The Role And Mechanism Of Autophagy In The Protection Of Trimetazidine Against Myocardial Ischemia-reperfusion Injury

PARTⅠPROTECTIVE EFFECT OF TRIMETAZIDINE ON MYOCARDIAL ISCHEMIA-REPERFUSION INJURY IN RATS AND ITS INHIBITION OF AUTOPHAGYObjective:To investigate the effect of trimetazidine（TMZ）on autophagy of cardiomyocytes in myocardial ischemia/reperfusion injury（MI/RI）.Methods:The MI/RI model was established using Sprague-Dawley（SD）rats.The modeling time was 30 min of ischemia and 2 h of reperfusion.Rats were divided into four groups:Sham group,ischemia/reperfusion（I/R）group,trimetazidine（TMZ）group,I/R+TMZ group.After the establishment of the model,the rats were given an echocardiogram,and then the blood was collected through the abdominal aorta,and then the heart tissue below the cross-section of the ligature was taken for further testing.Myocardial damage and oxidative stress were measured by serum ELISA.Myocardial infarct area was assessed by TTC staining.Myocardial cell apoptosis rate was observed by TUNEL staining.Myocardial ultrastructure and autophagosome were observed by transmission electron microscopy.LC3B and Beclin1 were detected by immunohistochemistry.The mRNA expression levels of LC3,Beclin1,P62,ATG5 and ATG7 were examined by RT-PCR.The expression of LC3-I/II,Beclin1,P62,ATG5 and ATG7 proteins and the expression of total protein and phosphorylated protein of AKT and mTOR were detected by Western Blot.Results:（1）Cardiac ultrasound:The heart rate of I/R and I/R+TMZ group was lower than that of Sham group and TMZ group,but the difference was not statistically significant（P>0.05）.Compared with EF and FS（83.975±1.483%,47.475±1.644%,respectively）in Sham group,EF and FS（60.063±3.448%,27.800±2.194%,respectively）of I/R group were pointedly lower（P<0.01）;after TMZ treatment,it was significantly improved（68.925±2.910%,33.850±2.220%;P<0.01）.Moreover,the ESV（0.219±0.036 ml）and EDV（0.545±0.053 ml）in the I/R group were significantly greater than those in the Sham group（0.070±0.009 ml,0.439±0.026 ml,respectively,P<0.01）;it was obviously reduced（0.146±0.028 ml,0.471±0.071 ml;P<0.01）after TMZ treatment.The LVIDs and LVIDd（0.445±0.028 cm,0.616±0.022 cm,respectively）in the I/R group were significantly higher than those in the Sham group（0.300±0.013 cm and 0.571±0.012 cm,respectively,P<0.01）,and were decreased to（0.386±0.024 cm,P<0.01）and（0.584±0.032 cm,P<0.05）respectively after TMZ treatment.In addition,there was no significant difference in the above-mentioned cardiac function index between the Sham group and the TMZ group（P>0.05）.（2）Serum biochemistry:Serum levels of LDH,CK-MB,ROS and MDA in I/R group were distinctly higher than those in Sham group（P<0.01）,and remarkedly declined after TMZ treatment（P<0.01）.The levels of SOD and GSH-px in I/R group were evidently lower than those in Sham group（P<0.01）,and the levels of them were significantly increased after TMZ intervention（P<0.01）.At the same time,the TMZ group had no significant effect on the above indicators compared with the Sham group（P>0.05）.（3）HE staining:The cardiomyocytes of the Sham group were arranged neatly,and the nucleus was located at the center of the muscle fibers;significant changes such as severe myocardial structural deformation,interstitial edema,myocardial necrosis and nuclear lysis were observed in the I/R group.In the I/R+TMZ group,most of the normal myocardial structures were preserved,and interstitial edema and myocardial necrosis were reduced compared with the I/R group.（4）TTC staining:No myocardial infarction was found in Sham group and TMZ group.Myocardial infarct size was significantly increased in I/R group（P<0.01）,and TMZ+I/R group was remarkably lesser than I/R group（P<0.01）.（5）TUNEL staining:The number of positive myocardial cells in the I/R group was significantly higher than that in the Sham group（P<0.01）,and drastically diminished after TMZ intervention（P<0.01）.（6）Transmission electron microscopy:Compared with Sham group,the number of autophagosomes in I/R group increased,mitochondrial ultrastructure changed such as swelling,disintegration,and sputum decreased or even disappeared;the number of autophagosomes was decreased and the shape of mitochondria was improved after TMZ treatment.（7）Immunohistochemistry:Compared with the Sham group,the protein levels of Beclin1 and LC3B in the I/R group were significantly increased,and considerably reduced after TMZ treatment.（8）Western Blot and RT-PCR:Western Blot showed that the expression of LC3-II,Beclin1,ATG5 and ATG7 proteins in I/R group was significantly upper than those in Sham group（P<0.01）,and remarkably declined after TMZ treatment（P<0.01）;the expression of P62 protein in I/R group was significantly lower than that in Sham group（P<0.01）,and increased after TMZ treatment（P<0.01）.RT-PCR showed that the expression of LC3,Beclin1,ATG5 and ATG7 mRNA was consistent with its protein level,while P62 mRNA level did not change significantly.Compared with the Sham group,the expression of p-AKT^Ser473er473 and p-mTOR^Ser2448er2448 in the I/R group was significantly lower（P<0.01）,which were drastically raised（P<0.01）after TMZ treatment.Conclusion:TMZ can improve cardiac function in MI/RI rats,reduce oxidative stress,diminish myocardial infarction and myocardial cell apoptosis,and improve myocardial tissue structure.TMZ can inhibits MI/R-induced excessive autophagy and may play a role by activating the AKT/mTOR pathway.PART Ⅱ THE ROLE AND MECHANISM OF AUTOPHAGY IN THE PROTECTION OF TRIMETAZIDINE AGAINST HYPOXIA-REOXYGENATION INJURY OF H9C2 MYOCARDIAL CELLSObjective: To explore the role and mechanism of autophagy in the protection of trimetazidine against hypoxia-reoxygenation injury of H9c2 myocardial cells.Methods: Hypoxia/reoxygenation（H/R）model was made by using rat H9c2 cardiomyocytes to imitate in vivo I/R model.First,the cells were treated with hypoxia time gradient of 0,1,2,3,4,6,and 8 h to detect the expression of LC3-I/II and Beclin1 proteins,and the time of hypoxia was screened;then the cells were treated with reoxygenation time gradient of 0,1,2,3,4,6 h to detect the expression of LC3-I/II and Beclin1 proteins and the reoxygenation time was screened;then the subsequent experiments were carried out according to the selected hypoxia and reoxygenation time.The viability of cells was determined using the CCK-8 kit.LDH and SOD levels were measured by microplate method.Apoptosis rate was tested by flow cytometry.The number of ultrastructures and autophagosomes were observed by transmission electron microscopy.Confocal laser scanning microscopy was used to observe autophagic fluorescence.Western Blot was used to detect expression levels of LC3-I/II,Beclin1,P62,ATG5,ATG7,Bax,Bcl-2,Caspase-3,Cleaved Caspase-3 proteins and AKT,m TOR total proteins and its phosphorylation.Results:（1）Screening for hypoxia-reoxygenation time: H9c2 cells were hypoxic for 0,1,2,3,4,6,and 8 h,respectively.The levels of LC3-II and Beclin1 proteins gradually decreased after peaking at 2 h of hypoxia.So,2 h of hypoxia was selected for subsequent study.H9c2 cells were respectively reoxygenated for 0,1,2,3,4,and 6 h.The levels of LC3-II and Beclin1 increased with time,and decreased after reoxygenation at 4 h.So,4 h of reoxygenation was used for subsequent studies.Therefore,the hypoxia and reoxygenation time of the subsequent experiments were 2 h and 4 h,respectively.（2）CCK-8 experiment: The cell viability of the H/R group was significantly lower than that of the Control group（P < 0.01）,and it noticeably improved after TMZ pretreatment（P < 0.01）.The effect of TMZ was significantly inhibited by LY294002（P < 0.01）.（3）Microplate method: LDH level in cell culture medium of the H/R group was higher than that of the Control group（P <0.01）,and remarkedly decreased after TMZ intervention（P <0.01）;LY294002 significantly inhibited this effect（P<0.01）.Compared with the Control group,the intracellular SOD level of the H/R group was notedly declined（P < 0.01）,and observably augmented after TMZ treatment（P < 0.01）.LY294002 markedly eliminated this effect（P < 0.01）.（4）Flow cytometry: Compared with the Control group,the apoptosis rate of the H/R group was notably raised（P <0.01）;the apoptosis rate was significantly decreased after TMZ intervention（P <0.01）,which was eliminated by LY294002（P <0.01）.（5）Transmission electron microscope: Transmission electron microscope showed that most autophagosomes had a bilayer membrane structure surrounded by mitochondria or other organelles in the cytoplasm;the number of autophagosomes among the Control group,Control + TMZ group and Control + LY294002 group was the same and there was no significant difference（P > 0.05）;the number of autophagosomes in the H/R group was markedly greater than that in the Control group（P < 0.01）,and it was significantly reduced after TMZ pretreatment（P < 0.01）,but this effect was obviously inhibited by LY294002（P < 0.01）.（6）CLSM observation:autophagic fluorescence was significantly accumulated in the H/R group,which was significantly reduced after TMZ intervention;and autophagic fluorescence was increased after TMZ and LY294002 co-treatment.（7）Western Blot assay: The expression levels of LC3-II,Beclin1,ATG5 and ATG7 in the H/R group were significantly higher than those in the Control group（P < 0.01）;the levels of these proteins in the H/R + TMZ group were significantly lower than those in the H/R group（P < 0.01）;but LY294002 attenuated this effect of TMZ（P < 0.01）;however,the expression of P62 protein in the H/R group was significantly lower than that in the Control group（P < 0.01）,and it was significantly increased after TMZ treatment（P< 0.01）.This effect was inhibited by LY294002（P < 0.01）.The levels of LC3-II,Beclin1,P62,ATG5 and ATG7 proteins between the Control group and Control + TMZ group were no significant difference（P > 0.05）;in addition,there was also no significant difference in the expression of these proteins in the H/R group,H/R + LY294002 group and H/R + TMZ +LY294002 group.At the same time,TMZ pretreatment reduced the levels of Cleaved caspase-3 and Bax protein,increased Bcl-2 protein level,and increased Bcl-2/Bax ratio（P < 0.01）,but this effect was inhibited by LY294002（P < 0.01）.The expression of p-AKTSer473 and p-mTOR^Ser2448 protein in the H/R group was significantly lower than those in the Control group（P <0.01）;compared with the H/R group,the expression levels of p-AKTSer473 and p-mTOR^Ser2448 were significantly increased in the H/R +TMZ group（P < 0.01）,but LY294002 noticeably diminished this effect of TMZ（P < 0.01）.Conclusion: TMZ may inhibit H/R-induced autophagy in H9c2 cardiomyocytes through AKT/m TOR signaling pathway,reduce cell damage and apoptosis,and increase cell survival.