573 2005

[75] F Ali N S Ali A Bauer et al ldquoPPAR120590 and PGC1120572 act coop-eratively to induce haem oxygenase-1 and enhance vascularendothelial cell resistance to stressrdquo Cardiovascular Researchvol 85 no 4 pp 701ndash710 2010

[76] H Cai ldquoHydrogen peroxide regulation of endothelial func-tion origins mechanisms and consequencesrdquo CardiovascularResearch vol 68 no 1 pp 26ndash36 2005

[77] Y-M Liu B Jiang Y-M Bao and L-J An ldquoProtocatechuic acidinhibits apoptosis by mitochondrial dysfunction in rotenone-induced PC12 cellsrdquo Toxicology in Vitro vol 22 no 2 pp 430ndash437 2008

[78] H Huang and K G Manton ldquoThe role of oxidative damage inmitochondria during aging a reviewrdquo Frontiers in Biosciencevol 9 pp 1100ndash1117 2004

[79] F M Yakes and B van Houten ldquoMitochondrial DNA damage ismore extensive and persists longer than nuclear DNA damagein human cells following oxidative stressrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 94 no 2 pp 514ndash519 1997

[80] B G Hill G A Benavides J R Lancaster Jr et al ldquoIntegrationof cellular bioenergetics withmitochondrial quality control andautophagyrdquo Biological Chemistry vol 393 no 12 pp 1485ndash15122012

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


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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OphthalmologyJournal of


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Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


mitochondrial biogenesis also appears to be a key event in thedevelopment of aging-related vascular pathologies [13 21 22]identification of mechanisms that promote mitochondrialbiogenesis in the endothelial cells may provide new clues onthe pathogenesis of vascular disease

The health of mitochondria is in part regulated by theirbiogenesis and peroxisome proliferator-activated receptorgamma-coactivator-alpha (PGC-1120572) is regarded as the keyregulator [23] In endothelial cells PGC-1120572 also orchestratescellular defenses against oxidative stress [24] Mitochondrialtranscription factor A (TFAM) is responsible for the tran-scriptional control of mtDNA and its translocation to themitochondria is important to initiate mtDNA transcriptionand replication [25]

As described above mitochondria are highly dynamicorganelles and their biogenesis is likely to be involved in theregulation of endothelial cell metabolism redox regulationand signal transduction [6 16 26] Pathways that regulatemitochondrial biogenesis are potential therapeutic targetsfor the amelioration of endothelial dysfunction and vasculardiseases [27]

Salidroside (SAL) is an active ingredient of the rootof Rhodiola rosea a well-known herb used to relieve highaltitude sickness [28] SALhas also been used to enhance boththe physical and mental performance SAL upregulates thelevels of antioxidative enzymes glutathione peroxidase-1 andthioredoxin-1 to counteract oxidative stress [29] Previousstudies have shown that SAL promotes DNA repair enzymeParp-1 to counteract oxidative stress [30] Meanwhile arecent study reported that SAL attenuated homocysteine-induced endothelial dysfunction by reducing oxidative stress[31] As noted above there is tightly relationship betweenreduction of mitochondrial biogenesis and endothelial dys-function the present study was conducted to determinewhether SAL recovers the endothelial dysfunction inducedby H2O2through stimulating mitochondrial biogenesis and

counteracting the oxidative stress-related eNOS and NF-120581Bsignaling pathways

2 Materials and Methods

21 Animals Animals were treated in accordance with theguide for the Care and Use of Laboratory Animals publishedby the US National Institutes of Health and approved bythe Local Animal Care Committee Healthy Wistar rats(200ndash250 g) were purchased from the Center of Experimen-tal Animals (Tongji Medical College Huazhong Universityof Science and Technology China) and maintained in acontrolled environment with a lightdark cycle of 12 h atemperature of 20 plusmn 2∘C and humidity of 50 plusmn 2

22 Cell Culture The collection of human umbilical cordswas approved by the Ethics Committee of Tongji MedicalCollege Huazhong University of Science and Technology(Wuhan China) and conducted in accordance with theDeclaration of [32] Primary cultured human umbilical veinendothelial cells (HUVECs) were prepared as described in[33] In brief the umbilical cord was washed with cold

PBS and then infused with 025 trypsin After digestionstopped the cells were collected by centrifuging for 10minat 1 000 rpm The cells were resuspended and then culturedin endothelial cell medium (ECM Sciencell Carlsbad CA)at 37∘C in an incubator with a humidified atmosphere of 5CO2 In all experiments cells were used at passages 2ndash7

23 Cell Viability Assays To study the effect of SAL onH2O2-

induced cytotoxicity HUVECs were inoculated at a densityof 2 times 104 per well in 96-well plates and cultured overnightthe cell viability was evaluated using the Cell CountingKit-8 (Dojindo Laboratories Kumamoto) [34] Briefly theHUVECs treated with H

2O2(Sigma 4 h) or SAL (National

Institute for Food and Drug Control purity gt 98 24 h) atthe indicated concentration in OPTI-MEM (Gibco) beforecell viability was measured Moreover to study the effect ofSAL on H

2O2-induced inhibition of cell viability the cells

were cultured as described above after treatment with SALfor 20 h the H

2O2(100 120583M) was added to the medium for

another 4 hours At the end of the time period the culturemedium was removedThe cells were washed twice with PBSand incubated with CCK-8 solution at 37∘C for 30min Theabsorbance was measured using a microplate reader with atest wavelength of 450 nm

24 Free RadicalMeasurement in Cell Free System Theeffectsof SAL on scavenging hydroxyl radical (OH∙) superoxideradical (O

2

minus) and H2O2were measured with commercially

available kits (Nanjing Jiancheng Bioengineering InstituteChina) according to the manufacturerrsquos instructions In briefthe OH∙ was generated by the Fenton reaction and thentreated with a chromogenic substrate nitrotetrazolium bluechloride (NBT) to yield a stable colored substance which wasmeasured using a microplate reader with a test wavelengthof 450 nm O

2

minus was generated by the xanthinexanthineoxidase system O

2

minus was detected by nitrite method and theabsorbance at 550 nm was measured The reaction productof H2O2and molybdic acid can be detected at 405 nm

Deionized water and ascorbic acid (Vc) were used as theblank and positive controls respectivelyThe inhibition rate =(optical density of blank control groupsmdashoptical density oftreatment groups)optical density of blank control groups

25 Vascular Function Measurement The thoracic segmentsof the rat aorta were dissected and the surrounding con-nective tissues were cleaned off Each aorta was cut intoring segments of 2sim3mm in length The aortic rings weremounted in organ chambers filledwithKrebs-Henseleit (KH)solution at 37∘C with constant bubbling of 95 oxygen5carbon dioxide KH solution contained (mM) 133 NaCl 475KCl 15 CaCl

2 125 MgCl

2 25 NaHCO

3 and 11 D-glucose

Isometric tension was recorded with a force transducer(RM6240C Chengdu Instrument Factory) A basal tensionof 20mN was applied to each vascular ring After beingplaced in organ baths for 90min 05120583M phenylephrine(PE Sigma) was first administered to the rings to test theircontractility and then 1 120583M acetylcholine (ACh Sigma) wasadministered to assess the integrity of the endothelial layer







2



2














16

12

08

04

0Ctr 01 1 10 100 1000

Rela

tive v

iabi

lity

lowast lowast

lowastlowast

lowastlowast

(120583M)

(a)

12

1

08

06

04

02

0

Rela

tive v

iabi

lity

Ctr 01 1 10 100 1000

(120583M)

lowastlowast

(b)

12

1

08

06

04

02

0

Rela

tive v

iabi

lity

lowast

dagger dagger

H2O2

1001minusminus

minusSAL (120583M)+ + + +

1

(c)


2O2



lowastlowast



3 Results





2O2in a



2







2







70

50

30

10

minus1001 1 10 100 1000

Vc

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

(120583M)

Inhi

bitio

n ra

te (

)

SAL

(a)

Vc

01 1 10 100 1000

(120583M)

80

60

40

20

0

minus20

Inhi

bitio

n ra

te (

)

lowastlowast

SAL

(b)

01 1 10 100 1000

Vc(120583M)

6

2

minus2

minus6

minus10

minus14

Inhi

bitio

n ra

te (

)

SAL

(c)









2O2could













minus20

0

20

40

60

80

100


Rela

xatio

n (

)

CtrH2O2

SAL + H2O2

lowastlowast

lowastlowast

lowastlowast


lowast

daggerdaggerdagger

daggerdagger

daggerdagger

daggerdagger

daggerdagger

ACh (lgM)

(a)

CtrH2O2

SAL + H2O2

0 3 6 9 12 15 18 21 24 27 30

(min)

130

120

110

100

90

lowastlowast lowast

lowast

lowast

lowastlowast

daggerdagger

daggerdagger


dagger

lowastlowast

lowastlowast

Nor

mal

ized

DH

E flu

ores

cenc

e (

)

(b)

200

160

120

80

CtrH2O2

SAL + H2O2

0 3 6 9 12 15 18 21 24 27 30

(min)

Nor

mal

ized

DA

F-FM

-DA

fluor

esce

nce (

)

lowast

lowast

lowast lowast

lowastlowast

lowastlowast

daggerdaggerdagger

dagger

(c)









2O2 119899 = 4



4 Discussion





2O2




2O2(Figure 2)



p-eNOS (ser1177)

eNOS

p-AMPK120572 (thr172)

AMPK120572

p-Akt (ser473)

Akt

H2O2

10 10 10

01 01 100 100

minus minus minus


120573-Actin

(a)

3

25

2

15

1

05

0

p-eNOSeNOS

lowastlowast

lowast lowast

dagger

H2O2

10 10 10

01 01 100 100


SAL (120583M)

Expr

essio

n ra

tio (f

old)

(b)

3

25

2

15

1

05

0


H2O2

10 10 10

01 01 100 100


SAL (120583M)

lowastlowast

dagger

Expr

essio

n ra

tio (f

old)

(c)

H2O2

10 10 10

01 01 100 100

minus minus minus

minus minus

SAL (120583M)

2

15

1

05

0

p-AktAkt

lowast

daggerlowastlowast

Expr

essio

n ra

tio (f

old)

(d)


2O2(01 120583M



2O2elicits







2





2O2







Ctr 10 20 30 60 240

(min)

16

14

12

1

08

06

04

02

0

lowast

Rela

tive a

ctiv

ity o

f NF-120581

B

(a)

12

1

08

06

04

02

0Ctr 10 20 30 60 240

(min)

Rela

tive a

ctiv

ity o

f NF-120581

B


(b)

14

12

1

08

06

04

02

0

Rela

tive a

ctiv

ity o

f NF-120581

B

H2O2

minus minus minus

minus minus minus

SAL (120583M) 1 1 110 10 10

01 01 01 100 100 100

lowast

lowast

lowastlowast

dagger

dagger dagger

daggerdagger

(c)

35

3

25

2

15

1

05

0

H2O2 (120583M) 01 1 10 100

+ + + + +minus

minus minus

Rela

tive a

ctiv

ity o

f NF-120581

B

lowastlowast


TNF-120572

(d)


2O2(01 120583M) (a) and SAL (10 120583M) (b) on




lowastlowast


2O2(01 1 10 and 100120583M) for 30min and




2O2 Our




2O2per se induces





2O2(01 1 10





Ctr SAL (1120583M)

SAL (10120583M)2

16

12

08

04

0

Rela

tive

fluor

esce

nce i

nten

sity

Ctr SAL (1120583M) SAL (10120583M)

lowast

(a)

Ctr SAL (1120583M) SAL (10120583M)

PGC-1120572

TFAM

120573-Actin

(b)

Ctr SAL (1120583M) SAL (10120583M)

2

16

12

08

04

0

lowast

lowast

PGC-1120572TFAM

Expr

essio

n ra

tio (f

old)

(c)






12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

JC-1

(RG

) (fo

ld)

(a)

12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

Cel

lula

r ATP

leve

l (fo

ld)

(b)




eNOS

NO

OO



Nucleus

NF-120581B

HO

HO

HOOH

Salidroside

OH

TFAM

Akt

p65

p50

PGC-1120572

















Acknowledgments


References






























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















2



2














16

12

08

04

0Ctr 01 1 10 100 1000

Rela

tive v

iabi

lity

lowast lowast

lowastlowast

lowastlowast

(120583M)

(a)

12

1

08

06

04

02

0

Rela

tive v

iabi

lity

Ctr 01 1 10 100 1000

(120583M)

lowastlowast

(b)

12

1

08

06

04

02

0

Rela

tive v

iabi

lity

lowast

dagger dagger

H2O2

1001minusminus

minusSAL (120583M)+ + + +

1

(c)


2O2



lowastlowast



3 Results





2O2in a



2







2







70

50

30

10

minus1001 1 10 100 1000

Vc

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

(120583M)

Inhi

bitio

n ra

te (

)

SAL

(a)

Vc

01 1 10 100 1000

(120583M)

80

60

40

20

0

minus20

Inhi

bitio

n ra

te (

)

lowastlowast

SAL

(b)

01 1 10 100 1000

Vc(120583M)

6

2

minus2

minus6

minus10

minus14

Inhi

bitio

n ra

te (

)

SAL

(c)









2O2could













minus20

0

20

40

60

80

100


Rela

xatio

n (

)

CtrH2O2

SAL + H2O2

lowastlowast

lowastlowast

lowastlowast


lowast

daggerdaggerdagger

daggerdagger

daggerdagger

daggerdagger

daggerdagger

ACh (lgM)

(a)

CtrH2O2

SAL + H2O2

0 3 6 9 12 15 18 21 24 27 30

(min)

130

120

110

100

90

lowastlowast lowast

lowast

lowast

lowastlowast

daggerdagger

daggerdagger


dagger

lowastlowast

lowastlowast

Nor

mal

ized

DH

E flu

ores

cenc

e (

)

(b)

200

160

120

80

CtrH2O2

SAL + H2O2

0 3 6 9 12 15 18 21 24 27 30

(min)

Nor

mal

ized

DA

F-FM

-DA

fluor

esce

nce (

)

lowast

lowast

lowast lowast

lowastlowast

lowastlowast

daggerdaggerdagger

dagger

(c)









2O2 119899 = 4



4 Discussion





2O2




2O2(Figure 2)



p-eNOS (ser1177)

eNOS

p-AMPK120572 (thr172)

AMPK120572

p-Akt (ser473)

Akt

H2O2

10 10 10

01 01 100 100

minus minus minus


120573-Actin

(a)

3

25

2

15

1

05

0

p-eNOSeNOS

lowastlowast

lowast lowast

dagger

H2O2

10 10 10

01 01 100 100


SAL (120583M)

Expr

essio

n ra

tio (f

old)

(b)

3

25

2

15

1

05

0


H2O2

10 10 10

01 01 100 100


SAL (120583M)

lowastlowast

dagger

Expr

essio

n ra

tio (f

old)

(c)

H2O2

10 10 10

01 01 100 100

minus minus minus

minus minus

SAL (120583M)

2

15

1

05

0

p-AktAkt

lowast

daggerlowastlowast

Expr

essio

n ra

tio (f

old)

(d)


2O2(01 120583M



2O2elicits







2





2O2







Ctr 10 20 30 60 240

(min)

16

14

12

1

08

06

04

02

0

lowast

Rela

tive a

ctiv

ity o

f NF-120581

B

(a)

12

1

08

06

04

02

0Ctr 10 20 30 60 240

(min)

Rela

tive a

ctiv

ity o

f NF-120581

B


(b)

14

12

1

08

06

04

02

0

Rela

tive a

ctiv

ity o

f NF-120581

B

H2O2

minus minus minus

minus minus minus

SAL (120583M) 1 1 110 10 10

01 01 01 100 100 100

lowast

lowast

lowastlowast

dagger

dagger dagger

daggerdagger

(c)

35

3

25

2

15

1

05

0

H2O2 (120583M) 01 1 10 100

+ + + + +minus

minus minus

Rela

tive a

ctiv

ity o

f NF-120581

B

lowastlowast


TNF-120572

(d)


2O2(01 120583M) (a) and SAL (10 120583M) (b) on




lowastlowast


2O2(01 1 10 and 100120583M) for 30min and




2O2 Our




2O2per se induces





2O2(01 1 10





Ctr SAL (1120583M)

SAL (10120583M)2

16

12

08

04

0

Rela

tive

fluor

esce

nce i

nten

sity

Ctr SAL (1120583M) SAL (10120583M)

lowast

(a)

Ctr SAL (1120583M) SAL (10120583M)

PGC-1120572

TFAM

120573-Actin

(b)

Ctr SAL (1120583M) SAL (10120583M)

2

16

12

08

04

0

lowast

lowast

PGC-1120572TFAM

Expr

essio

n ra

tio (f

old)

(c)






12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

JC-1

(RG

) (fo

ld)

(a)

12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

Cel

lula

r ATP

leve

l (fo

ld)

(b)




eNOS

NO

OO



Nucleus

NF-120581B

HO

HO

HOOH

Salidroside

OH

TFAM

Akt

p65

p50

PGC-1120572

















Acknowledgments


References






























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















16

12

08

04

0Ctr 01 1 10 100 1000

Rela

tive v

iabi

lity

lowast lowast

lowastlowast

lowastlowast

(120583M)

(a)

12

1

08

06

04

02

0

Rela

tive v

iabi

lity

Ctr 01 1 10 100 1000

(120583M)

lowastlowast

(b)

12

1

08

06

04

02

0

Rela

tive v

iabi

lity

lowast

dagger dagger

H2O2

1001minusminus

minusSAL (120583M)+ + + +

1

(c)


2O2



lowastlowast



3 Results





2O2in a



2







2







70

50

30

10

minus1001 1 10 100 1000

Vc

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

(120583M)

Inhi

bitio

n ra

te (

)

SAL

(a)

Vc

01 1 10 100 1000

(120583M)

80

60

40

20

0

minus20

Inhi

bitio

n ra

te (

)

lowastlowast

SAL

(b)

01 1 10 100 1000

Vc(120583M)

6

2

minus2

minus6

minus10

minus14

Inhi

bitio

n ra

te (

)

SAL

(c)









2O2could













minus20

0

20

40

60

80

100


Rela

xatio

n (

)

CtrH2O2

SAL + H2O2

lowastlowast

lowastlowast

lowastlowast


lowast

daggerdaggerdagger

daggerdagger

daggerdagger

daggerdagger

daggerdagger

ACh (lgM)

(a)

CtrH2O2

SAL + H2O2

0 3 6 9 12 15 18 21 24 27 30

(min)

130

120

110

100

90

lowastlowast lowast

lowast

lowast

lowastlowast

daggerdagger

daggerdagger


dagger

lowastlowast

lowastlowast

Nor

mal

ized

DH

E flu

ores

cenc

e (

)

(b)

200

160

120

80

CtrH2O2

SAL + H2O2

0 3 6 9 12 15 18 21 24 27 30

(min)

Nor

mal

ized

DA

F-FM

-DA

fluor

esce

nce (

)

lowast

lowast

lowast lowast

lowastlowast

lowastlowast

daggerdaggerdagger

dagger

(c)









2O2 119899 = 4



4 Discussion





2O2




2O2(Figure 2)



p-eNOS (ser1177)

eNOS

p-AMPK120572 (thr172)

AMPK120572

p-Akt (ser473)

Akt

H2O2

10 10 10

01 01 100 100

minus minus minus


120573-Actin

(a)

3

25

2

15

1

05

0

p-eNOSeNOS

lowastlowast

lowast lowast

dagger

H2O2

10 10 10

01 01 100 100


SAL (120583M)

Expr

essio

n ra

tio (f

old)

(b)

3

25

2

15

1

05

0


H2O2

10 10 10

01 01 100 100


SAL (120583M)

lowastlowast

dagger

Expr

essio

n ra

tio (f

old)

(c)

H2O2

10 10 10

01 01 100 100

minus minus minus

minus minus

SAL (120583M)

2

15

1

05

0

p-AktAkt

lowast

daggerlowastlowast

Expr

essio

n ra

tio (f

old)

(d)


2O2(01 120583M



2O2elicits







2





2O2







Ctr 10 20 30 60 240

(min)

16

14

12

1

08

06

04

02

0

lowast

Rela

tive a

ctiv

ity o

f NF-120581

B

(a)

12

1

08

06

04

02

0Ctr 10 20 30 60 240

(min)

Rela

tive a

ctiv

ity o

f NF-120581

B


(b)

14

12

1

08

06

04

02

0

Rela

tive a

ctiv

ity o

f NF-120581

B

H2O2

minus minus minus

minus minus minus

SAL (120583M) 1 1 110 10 10

01 01 01 100 100 100

lowast

lowast

lowastlowast

dagger

dagger dagger

daggerdagger

(c)

35

3

25

2

15

1

05

0

H2O2 (120583M) 01 1 10 100

+ + + + +minus

minus minus

Rela

tive a

ctiv

ity o

f NF-120581

B

lowastlowast


TNF-120572

(d)


2O2(01 120583M) (a) and SAL (10 120583M) (b) on




lowastlowast


2O2(01 1 10 and 100120583M) for 30min and




2O2 Our




2O2per se induces





2O2(01 1 10





Ctr SAL (1120583M)

SAL (10120583M)2

16

12

08

04

0

Rela

tive

fluor

esce

nce i

nten

sity

Ctr SAL (1120583M) SAL (10120583M)

lowast

(a)

Ctr SAL (1120583M) SAL (10120583M)

PGC-1120572

TFAM

120573-Actin

(b)

Ctr SAL (1120583M) SAL (10120583M)

2

16

12

08

04

0

lowast

lowast

PGC-1120572TFAM

Expr

essio

n ra

tio (f

old)

(c)






12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

JC-1

(RG

) (fo

ld)

(a)

12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

Cel

lula

r ATP

leve

l (fo

ld)

(b)




eNOS

NO

OO



Nucleus

NF-120581B

HO

HO

HOOH

Salidroside

OH

TFAM

Akt

p65

p50

PGC-1120572

















Acknowledgments


References






























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















70

50

30

10

minus1001 1 10 100 1000

Vc

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

(120583M)

Inhi

bitio

n ra

te (

)

SAL

(a)

Vc

01 1 10 100 1000

(120583M)

80

60

40

20

0

minus20

Inhi

bitio

n ra

te (

)

lowastlowast

SAL

(b)

01 1 10 100 1000

Vc(120583M)

6

2

minus2

minus6

minus10

minus14

Inhi

bitio

n ra

te (

)

SAL

(c)









2O2could













minus20

0

20

40

60

80

100


Rela

xatio

n (

)

CtrH2O2

SAL + H2O2

lowastlowast

lowastlowast

lowastlowast


lowast

daggerdaggerdagger

daggerdagger

daggerdagger

daggerdagger

daggerdagger

ACh (lgM)

(a)

CtrH2O2

SAL + H2O2

0 3 6 9 12 15 18 21 24 27 30

(min)

130

120

110

100

90

lowastlowast lowast

lowast

lowast

lowastlowast

daggerdagger

daggerdagger


dagger

lowastlowast

lowastlowast

Nor

mal

ized

DH

E flu

ores

cenc

e (

)

(b)

200

160

120

80

CtrH2O2

SAL + H2O2

0 3 6 9 12 15 18 21 24 27 30

(min)

Nor

mal

ized

DA

F-FM

-DA

fluor

esce

nce (

)

lowast

lowast

lowast lowast

lowastlowast

lowastlowast

daggerdaggerdagger

dagger

(c)









2O2 119899 = 4



4 Discussion





2O2




2O2(Figure 2)



p-eNOS (ser1177)

eNOS

p-AMPK120572 (thr172)

AMPK120572

p-Akt (ser473)

Akt

H2O2

10 10 10

01 01 100 100

minus minus minus


120573-Actin

(a)

3

25

2

15

1

05

0

p-eNOSeNOS

lowastlowast

lowast lowast

dagger

H2O2

10 10 10

01 01 100 100


SAL (120583M)

Expr

essio

n ra

tio (f

old)

(b)

3

25

2

15

1

05

0


H2O2

10 10 10

01 01 100 100


SAL (120583M)

lowastlowast

dagger

Expr

essio

n ra

tio (f

old)

(c)

H2O2

10 10 10

01 01 100 100

minus minus minus

minus minus

SAL (120583M)

2

15

1

05

0

p-AktAkt

lowast

daggerlowastlowast

Expr

essio

n ra

tio (f

old)

(d)


2O2(01 120583M



2O2elicits







2





2O2







Ctr 10 20 30 60 240

(min)

16

14

12

1

08

06

04

02

0

lowast

Rela

tive a

ctiv

ity o

f NF-120581

B

(a)

12

1

08

06

04

02

0Ctr 10 20 30 60 240

(min)

Rela

tive a

ctiv

ity o

f NF-120581

B


(b)

14

12

1

08

06

04

02

0

Rela

tive a

ctiv

ity o

f NF-120581

B

H2O2

minus minus minus

minus minus minus

SAL (120583M) 1 1 110 10 10

01 01 01 100 100 100

lowast

lowast

lowastlowast

dagger

dagger dagger

daggerdagger

(c)

35

3

25

2

15

1

05

0

H2O2 (120583M) 01 1 10 100

+ + + + +minus

minus minus

Rela

tive a

ctiv

ity o

f NF-120581

B

lowastlowast


TNF-120572

(d)


2O2(01 120583M) (a) and SAL (10 120583M) (b) on




lowastlowast


2O2(01 1 10 and 100120583M) for 30min and




2O2 Our




2O2per se induces





2O2(01 1 10





Ctr SAL (1120583M)

SAL (10120583M)2

16

12

08

04

0

Rela

tive

fluor

esce

nce i

nten

sity

Ctr SAL (1120583M) SAL (10120583M)

lowast

(a)

Ctr SAL (1120583M) SAL (10120583M)

PGC-1120572

TFAM

120573-Actin

(b)

Ctr SAL (1120583M) SAL (10120583M)

2

16

12

08

04

0

lowast

lowast

PGC-1120572TFAM

Expr

essio

n ra

tio (f

old)

(c)






12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

JC-1

(RG

) (fo

ld)

(a)

12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

Cel

lula

r ATP

leve

l (fo

ld)

(b)




eNOS

NO

OO



Nucleus

NF-120581B

HO

HO

HOOH

Salidroside

OH

TFAM

Akt

p65

p50

PGC-1120572

















Acknowledgments


References






























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















minus20

0

20

40

60

80

100


Rela

xatio

n (

)

CtrH2O2

SAL + H2O2

lowastlowast

lowastlowast

lowastlowast


lowast

daggerdaggerdagger

daggerdagger

daggerdagger

daggerdagger

daggerdagger

ACh (lgM)

(a)

CtrH2O2

SAL + H2O2

0 3 6 9 12 15 18 21 24 27 30

(min)

130

120

110

100

90

lowastlowast lowast

lowast

lowast

lowastlowast

daggerdagger

daggerdagger


dagger

lowastlowast

lowastlowast

Nor

mal

ized

DH

E flu

ores

cenc

e (

)

(b)

200

160

120

80

CtrH2O2

SAL + H2O2

0 3 6 9 12 15 18 21 24 27 30

(min)

Nor

mal

ized

DA

F-FM

-DA

fluor

esce

nce (

)

lowast

lowast

lowast lowast

lowastlowast

lowastlowast

daggerdaggerdagger

dagger

(c)









2O2 119899 = 4



4 Discussion





2O2




2O2(Figure 2)



p-eNOS (ser1177)

eNOS

p-AMPK120572 (thr172)

AMPK120572

p-Akt (ser473)

Akt

H2O2

10 10 10

01 01 100 100

minus minus minus


120573-Actin

(a)

3

25

2

15

1

05

0

p-eNOSeNOS

lowastlowast

lowast lowast

dagger

H2O2

10 10 10

01 01 100 100


SAL (120583M)

Expr

essio

n ra

tio (f

old)

(b)

3

25

2

15

1

05

0


H2O2

10 10 10

01 01 100 100


SAL (120583M)

lowastlowast

dagger

Expr

essio

n ra

tio (f

old)

(c)

H2O2

10 10 10

01 01 100 100

minus minus minus

minus minus

SAL (120583M)

2

15

1

05

0

p-AktAkt

lowast

daggerlowastlowast

Expr

essio

n ra

tio (f

old)

(d)


2O2(01 120583M



2O2elicits







2





2O2







Ctr 10 20 30 60 240

(min)

16

14

12

1

08

06

04

02

0

lowast

Rela

tive a

ctiv

ity o

f NF-120581

B

(a)

12

1

08

06

04

02

0Ctr 10 20 30 60 240

(min)

Rela

tive a

ctiv

ity o

f NF-120581

B


(b)

14

12

1

08

06

04

02

0

Rela

tive a

ctiv

ity o

f NF-120581

B

H2O2

minus minus minus

minus minus minus

SAL (120583M) 1 1 110 10 10

01 01 01 100 100 100

lowast

lowast

lowastlowast

dagger

dagger dagger

daggerdagger

(c)

35

3

25

2

15

1

05

0

H2O2 (120583M) 01 1 10 100

+ + + + +minus

minus minus

Rela

tive a

ctiv

ity o

f NF-120581

B

lowastlowast


TNF-120572

(d)


2O2(01 120583M) (a) and SAL (10 120583M) (b) on




lowastlowast


2O2(01 1 10 and 100120583M) for 30min and




2O2 Our




2O2per se induces





2O2(01 1 10





Ctr SAL (1120583M)

SAL (10120583M)2

16

12

08

04

0

Rela

tive

fluor

esce

nce i

nten

sity

Ctr SAL (1120583M) SAL (10120583M)

lowast

(a)

Ctr SAL (1120583M) SAL (10120583M)

PGC-1120572

TFAM

120573-Actin

(b)

Ctr SAL (1120583M) SAL (10120583M)

2

16

12

08

04

0

lowast

lowast

PGC-1120572TFAM

Expr

essio

n ra

tio (f

old)

(c)






12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

JC-1

(RG

) (fo

ld)

(a)

12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

Cel

lula

r ATP

leve

l (fo

ld)

(b)




eNOS

NO

OO



Nucleus

NF-120581B

HO

HO

HOOH

Salidroside

OH

TFAM

Akt

p65

p50

PGC-1120572

















Acknowledgments


References






























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















p-eNOS (ser1177)

eNOS

p-AMPK120572 (thr172)

AMPK120572

p-Akt (ser473)

Akt

H2O2

10 10 10

01 01 100 100

minus minus minus


120573-Actin

(a)

3

25

2

15

1

05

0

p-eNOSeNOS

lowastlowast

lowast lowast

dagger

H2O2

10 10 10

01 01 100 100


SAL (120583M)

Expr

essio

n ra

tio (f

old)

(b)

3

25

2

15

1

05

0


H2O2

10 10 10

01 01 100 100


SAL (120583M)

lowastlowast

dagger

Expr

essio

n ra

tio (f

old)

(c)

H2O2

10 10 10

01 01 100 100

minus minus minus

minus minus

SAL (120583M)

2

15

1

05

0

p-AktAkt

lowast

daggerlowastlowast

Expr

essio

n ra

tio (f

old)

(d)


2O2(01 120583M



2O2elicits







2





2O2







Ctr 10 20 30 60 240

(min)

16

14

12

1

08

06

04

02

0

lowast

Rela

tive a

ctiv

ity o

f NF-120581

B

(a)

12

1

08

06

04

02

0Ctr 10 20 30 60 240

(min)

Rela

tive a

ctiv

ity o

f NF-120581

B


(b)

14

12

1

08

06

04

02

0

Rela

tive a

ctiv

ity o

f NF-120581

B

H2O2

minus minus minus

minus minus minus

SAL (120583M) 1 1 110 10 10

01 01 01 100 100 100

lowast

lowast

lowastlowast

dagger

dagger dagger

daggerdagger

(c)

35

3

25

2

15

1

05

0

H2O2 (120583M) 01 1 10 100

+ + + + +minus

minus minus

Rela

tive a

ctiv

ity o

f NF-120581

B

lowastlowast


TNF-120572

(d)


2O2(01 120583M) (a) and SAL (10 120583M) (b) on




lowastlowast


2O2(01 1 10 and 100120583M) for 30min and




2O2 Our




2O2per se induces





2O2(01 1 10





Ctr SAL (1120583M)

SAL (10120583M)2

16

12

08

04

0

Rela

tive

fluor

esce

nce i

nten

sity

Ctr SAL (1120583M) SAL (10120583M)

lowast

(a)

Ctr SAL (1120583M) SAL (10120583M)

PGC-1120572

TFAM

120573-Actin

(b)

Ctr SAL (1120583M) SAL (10120583M)

2

16

12

08

04

0

lowast

lowast

PGC-1120572TFAM

Expr

essio

n ra

tio (f

old)

(c)






12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

JC-1

(RG

) (fo

ld)

(a)

12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

Cel

lula

r ATP

leve

l (fo

ld)

(b)




eNOS

NO

OO



Nucleus

NF-120581B

HO

HO

HOOH

Salidroside

OH

TFAM

Akt

p65

p50

PGC-1120572

















Acknowledgments


References






























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Ctr 10 20 30 60 240

(min)

16

14

12

1

08

06

04

02

0

lowast

Rela

tive a

ctiv

ity o

f NF-120581

B

(a)

12

1

08

06

04

02

0Ctr 10 20 30 60 240

(min)

Rela

tive a

ctiv

ity o

f NF-120581

B


(b)

14

12

1

08

06

04

02

0

Rela

tive a

ctiv

ity o

f NF-120581

B

H2O2

minus minus minus

minus minus minus

SAL (120583M) 1 1 110 10 10

01 01 01 100 100 100

lowast

lowast

lowastlowast

dagger

dagger dagger

daggerdagger

(c)

35

3

25

2

15

1

05

0

H2O2 (120583M) 01 1 10 100

+ + + + +minus

minus minus

Rela

tive a

ctiv

ity o

f NF-120581

B

lowastlowast


TNF-120572

(d)


2O2(01 120583M) (a) and SAL (10 120583M) (b) on




lowastlowast


2O2(01 1 10 and 100120583M) for 30min and




2O2 Our




2O2per se induces





2O2(01 1 10





Ctr SAL (1120583M)

SAL (10120583M)2

16

12

08

04

0

Rela

tive

fluor

esce

nce i

nten

sity

Ctr SAL (1120583M) SAL (10120583M)

lowast

(a)

Ctr SAL (1120583M) SAL (10120583M)

PGC-1120572

TFAM

120573-Actin

(b)

Ctr SAL (1120583M) SAL (10120583M)

2

16

12

08

04

0

lowast

lowast

PGC-1120572TFAM

Expr

essio

n ra

tio (f

old)

(c)






12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

JC-1

(RG

) (fo

ld)

(a)

12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

Cel

lula

r ATP

leve

l (fo

ld)

(b)




eNOS

NO

OO



Nucleus

NF-120581B

HO

HO

HOOH

Salidroside

OH

TFAM

Akt

p65

p50

PGC-1120572

















Acknowledgments


References






























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Ctr SAL (1120583M)

SAL (10120583M)2

16

12

08

04

0

Rela

tive

fluor

esce

nce i

nten

sity

Ctr SAL (1120583M) SAL (10120583M)

lowast

(a)

Ctr SAL (1120583M) SAL (10120583M)

PGC-1120572

TFAM

120573-Actin

(b)

Ctr SAL (1120583M) SAL (10120583M)

2

16

12

08

04

0

lowast

lowast

PGC-1120572TFAM

Expr

essio

n ra

tio (f

old)

(c)






12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

JC-1

(RG

) (fo

ld)

(a)

12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

Cel

lula

r ATP

leve

l (fo

ld)

(b)




eNOS

NO

OO



Nucleus

NF-120581B

HO

HO

HOOH

Salidroside

OH

TFAM

Akt

p65

p50

PGC-1120572

















Acknowledgments


References






























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

JC-1

(RG

) (fo

ld)

(a)

12

08

06

04

02

Ctr H2O2 SAL + H2O2

lowast

dagger

0

1

Cel

lula

r ATP

leve

l (fo

ld)

(b)




eNOS

NO

OO



Nucleus

NF-120581B

HO

HO

HOOH

Salidroside

OH

TFAM

Akt

p65

p50

PGC-1120572

















Acknowledgments


References






























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























Acknowledgments


References






























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article salidroside stimulates mitochondrial...

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