Wenyang Yiqi Formula Regulates Autophagy In Cardiomyocytes Induced By Oxygen Glucose Deprivation Via Mediating MTOR

Objective: To observe the effect of Wenyang Yiqi Formula(WYYQ)on autophagy induced by oxygen glucose deprivation(OGD)in H9c2 cells,and to explore its mechanism of regulation of autophagy against heart failure.Methods:1 Prepare OGD-induced autophagy model of H9c2 cells,and determine the intervention conditions for the establishment of the model.2 Each experimental control group(equal volume complete medium)was cultured in a common incubator.The rapamycin group and the WYYQ group were replaced with the same volume as the model group after 24 hours of intervention with the drug-containing medium.Serum and sugar-free medium was transferred to an hypoxic incubator for 6 h.3 CCK-8 analysis of gradient concentration of WYYQ on H9c2 cell activity,flow cytometry detection of H9c2 cell apoptosis,qRT-PCR detection of H9c2 cell Beclin-1 and LC3 expression levels,transmission electron microscopy observation of autophagosome formation,Western blot was used to detect the expression of Beclin-1,LC3-II/LC3-I,p62 and activation of mTOR pathway in H9c2 cells.Results:1 In the OGD-induced autophagy of H9c2 cells experiment,compared with OGD 0 h,the expression of Beclin-1,LC3 m RNA and Beclin-1 protein,LC3-II/LC3-I were increased maximally at the 6th hour after OGD intervention,and the expression of p62 was decreased,the difference was statistically significant(P<0.01).2 In the CCK-8 analysis of the effect of gradient concentration of WYYQ on the activity of H9c2 cells experiment,the cell activity was decreased after pretreatment with 3.2、6.4 mg/ml WYYQ for 12 h and 24 h,and 6.4 mg/ml group was pretreated for 12 h and 24 h compared with the control group,the difference was statistically significant(P<0.05 or P<0.01).In the Annexin V-FITC/PI staining experiment,compared with OGD group,the apoptosis of H9c2 cells in WYYQ group was reduced,and the anti-apoptotic effects of the different doses of the intervention groups were compared,the difference was statistically significant(P<0.05 or P<0.01).3 In the qRT-PCR detection of Beclin-1 and LC3 expression in H9c2 cells experiment,compared with the OGD group,the expression of Beclin-1 and LC3 in WYYQ group was lower,and there were differences between the different dose intervention groups,the difference was statistically significant(P<0.05 or P<0.01).In the Western Blot detection of Beclin-1,LC3-II/LC3-I and p62 expression in H9c2 cells experiment,compared with the OGD group,the expression of Beclin-1,LC3-II/LC3-I was decreased and the expression of p62 was increased in WYYQ group,and there were differences between the different dose intervention groups,the difference was statistically significant(P<0.05 or P<0.01).4 In the transmission electron microscopy observation of autophagosome formation in H9c2 cells experiment,compared with the OGD group and the OGD + rapamycin group,WYYQ could reduce autophagosome formation.In the qRT-PCR detection of Beclin-1 and LC3 expression in H9c2 cells experiment,compared with the OGD group and the OGD + rapamycin group,the expression of Beclin-1 and LC3 was decreased in the intervention group of WYYQ,the difference was statistically significant(P<0.01).In the Western Blot detection of p-mTOR,mTOR,Beclin-1,LC3-II/LC3-I and p62 expression in H9c2 cells experiment,compared with OGD group and OGD + rapamycin group,the expression of Beclin-1,LC3-II/LC3-I was decreased and the expression of p62 was increased in the intervention group of WYYQ,the ratio of p-mTOR/mTOR was increased in OGD+WYYQ group,the difference was statistically significant(P<0.01),the ratio of p-mTOR/mTOR in OGD + rapamycin + WYYQ group did not significantly change(P>0.05).Conclusions:1 WYYQ significantly reversed the autophagy activity and p62 expression of H9c2 cells induced by OGD,and significantly inhibited OGD-induced apoptosis in a dose-dependent manner.2 WYYQ inhibited autophagy by mediating the mTOR signaling pathway.