沉默信息调节因子1对高糖诱导的足细胞凋亡的影响及机制研究

Effect and its mechanism of SIRT1 on apoptosis of podocytes induced by high glucose

目的研究沉默信息调节因子1(silence information regulator 1,SIRT1)对高糖诱导的足细胞凋亡的影响和机制。方法取足细胞分别给予不同处理,分为5组:Control组(5.6 mmol/L葡萄糖培养液培养)、Osmotic-NC组(葡萄糖5.6 mmol/L+甘露醇25 mmol/L培养液培养)、HG组(30 mmol/L葡萄糖培养液培养)、HG+SIRT1组[SIRT1过表达载体(pcDNA3.1-SIRT1)转染+30 mmol/L葡萄糖培养液培养]、HG+NC组[阴性对照载体(pcDNA3.1)转染+30 mmol/L葡萄糖培养液培养],采用qRT-PCR和Western blot技术检测各组SIRT1表达效果,以试剂盒分别检测各组细胞中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)、活性氧簇(ROS)水平,采用流式细胞术检测各组细胞凋亡,采用Western blot检测各组细胞中Cleaved Caspase-3和Cleaved Caspase-9蛋白表达变化。结果高糖培养后的足细胞中SIRT1 mRNA及蛋白水平均降低。转染SIRT1过表达载体后的足细胞经过高糖处理以后,细胞中的SIRT1 mRNA及蛋白水平升高。高糖处理后的足细胞中SOD、CAT、GSH-Px活性降低,MDA和ROS水平升高,细胞凋亡率升高,细胞中Cleaved Caspase-3和Cleaved Caspase-9蛋白水平升高。上调SIRT1可以提高高糖条件下足细胞SOD、CAT、GSH-Px活性,减少细胞中MDA和ROS水平,减少细胞凋亡率和Cleaved Caspase-3、Cleaved Caspase-9蛋白表达。结论上调SIRT1可以降低高糖诱导的足细胞凋亡,其作用机制可能与降低氧化损伤有关。

Objective To study the effect and its mechanism of SIRT1 on glucose-induced apoptosis of podocytes. Methods Human kidney podocytes were taken to treat with different methods, and divided into 5 groups:control group (culturing with the medium containing 5.6 mmol/L glucose), osmotic-NC group (culturing with the medium containing glucose 5.6 mmol/L+mannitol 25 mmol/L), HG group (culturing with the medium of 30 mmol/L glucose), HG+SIRT1 group[transfecting with SIRT1 over-expression vector (pcDNA3.1-SIRT1)+culturing with the medium containing 30 mmol/L glucose], and HG NC group[transfecting with the negative control vector (pcDNA3.1)+culturing with the medium containing 30 mmol/L glucose]. The expression of SIRT1 in each group was detected by qRT-PCR and Western blot. Activities of SOD, CAT and GSH-Px, and levels of MDA and ROS in the cells from each group were detected with various corresponding kits. Cell apoptosis in each group were detected by flow cytometry. Western blot was used to detect changes of the expression of Cleaved Caspase-3 and Cleaved Caspase-9 in the cells from each group. Results The levels of SIRT1 mRNA and protein in podocytes cultured with high glucose decreased. After high glucose treatment of podocytes transfected with pcDNA3.1-SIRT1, the levels of SIT1 mRNA and protein in podocytes increased, the activities of SOD, CAT and GSH-Px decreased, the levels of MDA and ROS increased, the apoptotic rate increased, and the levels of cleaved caspase-3 and cleaved caspase-9 protein increased in podocytes treated with high glucose. Upregulation of SIRT1 could increase SOD, CAT, GSH-Px activities, decrease MDA and ROS levels, and decrease apoptosis and expression of cleaved caspase-3 and cleaved caspase-9 in podocytes under high glucose conditions. Conclusions Upregulation of SIRT1 can reduce the apoptosis of podocytes induced by high glucose, and the mechanism may be related to reduction of oxidative damage.