Protective Effects And Mechanisms Of Pirfenidone On Hcy-induced Apoptosis In H9C2 Cardiomyocytes

Objective: In this study,homocysteine(Hcy)was used to simulate apoptosis of H9C2 rat cardiomyocytes,and the protective effect of pirfenidone(PFD)pretreatment on Hcy-induced apoptosis of H9C2 cells was observed,at the same time,Gap 26 and AAP10,the blocker and agonist of Connexin43(Cx43),were used to investigate the mechanism of Cx43 on the apoptosis of H9C2 cells induced by Hcy.Methods: The H9C2 cardiomyocytes were divided into Control group,Hcy group,Hcy+PFD treatment group(After 30 min pretreatment with PFD,3 mmol/l Hcy was added and incubated for 24 h).The effects of different concentrations of Hcy(0.1,0.5,1,2,3 mmol/l)and PFD(0.1,0.5,1,1.5 mg/ml)on the viability of H9C2 cardiomyocytes were detected by CCK-8 method,the state of H9C2 cardiomyocytes was observed by inverted microscope,and the apoptosis rate of H9C2 cardiomyocytes was detected by Flow cytometry.The localization and expression of Cx43,Bax,Bcl-2 and caspase-3 in H9C2 cardiomyocytes were observed by immunofluorescence.The m RNA and protein expression levels of Cx43,Bax,Bcl-2,and caspase-3 in H9C2 cardiomyocytes were detected by q RT-PCR and Western blot.Results:(1)The results of cck-8 showed that Hcy could decrease the activity of cardiac myocytes in a concentration-dependent manner.PFD at 0.1-1 mg/ml treatment alone showed no obvious cytotoxicity towards H9C2 cells,however,PFD could decreased cell viability at 1.5 mg/ml.Compared with Hcy group,the cell viability of Hcy+PFD group increased in a concentration-dependent manner.Cell morphology found that the cells in the Hcy treatment group showed significant disarrangement and extensive cell shrinkage,but the cell status in the PFD+Hcy group was significantly improved.(2)Hcy treatment group significantly increased the apoptosis rate of cardiomyocytes,while the PFD+Hcy group significantly reduced the apoptosis rate.Compared with the control group,Hcy group up-regulated the Bax/Bcl-2 ratio and increased the expression of cleaved caspase-3,compared with the Hcy group,the PFD+Hcy group significantly reduced the Bax/Bcl-2 ratio and the protein expression of cleaved caspase-3.The expression and distribution of Bax,Bcl-2 and caspase-3 in H9C2 cardiomyocytes were detected by immunofluorescence staining.The results showed that Bax and caspase-3 were mainly distributed in the cytoplasm;Bcl-2 was mainly distributed in the nuclear membrane.The expression changes of Bax,Bcl-2and caspase-3 in each group were consistent with the results of Western blot.At the same time,q RT-PCR showed an increase in the ratio of Bax/Bcl-2 and caspase-3 m RNA after Hcy treatment in H9C2 cells,whereas the effects were prevented after pretreatment with PFD.Real-time quantitative PCR showed that after Hcy treatment,the ratio of Bax/Bcl-2 and the expression of caspase-3 m RNA in H9C2 cells increased,while after PFD pretreatment,the expression of Bax/Bcl-2 and caspase-3 m RNA in the PFD+Hcy group significantly decreased compared with the Hcy group.(3)Compared with Hcy group,PFD +Hcy group significantly down-regulated Cx43 protein expression.The results of immunofluorescence of H9C2 showed that Cx43 was mainly distributed in the cytoplasm and cell membrane.The Cx43 fluorescence intensity and m RNA levels of each group were consistent with the results of Western blot.H9C2 cardiomyocytes were pretreated with Cx43 inhibitor Gap26 and then treated with Hcy for 24 h.The apoptosis rate of the Gap26 pretreatment group was significantly decreased than that of the Hcy group.The Bax/Bcl-2 ratio,cleaved caspase-3 and Cx43 protein expression of the Gap26 pretreated group were significantly lower than that of the Hcy group.At the same time,the expression changes of Bax,Bcl-2,caspase-3 and Cx43 m RNA levels in each group were consistent with the Western blot.(4)H9C2cardiomyocytes were pre-treated with the Cx43 agonist AAP10.Compared with the Hcy group,the apoptosis rate of the PFD+Hcy group was significantly reduced,while the AAP10 pretreatment group significantly increased the apoptosis rate compared with the PFD+Hcy group.Compared with the Hcy group,PFD+Hcy group significantly reduced the ratio of Bax/Bcl-2 and the protein expression of cleaved caspase-3 and Cx43.Compared with the PFD+Hcy group,the AAP10 pretreatment group significantly increased the m RNA expression levels of Bax/Bcl-2,caspase-3 and Cx43.Conclusion: PFD protects Hcy-induced H9C2 cardiomyocyte apoptosis by inhibiting Cx43 signaling pathway.