tetrahydroxystilbene glucoside (tsg) restores the effect of … · 2019. 7. 30. · researcharticle...

Research ArticleTetrahydroxystilbene Glucoside (TSG) Restores the Effect ofTransient Hypoxia on Reperfusion Injury in Senescent H9c2Cells by Regulating Mitochondrial Energy Metabolism

Yin Cheng1 Jia Song2 Pengfei Hu3 and Yun Zhu 1

1Department of Emergency Zhejiang Hospital Hangzhou 310012 China2Department of Emergency e Second Affiliated Hospital of Zhejiang Chinese Medical University Hangzhou 31005 China3Department of Cardiology e Second Affiliated Hospital of Zhejiang Chinese Medical University Hangzhou 31005 China

Correspondence should be addressed to Yun Zhu zy1011750869163com

Received 28 October 2018 Accepted 4 December 2018 Published 13 December 2018

Academic Editor Michał Tomczyk

Copyright copy 2018 Yin Cheng 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

Tetrahydroxystilbene glucoside (TSG) is extracted from a famous Chinese herbal medicine which is widely used as an antiagingagent in history Lots of studies gave evidence that TSG exhibited benefits to brain like improvement of learning and memory andsynaptic plasticity Moreover the polyphenolic structure of TSG enables its capability to prevent cerebral ischemiareperfusioninjury (IRI) by reducing apoptosis and ROSRNS generation Due to its antioxidant profile TSG had been demonstrated toalleviate cardiac toxicity by regulating biochemical indexes and ROS However whether TSG exhibited cardioprotective effectsvia mitochondrial energy metabolic functions which played crucial role in IRI remained unclear Here we used an in vitro agingmodel of cardiomyocytes to evaluate the effects of TSG on transient hypoxia-pretreated hypoxiareoxygenation (HR) injury andmitochondrial energy metaolism Our results showed that TSG enhanced cardioprotective effect of transient hypoxia on HR byreducing excessive ROS production and calcium overloading Significant improvements ofmitochondrial respiratory functions andketone body metabolism elucidated that TSG restored the effect of transient hypoxia on HR injury in aging cardiomyocytes viaupregulating mitochondrial energy metabolism

1 Introduction

Ischemic preconditioning (IPC) targeting to mitochondrialATP-sensitive potassium channel (KATP channel) was one ofthe effective clinicalexperimental interventions to preventischemiareperfusion injury (IRI) [1] Mechanistic studieshad verified selectively activating KATP channel by pharma-cological intervention performed the approximately effects ofIPC but not in aging hearts [2] One of natural compoundshad been discovered to restore the cardioprotective effects ofIPC in aging hearts was resveratrol with polyphenol struc-ture [3] Resveratrol was natural compound derived fromgrapes and red wine performing the anti-aging and anti-oxidant profiles [4ndash6] Taking these aspects as considerationanother natural compound tetrahydroxystilbene glucoside(TSG) derived from traditional Chinese herb Polygonummultiflorum with polyphenolic structure probably have theability to restore IPCrsquos effects in aging hearts [7 8]

The neuroprotective effects of TSG had shown that TSGsignificantly reduced cognitive impairments and improvedthe hippocampal synaptic plasticity [7 9] and furtherimproved brain memory by activating SIRT1 expressionand phosphorylation of ERKs CaMKII [10] Depending onthese activities TSG intervention restored learning memoryin APP transgenic mice a mouse model of Alzheimerrsquosdisease and increased neuron survival in Parkinsonrsquosmice viaPI3KAkt signaling [11 12] Based on the beneficial effects ofTSG on brain Chenrsquos group found pretreatment of TSG pre-vented cerebral ischemia injury via inhibition of ROSRNSgeneration involving JNK and NF-120581B signaling [13] All ofthese studies indicated TSG probably have beneficial effect oncardiac IPC against prolonged IR injury in aging hearts

Here we applied an in vitro model of IR to eval-uate whether TSG could restore the effect of transienthypoxia against HR injury in senescent H9c2 cardiomy-ocytes We stepped forward to detect mitochondrial energy

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 2545024 7 pageshttpsdoiorg10115520182545024

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Primer sequences of ACAT1 and OXCT1

Name Forward ReverseACAT1 51015840-ATGGCTGCCCTGGCGGTTCTA-31015840 51015840-CTACAGCTTCTCAATCAGCAC-31015840

OXCT1 51015840-CTGGAGTTTGAGGACGGCAT-31015840 51015840-TCCGCATCAGCTTCGTCTTT-31015840

120573-Actin 51015840-GCTACAGCTTCACCACCACA-31015840 51015840-ATCGTACTCCTGCTTGCTGA-31015840

metabolism including mitochondrial respiration and ketonebody metabolism and uncovered the role of both energeticmetabolisms in regulation of TSG in senescent H9c2 cellsFinally we demonstrated TSG restored the effect of transienthypoxia onHR injury by regulatingmitochondrialOXPHOSand ketone body metabolism

2 Materials and Methods

21 Cell Culture and Pharmacological Interventions H9c2cardiomyocyte cell line was purchased from the AmericanType Culture Collection (CRL1446 ATCC USA) and cul-tured in full Dulbeccorsquos Modified Eagle Medium (DMEM)composed of 10 fetal bovine serum (FBS) TSG (Sigma-Aldrich St Louis MO USA) was given 24 h before transienthypoxia and HR H9c2 cells were divided into 8 groups (i)control cultured in normoxia (ii) HR alone (iii) transienthypoxia (iv) transient hypoxia + HR (v) TSG alone (vi)TSG+HR (vii) transient hypoxia + TSG and (viii) transienthypoxia + TSG + HR

22 In Vitro Model of Senescence HR and Transient HypoxiaSenescent H9c2 cells were induced by D-galactose (D-gal)with 20 gL for 60 h after cell adherence and all the H9c2cells used in this studywere senescent H9c2 cells hypoxiawasinduced by replacing the normoxia medium with no glucoseDMEM and placing cells into a chemical hypoxia system(BD USA) After 12 h hypoxia H9c2 cells were subjected toreoxygenation by replacing with cultural medium for 24 hTransient hypoxia was performed 2 h before HR injury

23 Cell Viability and Mitochondrial Viability Cell viabilitiesof H9c2 cells after different treatments were determined byusing 3-(45-dimethylthiazol-2-yl)-25-diphe-nyltetrazoliumbromide (MTT) assay purchased from Sigma-Aldrich (StLouis MO USA) After reoxygenation cultural mediumcontaining MTT (500 120583gml) was added to the plates (100120583lwell) and incubated for 4 h at 37∘C Then 10 SDS-HClsolution was added to solubilize formazan crystals formedin viable cells The absorbance was measure optical densityat 570650 nm by multimode plate reader (SpectraMaxParadigm Molecular Devices USA) Cell viability of groupwithout any treatment was considered as 100

The mitochondrial viability was detected by the mito-chondrial viability assay kit (ab129732 Abcam USA) After4 h dye staining 96-well microplate was shaken gently for5 min then the fluorescence was detected at 550590 nm indark Mitochondrial viability of group without any treatmentwas considered as 100

24 Measurements of Reactive Oxygen Species (ROS) andCalcium (1198621198862+) ROS and calcium were measured by BD

FACSAria III flow cytometer (BD USA) using the DCFDAROS probe (Invitrogen USA) and Fluo-4 AM (InvitrogenUSA) fluorescent probe respectively according to the man-ufactoryrsquos methods Briefly cells were digested by Trypsin-EDTA and resuspended with PBS for washing Then cellswere incubated with DCFDA or Fluo-4 probe for 30 min at37∘C in darkTheROS production and calcium concentrationin each group were indicated by intensity of green fluores-cence (Ex 488nm Em535nm) Fluorescent intensity of groupwithout any treatment was considered as 100

25 Measurements of Oxygen Consumption Rate (OCR) andRespiratory Function Mitochondrial OCR and respiratoryfunction were detected by using the XF Cell Mito StressTestwith SeahorseXFpExtracellular FluxAnalyzer (SeahorseBioscience USA) H9c2 cells were seeded on the XFp cellculture miniplates (5000well) and the sensor cartridge washydrated in a non-CO2 incubator at 37∘C one day beforeassay After HR the port A on the sensor cartridge wasinjected with 15 120583M oligomycin (complex V inhibitor) 2120583M FCCP and 05 120583M rotenoneantimycin A (inhibitors ofcomplex I and complex III) were loaded to ports A B andC respectively After sensor calibration the sensor was puton the cell plate immediately and detected by Mito StressTest OCR ATP production basal respiration and maximalrespiration were calculated in according to protocol andnormalized to protein concentration in each well

26 RNA Extraction Reverse Transcription and QuantitativeReal-Time PCR Cellular RNAwas extracted by using Favor-Prep RNA purification kit (Favorgen Biotech Corp) Cellswere lysed in 300 120583l of lysis buffer and total RNAwas isolatedon spin columns following the manufacturerrsquos instructionsAfter that RNA was eluted with 30 120583l water without RNaseand purified RNA (1 120583g) was reversal transcribed to 20 120583lsystem Synthesized cDNAwas then detected byQuantitativereal-time PCR performing with the real-time PCR systemusing the FastStart Universal SYBR-Green kit according tothe manufacturerrsquos instructions Primer sequences directedagainst ACAT1 and OXCT1 were listed in Table 1 for mRNAquantification and 120573-actin was used as an endogenouscontrol The PCR mixture was comprised with 10 120583l SYBRmix 1 120583g template ddH2O 05 120583l forward primers and 05 120583lreverse primers making up to 20 120583l The procedure of RCRincluded 50∘C for 2min and 95∘C for 10min andwas followedby 45 cycles of 95∘C for 15 s and 60∘C for 60 s Gene expressionlevel was based on the comparative CT value and normalizedwith 120573-actin

27 Measurements of Cellular BHB and ACAC Cellular BHBwas detected by using a Caymanrsquos Colorimetric Assay KitBriefly cells were washed with PBS for 1 time and digested

Evidence-Based Complementary and Alternative Medicine 3

0

50

100

150

Cel

l Via

bilit

y(

of C

ontr

ol)

- + - + - + - +HRCtrl HYP TSG HYP+TSG

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

(a)

0

50

100

150

Mito

chon

dria

l Via

bilit

y(

of C

ontr

ol)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

0

100

200

300

400

Rela

tive i

nten

sity

ofG

reen

FL

( o

f Con

trol

)


lowastlowast

lowastlowast

lowastlowastlowast

lowast

(c)

0

200

400

600

Rela

tive i

nten

sity

ofG

reen

FL

( o

f Con

trol

)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)

Figure 1 TSG restored protective effect of transient hypoxia (TH) on senescent H9c2 cells against HR injury (a) Effect of TH and TSGon cell viability in senescent H9c2 cells against HR injury determined by MTT assay Data (n=5) were shown as the mean plusmn SEM lowastplt005and lowastlowastplt001 lowastlowastlowastplt0001 (b) Effect of TH and TSG on mitochondrial viability in senescent H9c2 cells against HR injury determined byMitochondrial Viability assay Data (n=5) were shown as the mean plusmn SEM lowastlowastlowastplt0001 (c d) Effects of TH and TSG on ROS production andCa2+ concentration in senescent H9c2 cells against HR injury determined by flow cytometry respectively stained by H

2DCFDA and Fluo-4

AM Data (n=3) were shown as the mean plusmn SEM lowastplt005 lowastlowastplt001 lowastlowastlowastplt0001

by trypsin and then collected by centrifuging at 2000 g for 10minutes Pellets were resuspended with 1 ml cold assay buffer(1times) and supersonicated with 1 second bursts for 20 times andcentrifuged at 10000 g for 10 minutes Then the pellets wereresuspended in 1 ml of cold assay buffer for further detection50 120583l of developer solution was added to 50 120583l of samples andplate was incubated in the dark for 30 minutes and read platewith the absorbance at 450 nm

The concentration of ACAC was detected after differenttreatments by using a Colorimetric Assay Kit (Abcam UK)Cells pellets were collected and resuspended with ddH

2O as

above Sample volumes (10-100 120583l) were adjusted to 110 120583l byddH2O 80 120583l ACAC assay buffer and 10 120583l ACAC substrate

were added and mixed well Plate was incubated at 25∘C for15 min and the values of absorbance at OD 550 nm weremeasured in a kinetic mode

28 Measurements of Cellular Acetyl-CoA Cellular acetyl-CoA concentrations were determined using a commercialKit (Abnova TaiWan) H9c2 cells were collected and super-sonicated rapidly on ice and centrifuged at 10000 times g 4∘CSupernatants were stayed on ice for 5 min and neutralizedpH to 6-8 with 3 M KHCO

3and diluted volume to 50 120583l

Then added reaction mix with 50 120583l to samples and incubatedfor 10 min at 37∘C The plate was measured with fluorescent

detection using ExEm= 535589 nm Fluorescent intensity ofgroup without any treatment was considered as 100

29 Statistics Data were analyzed using Student t-test be-tween 2 groups by GraphPad Prism 70 All data werepresented as the means plusmn SEM Plt005 was represented asstatistical significance

3 Results

31 TSG Restored the Effect of Transient Hypoxia on Reperfu-sion Injury in Senescent H9c2 Cells TSG had been reportedto alleviate aging-associated diseases and prevent IR injuryWhether TSG can restore the effect of transient hypoxia onlong-term HR injury in senescent H9c2 cells was tested inour study We firstly evaluated the cell viability and mito-chondrial viability in senescent cells HR injury significantlydecreased cell viability in control group and transient hypoxia(HYP) did not show any cardioprotective effect Excitinglycombined treatment of HYP and TSG showed an increasingcell viability andmitochondrial viability comparingwith con-trol group indicating TSG could restore the cardioprotectiveeffect of HYP Moreover HYP plus TSG group displayedhigher activity comparing with HYP only group and TSGonly group respectively (Figures 1(a) and 1(b))


156 808 145 211 277 342 408 473 538 604 669 735TIME (min)

Oligomycin FCCP Rotenone amp antimycin A

CtrlCtrl-HRHYPHYP-HR

TSGTSG-HRHYP+TSGHYP+TSG-HR

0

50

100

150

OCR

(pm

olm

in

g)

(a)

0

20

40

60

80

ATP

prod

uctio

n (p

mol

min

g)


lowast

lowastlowast

lowastlowast

lowast

(b)

0

20

40

60

80

Basa

l Res

pira

tion

(pm

olm

in

g)

- + - + - + - +HR

Ctrl HYP TSG HYP+TSG

lowast

lowastlowast

lowastlowast

lowast

(c)

0

50

100

150M

axim

al R

espi

ratio

n (p

mol

min

g)

- + - + - + - +HR


lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)

Figure 2 TSG restored protective effect of transient hypoxia (TH) on senescent H9c2 cells against HR injury by regulating mitochondrialOXPHOS (a) Effect of TH and TSG on OCR in senescent H9c2 cells against HR injury determined by mitochondrial respiration kit (b c d)Effect of TH and TSG on ATP production basal respiration and maximal respiration in senescent H9c2 cells against HR injury Data (n=3)were shown as the mean plusmn SEM lowastplt005 lowastlowastplt001 and lowastlowastlowastplt0001

To further demonstrate TSGrsquos ability to restore HYPrsquoscardioprotective effect on senescent H9c2 cells ROS andcalciumoverloading two crucialmarkers forHR injuryweredetected by flow cytometry The cellular concentrations ofROS and calcium increased significantly after HR injury (ge3 times) and HYP treatment had no effect on the excessiveproductions of ROS and calcium while combined treatmentsofHYP andTSGevidently reduced cytotoxicity by decreasingROS production and calcium overloading compared withHYP only groups and TSG only groups (Figures 1(c) and1(d))

Taken together TSG restored the cardiac beneficial effectof transient hypoxia in senescent H9c2 cells via reductionof cellular ROS calcium overloading and mitochondrialtoxicity based on its antiaging profile

32 TSG Restored the Effect of Transient Hypoxia on Reper-fusion Injury in Senescent H9c2 Cells via MitochondrialRespiratory Function Transient hypoxia stimuli activatedmitochondrial respiratory function against mitochondrialturnover and mitochondria-mediated cell apoptosis andorgan damage However mitochondrial functions were dis-rupted in aging progress especially in energy-consumption

organ such as heart Due to antiaging profile of TSGwe hypothesized TSG pretreatment had ability to alleviatebiological alterations of aging which probably can restorethe effect of transient hypoxia by regulation of mitochondrialrespiratory function

We tested living cell oxygen consumption rate (OCR)by XP Extracellular Flux Analyzer and results showed thatHYP did not enhance OCR when cells were exposed to HRinjury Pretreatment of HYP plus TSG enhancedOCR againstHR injury in senescent H9c2 cells (Figure 2(a)) We furtheranalyzed respiration-related parameters ATP productionbasal respiration and maximal respiration to understandTSGrsquos effect in detail Consistentwith the result ofOCR curveHYP could not upregulate ATP production basal respirationand maximal respiration in senescent cells against HRMeanwhile TSG only group and HYP plus TSG groupsignificantly upregulated ATP production basal respirationand maximal respiration the latter group displaying moreeffective mitochondrial respiratory functions than TSG onlygroup and restored the effect of HYP (Figures 2(b) 2(c) and2(d))

Collectively TSG restored the cardiac beneficial effect oftransient hypoxia in senescent H9c2 cells via enhancement of



lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

0

50

100

150

BHB

( o

f Con

trol

)

(a)

Ace

toac

etat

e


lowastlowast

lowastlowast

lowastlowastlowast

lowastlowast

0

50

100

150

( o

f Con

trol

)

(b)

Rela

tive l

evel

OXCT1ACAT1


lowastlowastlowast

lowastlowast

lowast

0

50

100

150

of m

RNA

(c)


lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

0

50

100

150

Ace

tyl C

oA (

of C

ontr

ol)

(d)

Figure 3 TSG restored protective effect of transient hypoxia (TH) on senescent H9c2 cells against HR injury by regulating ketone bodymetabolism (a b) The concentration of 120573-hydroxybutyrate (BHB) and acetoacetate (ACAC)in cell extractions Data (n=3) were presentedas meanplusmn SEM lowastplt005 lowastlowastplt001 lowastlowastlowastplt0001 (c) mRNA levels of OXCT1 and ACAT1 were quantified by real-time RT-PCR Data (n=3)were presented as mean plusmn SEM OXCT1 lowastplt005 and lowastlowastplt001 ACAT1 119901 lt 001 (d) The concentration of acetyl CoA in cell extractionsData (n=3) were presented as meanplusmn SEM lowastplt005 lowastlowastplt001 and lowastlowastlowastplt0001

mitochondrial respiratory functions based on its antiagingprofile

33 TSG Restored the Effect of Transient Hypoxia on Reper-fusion Injury in Senescent H9c2 Cells via Ketone BodyMetabolism Mitochondrial OXPHOS was important forcardiac energy demand even under reperfusion period inwhich peroxidative injury damaged mitochondrial respi-ration Therefore other energy metabolic utilizations likeketone body were essential for cardiac survival and workingwell We evaluated the level of two major components ofketone body 120573-hydroxybutyrate (BHB) and acetoacetate(ACAC) Firstly we found that the levels of BHB and ACACsignificantly decreased after HR injury in the control groupsuggesting that ketone body metabolism was damaged HYPhypoxia could not raise the levels of BHB and ACAChowever pretreatment of TSG had the ability to increasetheir levels in senescent cells Interestingly in group of HYPcombined with TSG the levels of BHB and ACAC evidently

increased comparing with the single treatment of HYP orTSG respectively suggesting TSG restored the HYPrsquos effecton ketone body in senescent cardiomyocytes (Figures 3(a)and 3(b))

Crucial regulators of ketone body metabolism wereOXCT1 and ACAT1 whose mRNA level was analyzed indifferent treatments After HR the mRNA level of bothproteins decreased significantly in control group as well asin HYP group indicating loss of cardioprotective effect ofHYP in senescent cardiomyocytes Single treatment of TSGevidently upregulatedmRNAexpression levels of OXCT1 andACAT1 against senescence Importantly TSG pretreatmentincreased the levels of OXCT1 and ACAT1 comparing withHYP and TSG single treatment group respectively suggest-ing the restoration of HYPrsquo ability in cardioprotection againstHR injury (Figure 3(c))

Acetyl-CoA was the final product in ketone body uti-lization in heart to supply the material for TCA cycleWe evaluated the concentration of Acetyl-CoA to examine


Loss of effect

Transient hypoxia

Aging cardiomyocyte

Acetyl CoA

BHBACAC

Acetone

Ketone body

Transient hypoxia

Restoration of effect

Aging cardiomyocyte

Acetyl CoA

BHBACAC

Acetone

Ketone body

TSG+

Figure 4 Schematic representation raised the effect of Tetrahydroxystilbene glucoside (TSG) on restoration of transient hypoxiarsquoscardioprotective effect in aging cardiomyocytes Left graph represented cells without TSG treatment transient hypoxia cannot upregulatemitochondrial respiration and ketone bodymetabolism in aging cardiomyocytes right graph represented cells with TSG treatment after TSGtreatment transient hypoxia restores the upregulation of mitochondrial respiration and ketone body metabolism in aging cardiomyocytes

whether the upregulations of ketone body and its relativeenzymes performed effective functions in cardiac energysupplementary when facing HR injury The results wereexactly as what we thought that combined treatment ofHYP and TSG significantly increased Acetyl-CoA comparingwith HYP only group and TSG only group respectively(Figure 3(d))

In summary TSG pretreatment restored cardioprotectiveeffects of HYP against HR injury via upregulations ofmitochondrial respiration and ketone body metabolism insenescent cardiomyocytes

4 Discussion

Our study amplified the knowledge of TSG application incardiac injury especially in senescent cardiomyocytes basedon its antiaging profiles We also gave insight into functionalregulation of these polyphenols or compounds with polyphe-nol structure in restoration of transient hypoxia or evenhypoxicischemic preconditioning We firstly used in vitromodel of aging by long-term treatment of D-gal accordingto previous studies [14ndash17] Then we found transient hypoxiacould not increase cell viability and mitochondrial viabilityagainst HR injury in senescent H9c2 cells which wasconsistent with previous reports [1 3 15 18] In the group ofTSG single treatment TSG increased cell and mitochondrialviability decreased ROS production and calcium overloadingagainst HR injury comparing with cells in control groupsuggesting TSGrsquos original antiaging and prevention fromHRinjury When cells were treated by TSG plus HYP significantenhancements of cell and mitochondrial viability were foundwith reductions of ROS production and calcium overloadingunder HR injury

As a compound extracted from an antiaging Chineseherb TSG could alleviated aging-associated phenotypes inlots of studies [11 19ndash21] However the relationship betweenTSG and mitochondria was not uncovered considering thecrucial role of mitochondria in aging progress and aging-related diseases Our study evaluated mitochondrial respira-tory function and found TSG single treatment had ability toenhance OCR and ATP production in senescent cardiomy-ocytes against HR injury Furthermore TSG restored theeffect of transient hypoxia to protect senescent cells fromHRinjury via upregulation of OCR ATP production and basaland maximal respiration That suggested that TSG contain-ing a polyphenol structure probably retain mitochondrialviability and respiratory function in aging cardiomyocytesfor restoration of transient hypoxiarsquos effect on long-term HRinjury

Ketone body composed with 120573-hydroxybutyrate (BHB)acetoacetate (AcAc) and acetone derived from liver andplayed an important role in energy supply when cell experi-enced glucose and oxygendeprivation in heart and brain [22]Hepatic ketogenesis applied acetyl-CoA to produce ketonebody and delivered to heart trough blood In heart ketonebody was transferred to acetyl-CoA by regulation of OXCT1and ACAT1 for utilization of TCA cycle inmitochondria [23]Therefore we detected the levels of BHB and ACAC twomajor components of ketone body andmRNAexpressions ofOXCT1 and ACAT1 in our study Interesting results were thatTSG single treatment significantly increased levels of BHBand ACAC and also the mRNA expressions of OXCT1 andACAT1 in cells exposed to HR More importantly combinedtreatments of TSG and HYP exhibited the higher levels ofthese parameters suggesting TSG restored HYPrsquos effect insenescent cardiomyocytes against HR injury in partly by


upregulating ketone body metabolism Finally the level ofacetyl-CoA increased in TSG plus HYP group indicatingmore production of acetyl-CoA for TCA cycle

In conclusion TSG assisted transient hypoxia to restoreits protective effect on senescent cardiomyocytes againstHR injury via reduction of ROS production and calciumoverloading enhancement mitochondrial respiratory func-tion and ATP production and ketone body metabolism(Figure 4)

Data Availability

The data used to support the findings of this study areincluded within the article in Figures 1ndash3

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This work was supported by grants from the Natural ScienceFoundation of Zhejiang Province (LY18H270008 PF Hu)

References

[1] P Abete G Testa F Cacciatore et al ldquoIschemic preconditioningin the younger and aged heartrdquo Aging and Disease (AampD) vol2 no 2 pp 138ndash148 2011

[2] R A Kloner and S H Rezkalla ldquoPreconditioning postcondi-tioning and their application to clinical cardiologyrdquo Cardiovas-cular Research vol 70 no 2 pp 297ndash307 2006

[3] H Zheng H Guo Y Hong F Zheng and J Wang ldquoTheeffects of age and resveratrol on the hypoxic precondition-ing protection against hypoxia-reperfusion injury studies inrat hearts and human cardiomyocytesrdquo European Journal ofCardio-oracic Surgery vol 45 pp 375ndash381 2015

[4] M D Knutson and C Leeuwenburgh ldquoResveratrol and novelpotent activators of SIRT1 Effects on aging and age-relateddiseasesrdquo Nutrition Reviews vol 66 no 10 pp 591ndash596 2008

[5] S J Park F Ahmad A Philp et al ldquoResveratrol amelioratesaging-related metabolic phenotypes by inhibiting cAMP phos-phodiesterasesrdquo Cell vol 148 no 3 pp 421ndash433 2012

[6] D R Valenzano E Terzibasi T Genade A Cattaneo LDomenici and A Cellerino ldquoResveratrol prolongs lifespanand retards the onset of age-related markers in a short-livedvertebraterdquoCurrent Biology vol 16 no 3 pp 296ndash300 2006

[7] T Wang Y J Yang P FWu et al ldquoTetrahydroxystilbene gluco-side a plant-derived cognitive enhancer promotes hippocam-pal synaptic plasticityrdquo European Journal of Pharmacology vol650 no 1 pp 206ndash214 2011

[8] X X Zhou Q Yang Y H Xie et al ldquoProtective effect oftetrahydroxystilbene glucoside against D-galactose inducedaging process in micerdquo Phytochemistry Letters vol 6 no 3 pp372ndash378 2013

[9] R Wang Y Tang B Feng et al ldquoChanges in hippocampalsynapses and learning-memory abilities in age-increasing ratsand effects of tetrahydroxystilbene glucoside in aged ratsrdquoNeuroscience vol 149 no 4 pp 739ndash746 2007

[10] T Chen Y-J Yang Y-K Li et al ldquoChronic administrationtetrahydroxystilbene glucoside promotes hippocampal mem-ory and synaptic plasticity and activates ERKs CaMKII andSIRT1miR-134 in vivordquo Journal of Ethnopharmacology vol 190pp 74ndash82 2016

[11] L Zhang L Huang L Chen D Hao and J Chen ldquoNeuropro-tection by tetrahydroxystilbene glucoside in the MPTP mousemodel of Parkinsonrsquos diseaserdquo Toxicology Letters vol 222 no 2pp 155ndash163 2013

[12] L Zhang Y Xing C-F Ye H-X Ai H-F Wei and L LildquoLearning-memory deficit with aging in APP transgenic miceof Alzheimerrsquos disease and intervention by using tetrahydrox-ystilbene glucosiderdquo Behavioural Brain Research vol 173 no 2pp 246ndash254 2006

[13] TWang J Gu P Wu et al ldquoProtection by tetrahydroxystilbeneglucoside against cerebral ischemia involvement of JNK SIRT1and NF-120581B pathways and inhibition of intracellular ROSRNSgenerationrdquo Free Radical Biology amp Medicine vol 47 no 3 pp229ndash240 2009

[14] Z Du Y Yang Y Hu et al ldquoA long-term high-fat diet increasesoxidative stress mitochondrial damage and apoptosis in theinner ear of d-galactose-induced aging ratsrdquo Hearing Researchvol 287 no 1-2 pp 15ndash24 2012

[15] H Li C Zhang W Sun et al ldquoExogenous hydrogen sulfiderestores cardioprotection of ischemic post-conditioning viainhibition of mPTP opening in the aging cardiomyocytesrdquo Cellamp Bioscience vol 5 no 43 2016

[16] X Ren L Chen J Xie et al ldquoResveratrol amelioratesmitochondrial elongation via Drp1ParkinPINK1 signaling insenescent-like cardiomyocytesrdquo Oxidative Medicine and Cellu-lar Longevity vol 2017 Article ID 4175353 20 pages 2017

[17] Y-Y Zhou X-F Ji J-P Fu et al ldquoGene transcriptional andmetabolic profile changes in mimetic aging mice induced by D-galactoserdquo PLoS ONE vol 10 no 7 Article ID e0132088 2015

[18] K Boengler R Schulz and G Heusch ldquoLoss of cardioprotec-tion with ageingrdquo Cardiovascular Research vol 83 no 2 pp247ndash261 2009

[19] C Shen F-L Sun R-Y Zhang et al ldquoTetrahydroxystilbene glu-coside ameliorates memory and movement functions protectssynapses and inhibits 120572-synuclein aggregation in hippocampusand striatum in aged micerdquo Restorative Neurology and Neuro-science vol 33 no 4 pp 531ndash541 2015

[20] L Zhang S Yu R Zhang Y Xing Y Li andL Li ldquoTetrahydrox-ystilbene glucoside antagonizes age-related120572-synuclein overex-pression in the hippocampus of APP transgenic mouse modelof Alzheimerrsquos diseaserdquoRestorative Neurology andNeurosciencevol 31 no 1 pp 41ndash52 2013

[21] L Zhou Y Hou Q Yang et al ldquoTetrahydroxystilbene glucosideimproves the learning and memory of amyloid-120573

1ndash42-injectedrats andmaybe connected to synaptic changes in the hippocam-pusrdquoCanadian Journal of Physiology and Pharmacology vol 90no 11 pp 1446ndash1455 2012

[22] T FukaoGMitchell J O Sass T Hori K Orii and Y AoyamaldquoKetone body metabolism and its defectsrdquo Journal of InheritedMetabolic Disease vol 37 pp 541ndash551 2014

[23] D G Cotter R C Schugar and P A Crawford ldquoKetone bodymetabolism and cardiovascular diseaserdquo American Journal ofPhysiology-Heart and Circulatory Physiology vol 304 no 8 ppH1060ndashH1076 2013

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Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

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Table 1 Primer sequences of ACAT1 and OXCT1

Name Forward ReverseACAT1 51015840-ATGGCTGCCCTGGCGGTTCTA-31015840 51015840-CTACAGCTTCTCAATCAGCAC-31015840

OXCT1 51015840-CTGGAGTTTGAGGACGGCAT-31015840 51015840-TCCGCATCAGCTTCGTCTTT-31015840

120573-Actin 51015840-GCTACAGCTTCACCACCACA-31015840 51015840-ATCGTACTCCTGCTTGCTGA-31015840

metabolism including mitochondrial respiration and ketonebody metabolism and uncovered the role of both energeticmetabolisms in regulation of TSG in senescent H9c2 cellsFinally we demonstrated TSG restored the effect of transienthypoxia onHR injury by regulatingmitochondrialOXPHOSand ketone body metabolism

2 Materials and Methods

21 Cell Culture and Pharmacological Interventions H9c2cardiomyocyte cell line was purchased from the AmericanType Culture Collection (CRL1446 ATCC USA) and cul-tured in full Dulbeccorsquos Modified Eagle Medium (DMEM)composed of 10 fetal bovine serum (FBS) TSG (Sigma-Aldrich St Louis MO USA) was given 24 h before transienthypoxia and HR H9c2 cells were divided into 8 groups (i)control cultured in normoxia (ii) HR alone (iii) transienthypoxia (iv) transient hypoxia + HR (v) TSG alone (vi)TSG+HR (vii) transient hypoxia + TSG and (viii) transienthypoxia + TSG + HR

22 In Vitro Model of Senescence HR and Transient HypoxiaSenescent H9c2 cells were induced by D-galactose (D-gal)with 20 gL for 60 h after cell adherence and all the H9c2cells used in this studywere senescent H9c2 cells hypoxiawasinduced by replacing the normoxia medium with no glucoseDMEM and placing cells into a chemical hypoxia system(BD USA) After 12 h hypoxia H9c2 cells were subjected toreoxygenation by replacing with cultural medium for 24 hTransient hypoxia was performed 2 h before HR injury

23 Cell Viability and Mitochondrial Viability Cell viabilitiesof H9c2 cells after different treatments were determined byusing 3-(45-dimethylthiazol-2-yl)-25-diphe-nyltetrazoliumbromide (MTT) assay purchased from Sigma-Aldrich (StLouis MO USA) After reoxygenation cultural mediumcontaining MTT (500 120583gml) was added to the plates (100120583lwell) and incubated for 4 h at 37∘C Then 10 SDS-HClsolution was added to solubilize formazan crystals formedin viable cells The absorbance was measure optical densityat 570650 nm by multimode plate reader (SpectraMaxParadigm Molecular Devices USA) Cell viability of groupwithout any treatment was considered as 100

The mitochondrial viability was detected by the mito-chondrial viability assay kit (ab129732 Abcam USA) After4 h dye staining 96-well microplate was shaken gently for5 min then the fluorescence was detected at 550590 nm indark Mitochondrial viability of group without any treatmentwas considered as 100

24 Measurements of Reactive Oxygen Species (ROS) andCalcium (1198621198862+) ROS and calcium were measured by BD

FACSAria III flow cytometer (BD USA) using the DCFDAROS probe (Invitrogen USA) and Fluo-4 AM (InvitrogenUSA) fluorescent probe respectively according to the man-ufactoryrsquos methods Briefly cells were digested by Trypsin-EDTA and resuspended with PBS for washing Then cellswere incubated with DCFDA or Fluo-4 probe for 30 min at37∘C in darkTheROS production and calcium concentrationin each group were indicated by intensity of green fluores-cence (Ex 488nm Em535nm) Fluorescent intensity of groupwithout any treatment was considered as 100

25 Measurements of Oxygen Consumption Rate (OCR) andRespiratory Function Mitochondrial OCR and respiratoryfunction were detected by using the XF Cell Mito StressTestwith SeahorseXFpExtracellular FluxAnalyzer (SeahorseBioscience USA) H9c2 cells were seeded on the XFp cellculture miniplates (5000well) and the sensor cartridge washydrated in a non-CO2 incubator at 37∘C one day beforeassay After HR the port A on the sensor cartridge wasinjected with 15 120583M oligomycin (complex V inhibitor) 2120583M FCCP and 05 120583M rotenoneantimycin A (inhibitors ofcomplex I and complex III) were loaded to ports A B andC respectively After sensor calibration the sensor was puton the cell plate immediately and detected by Mito StressTest OCR ATP production basal respiration and maximalrespiration were calculated in according to protocol andnormalized to protein concentration in each well

26 RNA Extraction Reverse Transcription and QuantitativeReal-Time PCR Cellular RNAwas extracted by using Favor-Prep RNA purification kit (Favorgen Biotech Corp) Cellswere lysed in 300 120583l of lysis buffer and total RNAwas isolatedon spin columns following the manufacturerrsquos instructionsAfter that RNA was eluted with 30 120583l water without RNaseand purified RNA (1 120583g) was reversal transcribed to 20 120583lsystem Synthesized cDNAwas then detected byQuantitativereal-time PCR performing with the real-time PCR systemusing the FastStart Universal SYBR-Green kit according tothe manufacturerrsquos instructions Primer sequences directedagainst ACAT1 and OXCT1 were listed in Table 1 for mRNAquantification and 120573-actin was used as an endogenouscontrol The PCR mixture was comprised with 10 120583l SYBRmix 1 120583g template ddH2O 05 120583l forward primers and 05 120583lreverse primers making up to 20 120583l The procedure of RCRincluded 50∘C for 2min and 95∘C for 10min andwas followedby 45 cycles of 95∘C for 15 s and 60∘C for 60 s Gene expressionlevel was based on the comparative CT value and normalizedwith 120573-actin

27 Measurements of Cellular BHB and ACAC Cellular BHBwas detected by using a Caymanrsquos Colorimetric Assay KitBriefly cells were washed with PBS for 1 time and digested


0

50

100

150

Cel

l Via

bilit

y(

of C

ontr

ol)


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

(a)

0

50

100

150

Mito

chon

dria

l Via

bilit

y(

of C

ontr

ol)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

0

100

200

300

400

Rela

tive i

nten

sity

ofG

reen

FL

( o

f Con

trol

)


lowastlowast

lowastlowast

lowastlowastlowast

lowast

(c)

0

200

400

600

Rela

tive i

nten

sity

ofG

reen

FL

( o

f Con

trol

)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)


2DCFDA and Fluo-4




2O as








3 Results



156 808 145 211 277 342 408 473 538 604 669 735TIME (min)




0

50

100

150

OCR

(pm

olm

in

g)

(a)

0

20

40

60

80

ATP

prod

uctio

n (p

mol

min

g)


lowast

lowastlowast

lowastlowast

lowast

(b)

0

20

40

60

80

Basa

l Res

pira

tion

(pm

olm

in

g)

- + - + - + - +HR


lowast

lowastlowast

lowastlowast

lowast

(c)

0

50

100

150M

axim

al R

espi

ratio

n (p

mol

min

g)

- + - + - + - +HR


lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)










lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

0

50

100

150

BHB

( o

f Con

trol

)

(a)

Ace

toac

etat

e


lowastlowast

lowastlowast

lowastlowastlowast

lowastlowast

0

50

100

150

( o

f Con

trol

)

(b)

Rela

tive l

evel

OXCT1ACAT1


lowastlowastlowast

lowastlowast

lowast

0

50

100

150

of m

RNA

(c)


lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

0

50

100

150

Ace

tyl C

oA (

of C

ontr

ol)

(d)








Loss of effect

Transient hypoxia

Aging cardiomyocyte

Acetyl CoA

BHBACAC

Acetone

Ketone body

Transient hypoxia


Aging cardiomyocyte

Acetyl CoA

BHBACAC

Acetone

Ketone body

TSG+




4 Discussion







Data Availability




Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

50

100

150

Cel

l Via

bilit

y(

of C

ontr

ol)


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

(a)

0

50

100

150

Mito

chon

dria

l Via

bilit

y(

of C

ontr

ol)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

0

100

200

300

400

Rela

tive i

nten

sity

ofG

reen

FL

( o

f Con

trol

)


lowastlowast

lowastlowast

lowastlowastlowast

lowast

(c)

0

200

400

600

Rela

tive i

nten

sity

ofG

reen

FL

( o

f Con

trol

)


lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)


2DCFDA and Fluo-4




2O as








3 Results



156 808 145 211 277 342 408 473 538 604 669 735TIME (min)




0

50

100

150

OCR

(pm

olm

in

g)

(a)

0

20

40

60

80

ATP

prod

uctio

n (p

mol

min

g)


lowast

lowastlowast

lowastlowast

lowast

(b)

0

20

40

60

80

Basa

l Res

pira

tion

(pm

olm

in

g)

- + - + - + - +HR


lowast

lowastlowast

lowastlowast

lowast

(c)

0

50

100

150M

axim

al R

espi

ratio

n (p

mol

min

g)

- + - + - + - +HR


lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)










lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

0

50

100

150

BHB

( o

f Con

trol

)

(a)

Ace

toac

etat

e


lowastlowast

lowastlowast

lowastlowastlowast

lowastlowast

0

50

100

150

( o

f Con

trol

)

(b)

Rela

tive l

evel

OXCT1ACAT1


lowastlowastlowast

lowastlowast

lowast

0

50

100

150

of m

RNA

(c)


lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

0

50

100

150

Ace

tyl C

oA (

of C

ontr

ol)

(d)








Loss of effect

Transient hypoxia

Aging cardiomyocyte

Acetyl CoA

BHBACAC

Acetone

Ketone body

Transient hypoxia


Aging cardiomyocyte

Acetyl CoA

BHBACAC

Acetone

Ketone body

TSG+




4 Discussion







Data Availability




Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















156 808 145 211 277 342 408 473 538 604 669 735TIME (min)




0

50

100

150

OCR

(pm

olm

in

g)

(a)

0

20

40

60

80

ATP

prod

uctio

n (p

mol

min

g)


lowast

lowastlowast

lowastlowast

lowast

(b)

0

20

40

60

80

Basa

l Res

pira

tion

(pm

olm

in

g)

- + - + - + - +HR


lowast

lowastlowast

lowastlowast

lowast

(c)

0

50

100

150M

axim

al R

espi

ratio

n (p

mol

min

g)

- + - + - + - +HR


lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)










lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

0

50

100

150

BHB

( o

f Con

trol

)

(a)

Ace

toac

etat

e


lowastlowast

lowastlowast

lowastlowastlowast

lowastlowast

0

50

100

150

( o

f Con

trol

)

(b)

Rela

tive l

evel

OXCT1ACAT1


lowastlowastlowast

lowastlowast

lowast

0

50

100

150

of m

RNA

(c)


lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

0

50

100

150

Ace

tyl C

oA (

of C

ontr

ol)

(d)








Loss of effect

Transient hypoxia

Aging cardiomyocyte

Acetyl CoA

BHBACAC

Acetone

Ketone body

Transient hypoxia


Aging cardiomyocyte

Acetyl CoA

BHBACAC

Acetone

Ketone body

TSG+




4 Discussion







Data Availability




Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

0

50

100

150

BHB

( o

f Con

trol

)

(a)

Ace

toac

etat

e


lowastlowast

lowastlowast

lowastlowastlowast

lowastlowast

0

50

100

150

( o

f Con

trol

)

(b)

Rela

tive l

evel

OXCT1ACAT1


lowastlowastlowast

lowastlowast

lowast

0

50

100

150

of m

RNA

(c)


lowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

0

50

100

150

Ace

tyl C

oA (

of C

ontr

ol)

(d)








Loss of effect

Transient hypoxia

Aging cardiomyocyte

Acetyl CoA

BHBACAC

Acetone

Ketone body

Transient hypoxia


Aging cardiomyocyte

Acetyl CoA

BHBACAC

Acetone

Ketone body

TSG+




4 Discussion







Data Availability




Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Loss of effect

Transient hypoxia

Aging cardiomyocyte

Acetyl CoA

BHBACAC

Acetone

Ketone body

Transient hypoxia


Aging cardiomyocyte

Acetyl CoA

BHBACAC

Acetone

Ketone body

TSG+




4 Discussion







Data Availability




Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















Data Availability




Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















tetrahydroxystilbene glucoside (tsg) restores the effect of … · 2019. 7. 30. · researcharticle...

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