同型半胱氨酸致足细胞凋亡中FoxO1 DNA甲基化水平增高

Increased FoxO1 DNA methylation level in homocysteine-induced podocyte apoptosis

背景:同型半胱氨酸增多会引起肾损伤并导致足细胞凋亡,但是其具体机制还尚不清楚。目的:探讨叉头框转录因子O1(forkhead box O,FoxO1)及其DNA甲基化在同型半胱氨酸致足细胞凋亡中的作用。方法:体外培养小鼠肾脏足细胞(MPC-5),将其分为对照组(0μmol/L同型半胱氨酸)和同型半胱氨酸组(80μmol/L同型半胱氨酸)。干预细胞48 h后,采用免疫荧光技术检验足细胞凋亡相关蛋白Bax、caspase12和Bcl-2的表达情况;采用实时荧光定量PCR(qRT-PCR)检测FoxO1 mRNA水平;采用Western blot检测FoxO1和DNMT1蛋白表达水平;采用巢式降落式特异性PCR(nMS-PCR)测验FoxO1的DNA甲基化水平。结果与结论:①与对照组相比,同型半胱氨酸组足细胞中Bax和caspase12表达明显增高,Bcl-2表达明显降低;②FoxO1的mRNA和蛋白表达水平明显降低(P<0.01);③与对照组相比,同型半胱氨酸组FoxO1 DNA甲基化水平明显升高(P<0.01),同型半胱氨酸组足细胞中DNMT1蛋白表达明显增高(P<0.01);④结果表明:FoxO1 DNA高甲基化在同型半胱氨酸致足细胞凋亡中作用显著,而DNMT1参与同型半胱氨酸诱导的足细胞凋亡过程。

BACKGROUND:The increase of homocysteine can lead to renal injury and podocyte apoptosis,but the specific mechanism is not clear.OBJECTIVE:To investigate the effect of Forkhead box O1(FoxO1)and its DNA methylation in podocyte apoptosis induced by homocysteine.METHODS:Mouse renal podocytes(MPC-5)were cultured in vitro and divided into control group(0μmol/L homocysteine)and homocysteine group(80μmol/L homocysteine).After 48 hours of intervention,the expression of podocyte apoptosis-related proteins Bax,caspase12 and Bcl-2 was detected by immunofluorescence technique;the expression level of FoxO1 mRNA was detected by real-time fluorescence quantitative PCR;the protein expression levels of FoxO1 and DNMT1 were detected by western blot;DNA methylation level of FoxO1 was detected by nested methylation-specific PCR.RESULTS AND CONCLUSION:Compared with the control group,the expression levels of Bax and caspase12 protein in podocytes of the homocysteine group were significantly increased,while the expression of Bcl-2 protein was significantly decreased.The expression levels of FoxO1 mRNA and protein were significantly decreased in the homocysteine group compared with the control group(P<0.01).At the same time,the methylation level of FoxO1 DNA in the homocysteine group was significantly higher than that in the control group(P<0.01),and the expression of DNMT1 protein in podocytes in the homocysteine group was significantly higher than that in the control group(P<0.01).To conclude,FoxO1 DNA hypermethylation plays a significant role in podocyte apoptosis induced by homocysteine,whereas DNMT1 participates in homocysteine-induced podocyte apoptosis.