高磷通过SET8-shRNA调控AKT/Caspase-3通路促进血管平滑肌细胞凋亡

High phosphorus promotes apoptosis of vascular smooth muscle cells by regulating AKT/Caspase-3 pathway via SET8-shRNA

目的探讨高磷通过SET8-shRNA调控AKT/Caspase-3通路促进大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMCs)发生钙化的机制。方法将血管平滑肌细胞随机分为两组:正常组和高磷组(10 mmol/Lβ-甘油磷酸)。采用茜素红染色及钙含量检测血管平滑肌细胞钙化,采用实时荧光定量及Western blot检测SET8、AKT、Caspase-3 mRAN和蛋白表达情况。为验证SET8在细胞钙化中的作用,将大鼠VSMCs分为6组:正常组、空质粒组、SET8低表达组(转染SET8-shRNA质粒)、高磷组(10 mmol/Lβ-甘油磷酸)、空质粒+高磷组(高磷基础上转染SET8过表达的对照质粒)、SET8过表达+高磷组(高磷基础上转染SET8过表达质粒)。检测各组血管平滑肌细胞的钙化、凋亡及AKT、Caspase-3表达情况。为进一步验证AKT在VSMCs钙化中的作用,将血管平滑肌细胞分为3组:AKT抑制剂组(1μl DMSO+AKT抑制剂LY294002)、对照组(1μl DMSO)和正常组,检测AKT、Caspase-3 mRAN和蛋白表达。结果与正常组比较,高磷组细胞钙化结节增多,钙含量增加(P<0.05),STE8和AKT表达降低,Caspase-3表达升高(P<0.05)。与正常组和空质粒组比较,SET8-shRNA组细胞钙化和凋亡增多,AKT表达降低,Caspase-3表达升高(P<0.05);与高磷组和空质粒+高磷组比较,SET8过表达+高磷组细胞钙化和凋亡减少,AKT表达升高,Caspase-3表达降低(P<0.05)。与正常组和对照组比较,AKT抑制剂组AKT表达显著降低,Caspase-3表达显著升高(P<0.05)。结论高磷通过SET8-shRNA抑制AKT活化,进而促进Caspase-3表达,增加VSMCs凋亡,从而影响细胞钙化的发生。

Objective To investigate the mechanism of high phosphorus promoting the calcification of vascular smooth muscle cells(VSMCs)through SET8-shRNA regulating AKT/Caspase-3 pathway.Methods The VSMCs were randomly divided into two groups:normal group and high phosphorus group(10 mmol/Lβ-glycerophosphate).Alizarin red staining and calcium content were used to detect the calcification of vascular smooth muscle cells.The mRAN and protein expression levels of SET8,AKT,Caspase-3 were detect-ed by real-time fluorescence quantification and Western blot.In order to verify the role of SET8 in cell calcification,rat VSMCs were divided into six groups:normal group,empty plasmid group(transfected with SET8-shRNA control plasmid),SET8 low expression group(transfection of SET8-shRNA plasmid),high phosphorus group(10 mmol/Lβ-glycerophosphate),empty plasmid+high phosphorus group(transfection of SET8 overexpression control plasmid),SET8 overexpression+high phosphorus group(transfection of SET8 overexpression plasmid).The calcification and the apoptosis of vascular smooth muscle cells and the expression levels of AKT and Caspase-3 were detected.In order to further verify the role of AKT in the calcification of VSMCs,vascular smooth muscle cells were divided into three groups:AKT inhibitor group(1μl DMSO+AKT inhibitor LY294002),control group(1μl DMSO)and normal group.The mRNA and protein expression levels of Akt and Caspase-3 were detected.Results Compared with normal group,the number of calcified nodules and the calcium content were increased in high phosphorus group(P<0.05),the expression of SET8 and AKT was decreased,and the expression of Caspase-3 was increased(all P<0.05).Compared with normal group and empty plasmid group,the calcification and the apoptosis were increased in SET8-shRNA group,the expression of AKT was decreased,and the expression of Caspase-3 was increased(all P<0.05).Compared with high phosphorus group and empty plasmid+high phosphorus group,the calcification and the apoptosis in SET8 overexpression+high phosphorus group were decreased,the expression of AKT was increased,and the expression of Caspase-3 was decreased(all P<0.05).Compared with normal group and control group,the expression of AKT was decreased significantly and the expression of Caspase-3 was increased significantly in AKT inhibitor group(all P<0.05).Conclusion High phosphorus can affect the cell calcification by inhibiting AKT activation through SET8-shRNA,promoting Caspase-3 expression,and increaing the apoptosis of VSMCs.