| Object:To study the effect of Ang ? on H9c2 cells,cell surface area,cell viability and apoptosis of H9c2 cells were detected.The optimal concentration of Ang ? in H9c2 cells was determined.At this concentration,SNF5 was first transfected into H9c2 cells to detect the effect of overexpression or knockdown SNF5 on SNF5 mRNA and protein expression in H9c2 cells in order to investigate the role of SNF5 in Ang ?-induced cardiac hypertrophy.Then H9c2 cells were treated with Ang for 24 hours,then the cell viability and apoptosis were detected.The expression of ANP and?-MHC protein was detected.In order to further explore the mechanism of SNF5 on angiotensin II-induced cardiac hypertrophy,the activity of MAPK/mTOR signaling pathway was detected.Methods:Firstly,H9c2 cells were cultured in vitro and cultured with 0 mol/L,10-9 mol/L,10-8mol/L,10-7 mol/L and 10-6 mol/L Ang ?.The surface area of cells was determined by Image-Pro Plus 7.0,the cell viability was determined by MTT assay and the apoptosis of cells were detected by flow cytometry.The optimum concentration was determined.Secondly,the assay was divided into four groups:control group?pc control?,transfected with SNF5 plasmid group?pc SNF5?,transfected with SNF5 inhibitor?si SNF5?and blank control group?si control?.m RNA and protein expression levels of SNF5were tested to verify the transfection efficiency.To study the effect of SNF5 expression on Ang ?-induced cardiac hypertrophy,H9c2 cells were treated with the optimum concentration Ang ? for 24 hours.MTT assay,flow cytometry and other related devices were used to evaluate the cell viability and apoptosis of four groups.We also examined the protein expression of ANP and?-MHC,which were involved in the regulation of cardiac hypertrophy.In order to further determine the effect of overexpression or knockdown of SNF5 on MAPK/mTOR signaling pathway,H9c2 cells were divided into four groups:H9c2 cell group,H9c2 cell+Ang ? group,H9c2 cells+pcSNF5+Ang ? group,H9c2 cells+si SNF5+Ang ? group,Western blot was used to detect the expression of P38 and mTOR protein in four groups of cell signaling pathways.Results:To determine the effect of differernt concentration of Ang ? on H9c2 cells:0 mol/L,10-9mol/L,10-8mol/L,10-7mol/L and 10-6mol/L Ang ? were added,surface area,cell viability and cell apoptosis were detected.We found that when the added concentration of Ang ? was 10-9 mol/L,in the cell surface area there was no significant difference compared with the addition of 0mol/L?P>0.05?.When added concentration was10-8 mol/L,10-7 mol/L and 10-6 mol/L,the surface area of??the cells was significantly increased?P<0.05?.When the concentration was 10-6 mol/L,the surface area of the cells was the highest?P<0.01?.When the increased of the concentration of Ang ?,the cell viability was decreased,the cell viability was the lowest when the concentration was 10-6 mol/L?P<0.05?.Compared with the addition of 0mol/L,the apoptosis rate of the cells was significantly increased?P<0.05?,and the apoptotic rate increased with the increase of the concentration.?P<0.01?.when the concentration was10-6mol/L,the apoptotic rate was the highest.Effects of transfection on the expression of SNF5:H9c2 cells were transfected with pc SNF5,si SNF5 and their corresponding blank control.They were divided into four groups.The transfection efficiency was verified by qPCR and Western blot analysis.The results showed that SNF5 mRNA expression in pc SNF5 group was significantly higher than that in pc control group?P<0.05?,while the expression of SNF5 mRNA in si SNF5group was significantly lower than that in control group?P<0.05?.Western blot analysis showed that transfection of SNF5 could increase the expression of SNF5 protein,and transfection of si SNF5 could inhibit the expression of SNF5 and decrease the expression of SNF5 protein.Effects of SNF5 overexpression on Ang ?-induced cardiac hypertrophy:Four groups of cells were treated with 10-6mol/L Ang ? for 24 hours.Cell viability and apoptosis were evaluated in four groups.The cell viability of pc SNF5 group was significantly higher than that of pc control group?P<0.05?,but the cell viability of si SNF5 group was significantly lower than that of si control group?P<0.05?.The cell viability was the highest in pc SNF5 group.The apoptotic rate of pc SNF5 group was significantly lower than that of control group?P<0.01?,but the apoptosis rate of si SNF5 group was significantly higher than that of control group?P<0.05?.The apoptotic rate of Si SNF5group was the highest.Expression of ANP and?-MHC protein was detected by western Blot.SNF5 overexpression could inhibit the protein expression of ANP and?-MHC,meanwhile knockout SNF5 could promote the protein expression of ANP and?-MHC.Effects of SNF5 on MAPK/m TOR signaling pathway in H9c2 cells:H9C2 cells were divided into four groups:blank cotrol group,without any treatment.Ang? treatment group,Ang?+pc SNF5 group and Ang?+si SNF5 group.The expression of p38 and mTOR protein in the three treatment groups containing Ang? were significantly increased by detecting the expression of p38 and mTOR phosphorylated protein?P<0.05?.The expression of p38 and mTOR in Ang?+pc SNF5 group was significantly down-regulated?P<0.01 or P<0.05?,while SNF5 inhibitor si SNF5 could block the effect of SNF5.The expression of p38 and mTOR protein in Ang?+si SNF5 group was significantly up-regulated compared with the blank control group?P<0.05?.Western blot showed that there was no significant difference in the expression of p38 and mTOR total protein.The expression of p38 and mTOR phosphorylated protein in pcSNF5 group was decreased,and the expression of p38 and mTOR phosphorylated protein in SNF5inhibitor si SNF5 group was up-regulated.Conclusions:We found that Ang ? can induce cardiomyocyte hypertrophy,and with the increase of Ang ? concentration,the cell surface area increases,the activity decreases and the apoptosis rate increases.When the added concentration was 10-6 mol/L,the cell ownedthe largest surface area,the lowest vitality and the highest rate of apoptosis.The optimal concentration of 10-6 mol/L was chosen as the follow-up study.Determining the optimal concentration of Ang ?,we transfected the cells to study effect of overexpression or knockdown SNF5 on the expression of SNF5 in H9c2 cells.The cells were divided into pc SNF5 group,pc control group,si control group and si SNF5 group.The expressions of SNF5 mRNA and protein in four groups were detected to verify the transfection efficiency.It was found that transfection of SNF5 could up-regulate the expression of SNF5 in cells,and SNF5 inhibitor could down-regulate the expression of SNF5 in cells.To evaluate the effect of SNF5 on Ang?-induced cardiomyocyte hypertrophy,the above four groups were treated with 10-6 mol/L Ang ? for 24 hours.Cell viability and cell apoptotic were tested.We found that SNF5 was able to improve cell activity,reduce cell apoptosis and reduce the expression of myocardial hypertrophy factor ANP and?-MHC protein.so SNF5 is a cardiomyocyte hypertrophy inhibitory factor,and when adding inhibitor of SNF5,cell viability decreased,cell apoptosis increased and protein expression of cardiomyocyte hypertrophy factor ANP and?-MHC were increased.Further study of MAPK/mTOR signaling in cells revealed that overexpression of SNF5 could down regulate the expression of p38 and mTOR phosphorylated proteins in cells.The signal pathway activity was inhibited,the addition of SNF5 inhibitor could relieve this inhibition effect..In summary,SNF5 could decrease the activity of MAPK/mTOR signaling pathway,increase cell viability and reduce cell apoptosis by up-regulating SNF5 in H9c2cells,which provides new strategies and targets for our treatment of hypertrophic cardiomyopathy... |
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