Danna He,Ruiping Zhao,Xiurong Song,Wei Li,Jun Hu,Yaojun Lu

Abstract Objective:To explore the role of CTRP3 in cardiomyocyte injury induced by oxygen glucose deprivation/reperfusion (OGD/R) and its possible mechanism.Methods:Rat cardiomyocytes H9c2 were cultured in vitro,and OGD/R induced H9c2 cell injury.qRT-PCR was used to detect the expression of CTRP3,SIRT1 and FOXO3a mRNA in cells.Western blot was used to detect the expression of CTRP3,SIRT1,FOXO3a,Bax,Bcl-2 and cleaved caspase-3 protein in cells.CCK-8 was used to detect cell survival.Flow cytometry was used to detect cell apoptosis.The kit was used to detect the CK-MB,cTnI,SOD and MDA activity,and the GSH-Px and ROS production of cells.Results:Compared with the control group,the expressions of CTRP3,SIRT1 and FOXO3a mRNA and protein,the cell survival rate,Bcl-2 protein expression,and SOD and GSHPx activities were significantly reduced (P＜0.05),while the apoptosis rate,the expression of Bax and cleaved caspase-3 protein,the contents of CK-MB,cTnI and MDA,and ROS fluorescence intensity were significantly increased in OGD/Rgroup (P＜0.05).There were no significant differences in cell indicators in the oe-NC group and OGD/R group(P＞0.05).Compared with the oe-NC group,the CTRP3,SIRT1 and FOXO3a mRNA and protein expressions,cell survival rate,Bcl-2 protein expression,and SOD and GSH-Px activities were significantly increased,whereas the apoptosis rate,the expression of Bax and cleaved caspase-3 protein,the contents of CK-MB,cTnI and MDA,and ROS fluorescence intensity were significantly reduced in the oe-CTRP3 group(P＜0.05).Compared with the oe-CTRP3 group,the SIRT1,FOXO3a and Bcl-2 protein expression,cell survival rate,SOD and GSH-Px activities in the oe-CTRP3+EX527 group were significantly reduced,while the apoptosis rate,the expression of Bax and cleaved caspase-3 protein,the contents of CK-MB,cTnI and MDA,and ROS fluorescence intensity were increased significantly (P＜0.05).Compared with the oe-CTRP3+EX527 group,the FOXO3a and Bcl-2 protein expressions,the cell survival rate and SOD and GSH-Px activities were significantly increased,while the apoptosis rate,the expressions of Bax and cleaved caspase-3 protein,the contents of CK-MB,cTnI and MDA,and ROS fluorescence intensity were significantly reduced in the oe-CTRP3+EX527+oe-FOXO3a group(P＜0.05).Conclusion:CTRP3 promotes the survival of cardiomyocytes induced by OGD/R and inhibits apoptosis and oxidative stress by activating the SIRT1/FOXO3a pathway.

Keywords cardiomyocytes;glucose and oxygen deprivation/reperfusion;CTRP3;SIRT1;FOXO3a

Introduction

Myocardial ischaemia/reperfusion (I/R) injury is a common clinical and pathological condition of ischemic heart disease (IHD).It is one of the main reasons for the failure and deterioration of acute myocardial infarction (AMI) after percutaneous coronary intervention(PCI)in AMI[1].The blood supply of ischemic myocardium was restored after PCI,which greatly reduced the disability rate and mortality of AMI patients.However,PCI treatment cannot reduce or restore the ischemic injury of cardiomyocytes,but aggravate the degree of myocardial injury in some patients with AMI [2].At present,reducing myocardial injury caused by reperfusion therapy after myocardial infarction has become the hot issue of clinical research.C1q/TNF-related protein 3 (CTRP3) is a new adipose factor and a highly conserved CTRP member of the adiponectin family [3].Exogenous CTRP3 enhanced the survival and retention of mesenchymal stromal cells (MSCs) in vivo,and improved the myocardial repair effect of MSCs.CTRP3 may play a role as a favorable regulator of the heart [4].At present,the mechanism of CTRP3 in myocardial I/R injury is not clear.Silent silent information regulator 1 activates forkhead box O3a (FOXO3a),which has a significant protective effect on apoptosis and mitochondrial dysfunction induced by I/R [5].In addition,evidence has shown that CTRP3 plays a protective role in severe acute pancreatitis by activating SIRT1 to inhibit the release of inflammatory mediators and acinous cell apoptosis [6].The relationship among CTRP3,SIRT1 and FOXO3a in myocardial I/R injury is not clear.Therefore,the purpose of this study is to explore the role of CTRP3 in myocardial I/R injury and whether its mechanism is related to the regulation of SIRT1/FOXO3a pathway,so as to clarify the mechanism of CTRP3 in myocardial I/R injury and develop new therapeutic targets to reduce myocardial I/R injury.

Materials and methods

Main materials

The H9c2 of rat cardiomyocytes was purchased from the cell bank of the typical Culture Preservation Committee of the Chinese Academy of Sciences.EX527 was purchased from MedChemExpress Company.The overexpression vector negative control (oe-NC),CTRP3 overexpression vector pcDNA3.1-CTRP3(oe-CTRP3) and FOXO3a overexpression vector pcDNA3.1-FOXO3a (oe-FOXO3a) were purchased from Wuhan GeneCreate Biological Engineering Co.,Ltd.;Lipofectamine 2000 transfection reagent was purchased from Thermo Fishier Technology (China) Co.,Ltd.AceQ qPCR SYBR Green Master Mix was purchased from Vazyme Biotech Co.,Ltd.;CTRP3,SIRT1,FOXO3a and cleaved caspase-3 antibodies are purchased from Abcam.Bax,Bcl-2 and GAPDH antibodies were purchased from Wuhan Proteintech Biotechnology Co.,Ltd.;CCK-8 kit was purchased from American APExBIO Technology.Annexin VFITC/PI apoptosis detection kit was purchased from Beijing Solarbio Science &Technology Co.Creatine kinase isoenzyme (CK-MB) test kit,cardiac troponin I(cTnI)test kit,total superoxide dismutase(SOD)determination kit (WST-1 method),malondialdehyde(MDA) determination kit (TBA method),glutathione peroxidase (GSH-PX) determination kit (colorimetric method)were purchased from Nanjing Jiancheng Bioengineering Institute.The reactive oxygen species(ROS)detection kit was purchased from Shanghai Beyotime Biotechnology Co.,Ltd.

Cell culture and cell OGD/R treatment

H9c2 cells were cultured in normal DMEM medium containing 10% FBS in 5% CO2cell incubator at 37 ℃.The fresh culture medium was changed every 2 days,and when the degree of cell confluence reached 80%-90%,the cells were digested by trypsin,and the plate laying operation was carried out according to the needs of the experiment.In reference [7],the H9c2 cell culture medium was replaced by sugar-free DMEM medium without FBS,and the cells were cultured in 94%N2,5%CO2,and 1%O2cell incubator at 37 ℃,for 6 h,and the cell culture medium was replaced with normal DMEM medium containing 10%FBS for 6 h.The cells were cultured in 5% CO2cell incubator at 37 ℃for 6 h.The cells were collected and the follow-up experiments were carried out.

Detection of EX527 administration concentration

The logarithmically growing H9c2 cells were inoculated into 96-well plates with 5 × 103cells per well.After 24 hours of culture,the cells were randomly divided into 0 μmol/L group,0.25 μmol/L group,0.5 μmol/L group,1 μmol/L group,2 μmol/L group and 4 μmol/L group.Different concentrations of EX527 were added to the cells according to the groups.In addition,the zeroing group was set up without adding cells or any reagents.After the cells were cultured for 48 hours,the optical density(OD)value of the cells at 450 nm was detected by CCK-8 method.Cell survival rate (%)=(ODexperimentalgroup-ODzeroinggroup)/ (OD0μmol/Lgroup-ODzeroinggroup)×100%.

Cell grouping and transfection

The logarithmically growing H9c2 cells were inoculated into 6-well plates with 1×106cells per well.After 24 hours of culture,the cells were randomly divided into control group,OGD/R group,oe-NC group(transfection oe-NC),oe-CTRP3 group (transfection oe-CTRP3),oe-CTRP3+EX527 group (transfection oe-CTRP3),oe-CTRP3+EX527+oe-FOXO3a group(transfection oe-CTRP3 and oe-FOXO3a).According to the grouping,oe-NC,oe-CTRP3 and oe-FOXO3a were transfected according to the instructions of Lipofectamine 2000 transfection reagent.After the cells were cultured for 24 hours,the fresh medium was changed and 1 μmol/L EX527 was added to oe-CTRP3+EX527 group and oe-CTRP3+EX527+oe-FOXO3a group.After the cells were cultured for 48 hours,all the cells in the other groups except control group were treated with OGD/R,and then the followup experiment was carried out.

The expression of CTRP3,SIRT1 and FOXO3a mRNA was detected by qRT-PCR

H9c2 cells were collected and total RNA was extracted by Trizol reagent.The extracted RNA was immediately reverse transcribed into cDNA,and the cDNA was used as a template for qRT-PCR experiment.According to the instruction of AceQ qPCR SYBR Green Master Mix,the qRT-PCR experiment was carried out.Reaction procedure:95 ℃,5 min;95 ℃,10 s,60 ℃,30 s,a total of 40 cycles,95 ℃,15s,60 ℃,60 s,95 ℃,15 s.With GAPDH as the internal reference,the expressions of CTRP3,SIRT1 and FOXO3a mRNA were calculated by 2-ΔΔCtmethod.Primer sequences were as follows:CTRP3-Forward primer:5’-ATGGAGGTGAGCAGAAGAGC-3’,Reverse primer:5’-CACAGTCCCCGTTTTAGCAT-3’,SIRT1-Forward primer:5’-GCCTCATCTGCATTTTGATG-3’,Reverse primer:5’-TCTGGCATGTCCCACTATCA-3’,FOXO3a-Forward primer:5’-CTGGGGGAACCTGTCCTATG-3’,Reverse primer:5’-TCATTCTGAACGCGCATGAAG-3’,GAPDH-Forward primer:5’-GGTGAAGGTCGGAGTCAA-CG-3’,Reverse primer:5’-TGGGTGGAATCATATTGGAACA-3’.

Detection of the expression of CTRP3,SIRT1,FOXO3a,Bax,Bcl-2 and cleaved caspase-3 pro⁃teins by Western blot

H9c2 cells were collected,RIPA lysate was used to lyse cell precipitation,and the supernatant was collected,and the protein concentration of the supernatant was determined by BCA method.According to the determined concentration,30 μg protein was absorbed and placed in the upper sample hole for SDS-PAGE gel electrophoresis and membrane transfer operation.5% skimmed milk powder was sealed for 3 hours at room temperature,and then incubated overnight at 4 ℃by adding antibody diluents such as CTRP3 (1∶2,000),SIRT1 (1∶1,000),FOXO3a (1∶1,000),Bax(1∶1,000),Bcl-2(1∶2,000)and GAPDH(1∶2,000)at 4 ℃.The specific diluent of secondary antibody (1∶5,000) was added and incubated at room temperature for 1 h.After adding ECL luminescent solution,the protein bands were detected by gel imaging system,and the gray values of protein bands were analyzed by Image J software.

Detection of cell survival by CCK-8

After discarding the culture medium of H9c2 cells in 96-well plate and washing the cells with PBS for 3 times,CCK-8 solution (10 μL/well) was added to each group of cells.After the cells were incubated in an incubator at 37 ℃for 4 hours,the optical density(OD)value of the cells in each group was detected by enzyme labeling(450nm).The cell survival rate(%)=(ODexperimentalgroup-ODzeroinggroup)/ (ODcontrolgroup-ODzeroinggroup)×100% or cell survival rate (%)=(ODexperimentalgroup-ODzeroinggroup)/(ODoe-NCgroup-ODzeroinggroup)×100%.

Detection of apoptosis by flow cytometry

H9c2 cells were collected and washed with precooled PBS for 3 times and 1×Binding Buffer once.Then,the cells were resuscitated with 1×Binding Buffer and the cell density was adjusted to 1 × 106cells/mL.The 100 μL cell suspension was placed in a new centrifuge tube,5 μL Annexin V-FITC was added into the tube,then gently mixed and incubated at room temperature for 10 min.5 μL PI was added,mixed gently,and incubated at room temperature for 5 min.PBS was added to the tube to a total volume of 500 μL.after gently mixing,the cells were detected by flow cytometry immediately.

Detection of the activity of SOD and GSH-Px,and the content of MDA in cells by ELISA

H9c2 cells were collected,and the activities of SOD and GSH-Px and the content of MDA were detected according to the instructions of total superoxide dismutase (SOD) kit (WST-1 method),malondialdehyde(MDA) kit (TBA method) and glutathione peroxidase(GSH-PX)kit(colorimetric method).

Detection of ROS production by reactive oxygen species detection kit

H9c2 cell culture medium was added with 1 mL diluent with the concentration of 10 μmol/L of DCFHDA.The cells were incubated at 37 ℃in a cell incubator for 20 min.After washing the cells in serum-free medium for 3 times,the DCFH-DA positive cells were observed by fluorescence microscope and the fluorescence intensity was analyzed by Image J software.

Statistical analysis

The data were analyzed by SPSS21.0 software.The experimental data were expressed as mean±standard deviation.Independent samplet-test was used to compare the sample mean between the two groups.Oneway ANOVA was used to compare the sample mean between multiple groups,and LSD-ttest was used for pairwise comparison between the two groups.P＜0.05 was considered statistically significant.

Results

Effects of OGD/R on the expression of CTRP3,SIRT1 and FOXO3a in cardiomyocytes

Compared with control group,the expression of CTRP3,SIRT1 and FOXO3a mRNA in OGD/R group decreased significantly(P＜0.05),and the expression of CTRP3,SIRT1 and FOXO3a protein decreased significantly (P＜0.05).The results are shown in Figure 1.

Effect of overexpression of CTRP3 on cardiomyo⁃cyte injury induced by OGD/R

Compared with control group,the expression of CTRP3 mRNA and protein decreased significantly(P＜0.05).The cell survival rate and the expression of Bcl-2 protein in OGD/R group decreased significantly,while the apoptosis rate,the expression of Bax and cleaved caspase-3 protein,the content of CK-MB and cTnI increased significantly(P＜0.05).There were no significant differences in CTRP3 mRNA and protein expression,cell survival rate,apoptosis rate,CK-MB and cTnI contents,and the protein expressions of Bax,Bcl-2,cleaved caspase-3 between the oe-NC group and OGD/R group(P＞0.05).Compared with oe-NC group,the expression of CTRP3 mRNA and protein,cell survival rate and the expression of Bcl-2 protein in oe-CTRP3 group were significantly increased,while the apoptosis rate,Bax and cleaved caspase-3 protein expressions as well as CK-MB and cTnI contents were significantly decreased in oe-CTRP3 group(P＜0.05)(Figure 2).

Effects of overexpression of CTRP3 on oxidative stress induced by OGD/R in cardiomyocytes

Compared with control group,The SOD and GSH-Px activitieswere decreased significantly,whereas the MDA content and ROS fluorescence intensity were increased significantly in OGD/R group(P＜0.05).There was no significant difference in SOD and GSHPx activity,MDA content and ROS fluorescence intensity between oe-NC group and OGD/R group(P＞0.05).Compared with oe-NC group,the activities of SOD and GSH-Px in oe-CTRP3 group were significantly increased,while the MDA content and ROS fluorescence intensity were evidently decreased in oe-CTRP3 group(P＜0.05)(Figure 3).

Figure 1 Effects of OGD/R on the expression of CTRP3,SIRT1 and FOXO3a in cardiomyocytes.A:The expression of CTRP3,SIRT1 and FOXO3a mRNA was detected by qRT-PCR.B:The expression of CTRP3,SIRT1 and FOXO3a protein was detected by Western blot.Compared with control group,*P＜0.05.

Figure 2 Effect of CTRP3 overexpression on cardiomyocyte injury induced by OGD/R.A-B:The expression of CTRP3 mRNA and protein was detected by qRT-PCR and Western blot.C-D:The levels of cardiomyocyte injury markers CK-MB and cTnI.E:The cell survival rate was detected by CCK-8.F:The apoptosis was detected by flow cytometry.G:The expressions of Bax,Bcl-2 and cleaved caspase-3 protein were detected by Western blot.*P＜0.05 vs.Control group;#P＜0.05 vs.oe-NC group.

Figure 3 Effects of overexpression of CTRP3 on oxidative stress induced by OGD/R in cardiomyocytes.A-B:SOD and GSH-Px activities.C:MDA content.D-E:cellular ROS production(×200).*P＜0.05 vs.Control group;#P＜0.05 vs.oe-NC group.

Effects of overexpression of CTRP3 on the expres⁃sion of SIRT1 and FOXO3a in cardiomyocytes in⁃duced by OGD/R

Compared with control group,the expression of SIRT1 and FOXO3a mRNA,SIRT1 and FOXO3a protein decreased significantly in OGD/R group(P＜0.05),while there was no significant difference in SIRT1 and FOXO3a mRNA expression,SIRT1 and FOXO3a protein expression between oe-NC group and OGD/R group(P＞0.05).Compared with oe-NC group,SIRT1 and FOXO3a mRNA expression,SIRT1 and FOXO3a protein expression increased significantly in oe-CTRP3 group(P＜0.05)(Figure 4).

Effect of CTRP3/SIRT1/FOXO3a axis on cardio⁃myocyte injury induced by OGD/R

The cell survival rate was decreased after treated with 2 μmol/L and 4 μmol/L EX527(P＜0.05),but other concentrations had no significant effect on the cell survival rate (P＞0.05).Therefore,1 μmol/L EX527 was used in the follow-up experiment.Compared with oe-NC group,CTRP3,SIRT1 and FOXO3a protein expression,cell survival rate and Bcl-2 protein expression were significantly increased,while apoptosis rate,Bax and cleaved caspase-3 protein expressions,and CK-MB and cTnI contents were significantly decreased in oe-CTRP3 group(P＜0.05).There was no significant difference in CTRP3 protein expression,SIRT1 and FOXO3a protein expression,cell survival rate and Bcl-2 protein expression between oe-CTRP3+EX527 groupand oe-CTRP3 group(P＜0.05).Apoptosis rate,Bax and cleaved caspase-3 protein expressions,and CK-MB and cTnI contents were significantly increased in oe-CTRP3+EX527 group(P＜0.05).The expressions of CTRP3 and SIRT1 protein in oe-CTRP3+EX527+oe-FOXO3a group and oe-CTRP3+EX527 group were similar(P＞0.05).The expressionof FOXO3a and Bcl-2 protein and the cell survival rate were significantly increased,while the apoptosis rate,Bax and cleaved caspase-3 protein expressions,CK-MB and cTnI contents were significantly decreased in oe-CTRP3+EX527+oe-FOXO3a group(P＜0.05)(Figure 5).

Effect of CTRP3/SIRT1/FOXO3a axis on oxida⁃tive stress induced by OGD/R in cardiomyocytes

Compared with oe-NC group,the activities of SOD and GSH-Px in oe-CTRP3 group were significantly increased,while the MDA content and ROS fluorescence intensity were significantly decreased in oe-CTRP3 group (P＜0.05).Compared with oe-CTRP3 group,the activities of SOD and GSH-Px in oe-CTRP3+EX527 group were significantly decreased,while the MDA content and ROS fluorescence intensity were significantly increased(P＜0.05).Compared with oe-CTRP3+EX527 group,the activities of SOD and GSH-Px in oe-CTRP3+EX527+oe-FOXO3a group were significantly increased,while the MDA content and ROS fluorescence intensity were significantly decreased(P＜0.05)(Figure 6).

Figure 4 Effects of overexpression of CTRP3 on the expression of SIRT1 and FOXO3a in cardiomyocytes induced by OGD/R.A:SIRT1 and FOXO3a mRNA expressionswere detected by qRT-PCR.B:SIRT1 and FOXO3a protein expressionswere detected by Western blot.*P＜0.05 vs.Control group;#P＜0.05 vs.oe-NC group.

Figure 5 Effect of CTRP3/SIRT1/FOXO3a axis on cardiomyocyte injury induced by OGD/R.A:The cell survival rate was detected by CCK-8.Compared with 0 μmol/L group,*P＜0.05.B:The protein expressions of CTRP3,SIRT1 and FOXO3a were detected by Western blot.#P＜0.05 vs. oe-NC group;&P＜0.05 vs. oe-CTRP3 group;△P＜0.05 vs. oe-CTRP3+EX527 group.C:Effect of CTRP3/SIRT1/FOXO3a axis on the cell survival rate.D-E:The levels of cardiomyocyte injury markers CK-MB and cTnI.F:The apoptosis rate was detected by flow cytometry.G:The expression of Bax,Bcl-2 and caspase-3 protein was detected by Western blot.*P＜0.05 vs.oe-NC group;#P＜0.05 vs.oe-CTRP3 group;&P＜0.05 vs.oe-CTRP3+EX527 group.

Figure 6 Effect of CTRP3/SIRT1/FOXO3a axis on oxidative stress induced by OGD/R in cardiomyocytes.A-B:SOD and GSH-Px activities.C:MDA content.D-E:The production of ROS (×200).*P＜0.05 vs. oe-NC group;#P＜0.05 vs. oe-CTRP3 group;&P＜0.05 vs.oe-CTRP3+EX527 group.

Discussion

In the past 20 years,with the maturity of technology,PCI reperfusion therapy and thrombolytic therapy have been widely used in the world,and have become the main means of clinical treatment of AMI [8].Although timely and effective reperfusion therapy can save most of the damaged myocardium and improve cardiac function,reperfusion therapy will also be accompanied by myocardial I/R injury [9].Reperfusion injury can lead to cardiomyocyte death and irreversible damage of cardiac function,which mainly occurs in the early stage of reperfusion therapy [10].How to prevent and reduce the damage of I/R in AMI has become an urgent problem to be solved.

Studies have confirmed that oxidative stress and apoptosis play an important role in the pathological process of myocardial I/R injury.Inhibiting the level of oxidative stress and cardiomyocyte apoptosis after AMI I/R injury will be an important means for the prevention and treatment of myocardial injury caused by I/R [11].CTRP3 has a variety of biological functions in cells,which can promote cell differentiation and proliferation,increase liver lipid oxidation and lipid factor secretion,and reduce inflammatory response [12].The evidence shows that OGD/R treatment can down-regulate the expression of CTRP3 in hippocampal neurons.Overexpression of CTRP3 can increase the viability of hippocampal neurons induced by OGD/R,reduce the production of ROS and increase the activities of SOD and GSH-Px to reduce the oxidative stress induced by OGD/R.In addition,CTRP3 can up-regulate the expression of Bcl-2 and down-regulate the expression of Bax and the activity of caspase-3,thus inhibiting the apoptosis of hippocampal neurons induced by OGD/R.The results of this study suggest that CTRP3 may be an important target for improving tissue I/R damage [13].At present,studies have shown that CTRP3 has protective effects on a variety of heart diseases.For example,in lipopolysaccharide-induced sepsis,the overexpression of heart-specific CTRP3 improves myocardial dysfunction and inhibits lipopolysaccharide-induced cardiomyocyte apoptosis in mice [14].CTRP3 protects mice from pathological cardiac remodeling and left ventricular dysfunction caused by pressure overload by reducing endoplasmic reticulum stress,restoring left ventricular systolic function,reducing myocardial hypertrophy and fibrosis [15].In this study,H9c2 cells were induced by OGD/R to establish the model of myocardial I/R injury in vitro.The results showed that OGD/R treatment could inhibit the expression of CTRP3 mRNA and protein in H9c2 cells,inhibit the survival of H9c2 cells and promote apoptosis,and promote the production of oxidative stress injury and myocardial injury markers CK-MB and cTnI.Overexpression of CTRP3 can promote the survival of H9c2 cells and inhibit apoptosis and the production of CKMB and cTnI.In addition,overexpression of CTRP3 could up-regulate the activities of SOD and GSH-Px,and down-regulate the contents of MDA and ROS in cells.The above results show that CTRP3 can improve H9c2 cell injury induced by OGD/R and promote cell survival by inhibiting apoptosis and oxidative stress.

SIRT1 is a histone deacetylase dependent on nicotinamide adenine dinucleotides that regulates a variety of cellular functions,including cell differentiation,survival and metabolism [16].SIRT1 plays an important protective role in tissue I/R injury.For example,the results of Zhang YM et al.[17]show that activation of SIRT1 pathway can block inflammatory response and thus play a neuroprotective role in brain I/R injury.In addition,the level of SIRT1 in the kidney of rats decreased after I/R injury.SIRT1 agonists can improve renal I/R injury by inhibiting endoplasmic reticulum stress and cell pyrogenesis[18].Similarly,SIRT1 plays an important role in myocardial I/R injury.Studies by Niu X et al.[19]have shown that upregulation of SIRT1 can attenuate oxidative stress and cytotoxicity induced by OGD/R,inhibit mitochondrial-mediated apoptosis,and thus improve myocardial Ipar injury.At present,evidence has shown that CTRP3 can play an important role in the occurrence and development of doxorubicin cardiotoxicity by regulating the expression of SIRT1.Overexpression of CTRP3 can promote cardiomyocyte survival and inhibit cardiomyocyte apoptosis and inflammation by activating SIRT1,thus improving cardiac insufficiency induced by adriamycin[20].The results showed that OGD/R treatment could inhibit the expression of SIRT1 mRNA and protein in H9c2 cells,and overexpression of CTRP3 could promote the expression of SIRT1 in H9c2 cells.H9c2 cells overexpressing CTRP3 were treated with EX527,a specific inhibitor of SIRT1.The results showed that EX527 could partially reverse the effect of overexpressing CTRP3 on H9c2 cells,inhibit the survival of H9c2 cells overexpressing CTRP3,promote apoptosis,production of CK-MB and cTnI,and promote oxidative stress injury.The results show that CTRP3 can inhibit apoptosis and oxidative stress by up-regulating the expression of SIRT1,and improve the injury of H9c2 cells induced by OGD/R.

FOXO3a is a potential downstream target of SIRT1.SIRT1 activates FOXO3a by deacetylation and induces the expression of antioxidant factors including manganese superoxide dismutase and catalase,thus promoting the resistance of cells to oxidative stress[21].Studies have shown that FOXO3a is an important regulator of cardiomyocyte apoptosis under oxidative stress.Targeted inhibition of FOXO3a expression can induce oxidative stress and promote apoptosis of cardiomyocytes [22].The results suggest that FOXO3a may also play an important role in myocardial I/R injury.The results of Li S et al.[23]further proved the role of FOXO3a in myocardial I/R injury.The activation of FOXO3a can play an anti-apoptotic effect by inhibiting the production of ROS,the release of cytochrome c and the activation of caspase-3,so as to protect the myocardium of diabetic mice from I/R injury.At present,there is evidence that there is a close relationship between SIRT1/FOXO3a pathway and liver I/R injury.Activation of SIRT1/FOXO3a pathway may play a role in the treatment of liver I/R injury by promoting autophagy and improving mitochondrial dynamics [24].The relationship between SIRT1/FOXO3a pathway and myocardial I/R injury needs to be further explored.The results showed that OGD/R treatment could inhibit the expression of FOXO3a mRNA and protein in H9c2 cells,and overexpression of CTRP3 could promote the expression of FOXO3a in H9c2 cells.The H9c2 cells overexpressing CTRP3 were treated with EX527,a specific inhibitor of SIRT1.The results showed that EX527 could inhibit the expression of FOXO3a in H9c2 cells overexpressing CTRP3.Further experimental results showed that overexpression of FOXO3a had no significant effect on the expression of CTRP3 and SIRT1 in H9c2 cells,indicating that FOXO3a is a downstream factor of CTRP3 and SIRT1.In addition,overexpression of FOXO3a could partially reverse the effects of EX527 and overexpression of CTRP3 on H9c2 cells treated with OGD/R,promote cell survival,and inhibit apoptosis,production of CK-MB and cTnI and oxidative stress.The results show that CTRP3 can inhibit apoptosis and oxidative stress by activating SIRT1/FOXO3a pathway,and improve the injury of H9c2 cells induced by OGD/R.

To sum up,the results of this study show that CTRP3 can up-regulate SIRT1-mediated FOXO3a expression,improve OGD/R-induced H9c2 cell injury,and inhibit apoptosis and oxidative stress,which is a potential therapeutic target for myocardial I/R injury.The results of this study provide new scientific data for clarifying the mechanism of CTRP3 in myocardial I/R injury and developing new therapeutic strategies for myocardial I/R injury.

AcknowledgmentsThis study was funded by National Natural Science Foundation of China (No.81760077)