FGF-21 Post-treatment Alleviates Hypoxia/reoxygenation In H9c2 Cardiomyocytes By Promoting Autophagic Flux

Object:This study was designed to investigate the protective effects of FGF-21 in hypoxia/reoxygenation-induced injury of H9c2 cardiomyocytes,as well as the role of autophagy played in it.Methods:1.To observe the effects of FGF-21 on myocardial injury induced by hypoxia/reoxygenation in H9c2 cells,we divided the H9c2 cardiomyocytes into 3 groups: control group;hypoxia/reoxygenation(H/R)group;H/R+FGF-21 group.After 2 hours of hypoxia,H9c2 cells were incubated with FGF-21 at the final concentration of 0,12.5,25,50 and 100ng/ml for 1 hours.Myocardial viability were measured by CCK-8 Cell Counting Kit.Trypan blue staining was used to observe the number of cell death.The creatinine kinase(CK),creatinine kinase,MB isoenzyme(CK-MB),cardiac troponin T(cTnT),cardiac troponin I(cTnI)and lactate dehydrogenase(LDH)leverls in the cultured medium were detected by test kit or Automatic biochemical analyzer.2.To explore the mechanism of FGF-21 protect H9c2 cardiomyocytes against H/R injury.we divided the H9c2 cardiomyocytes into 5 groups:control group: H/R group;H/R + FGF-21 group;H/R + 3-MA group;H/R + FGF-21 + 3-MA group.LC3-II、Beclin-1 and p62 protein levels were detected by western blot.Autophagy flux was detected by tandem fluorescent mCherry-GFP-LC3.Myocardial viability were measured by CCK-8 Cell Counting Kit.Trypan blue staining was used to observe the number of cell death.The CK,CK-MB,cTnT,cTnI and LDH leverls in the cultured medium were detected by test kit or Automatic biochemical analyzer.P-mTOR,mTOR and Vps34 protein levels were detected by western blot.Results:1.The results showed that compared with normal control group,myocardial viability rate of H/R group was significantly decreased,and the member of death cells was significantly increased(P<0.05);the CK,CK-MB,cTnT,cTn I and LDH levels in the cultured medium in H/R group was significantly increased than H/R+FGF-21group(P<0.05).However,these injury change was statistically attenuated in H/R+FGF-21 group(P<0.05).2.Western blot results showed that compared with control group,the expression of LC3-II and Beclin-1 protein in H/R group was significantly increased,the expression of p62 was significantly decreased,and the autophagy flux was significantly enhanced(P<0.05);In H/R+FGF-21 group,the expression of LC3-II and Beclin-1 protein and autophagy flux was further enhanced,and the expression of p62 protein was further decreased(P<0.05);Compared with H/R+FGF-21 group,the increase of LC3-II and Beclin-1 protein and autophagy flux and the decrease of p62 protein in H/R+FGF-21+3-MA group was significantly attenuated(P<0.05).Compared with H/R group,myocardial viability rate of H/R+FGF-21 group was significantly increased,and the member of death cells was significantly decreased(P<0.05);the CK,CK-MB,cTnT,cTnI and LDH levels in the cultured medium in H/R+FGF-21 group was significantly decreased than H/R group(P<0.05).However,these protective effects was statistically attenuated in H/R+FGF-21+3-MA group(P<0.05).Western blot results showed that there are no differences between these three groups.The expression of mTOR and p-mTOR had no significant differences in these groups(P>0.05).The expression of Vps34 in H/R group was higher than normal control group(P<0.05),it was further enhanced in H/R+FGF-21 group,the difference was statistically significant(P<0.05);Compared with H/R+FGF-21 group,the increase of Vps34 in H/R+FGF-21+3-MA group was significantly attenuated(P<0.05).Conclusion: FGF-21 post-treatment alleviates hypoxia/reoxygenation injury in H9c2 cardiomyocytes by promoting autophagic flux via activation of the Beclin-1/Vps34 pathway.