抑制或过表达GRK5 基因对星形胶质细胞凋亡及IL-1β和TNF-α表达的影响

目的:探讨抑制或过表达G 蛋白偶联受体激酶5(GRK5)基因对星形胶质细胞(AS)凋亡及IL-1β和TNF-α 表达的影响。方法:培养原代AS,将干扰GRK5 表达的siRNA(GRK5-siRNA)及GRK5 的过表达质粒(pcDNA3.1-GRK5)分别转染AS,通过Western blot 检测转染24 h 后的细胞中GRK5 蛋白的表达;缺氧处理AS,细胞分为空白组、缺氧组、缺氧+pcDNA3.1-GRK5 组和缺氧+GRK5-siRNA 组,缺氧处理24 h 后通过流式细胞仪检测各组细胞的凋亡率及ROS 含量;RT-PCR检测IL-1β和TNF-α的mRNA 表达;Western blot 检测p53、信号转导与转录因子3(STAT3)和p-STAT3 蛋白表达。结果:与空白组比较,转染GRK5-siRNA 的AS 中GRK5 的表达显著降低,转染pcDNA3.1-GRK5 的细胞GRK5 的表达显著升高(P <0.05);与空白组比较,缺氧组细胞凋亡率、ROS 水平及IL-1β、TNF-α、p53 和p-STAT3 的表达均显著升高(P<0.05);与缺氧组比较,缺氧+pcDNA3.1-GRK5 组细胞凋亡率、ROS 水平及IL-1β、TNF-α、p53 和p-STAT3 的表达均显著降低,缺氧+GRK5-siRNA组细胞凋亡率、ROS 水平及IL-1β、TNF-α、p53 和p-STAT3 的表达均显著升高(P<0.05)。结论:过表达GRK5 可降低AS 凋亡和ROS 水平,下调IL-1β、TNF-α、p53 表达及STAT3 信号通路,抑制GRK5表达反之。

Inhibition or overexpression of GRK5 gene on apoptosis of astrocytes and expression of IL-1βand TNF-α

Objective:To investigate the effect of inhibition or overexpression of GRK5 gene on the apoptosis of astrocytes(AS) and the expression of IL-1βand TNF-α.Methods:Primary astrocytes was cultured,siRNA interference with GRK5 expression(GRK5-siRNA) and overexpressed plasmids of GRK5 (pcDNA3.1-GRK5) were transfected into astrocytes,GRK5 protein expression was detected in cells transfected with 24 h by Western blot,astrocytes were divided into blank group,hypoxia group,hypoxia+pcDNA3.1-GRK5 group and hypoxia group+GRK5-siRNA group,apoptosis rate and ROS content were detected by flow cytometry;IL-1βand TNF-αexpression were detected by RT-PCR;the expression of p53,STAT3 and p-STAT3 protein were detected by Western blot.Results:Compared with the blank group,the expression of GRK5 in the astrocytes transfected with GRK5-siRNA was reduced significantly,was increased significantly in pcDNA3.1-GRK5 group(P < 0.05);compared with the blank group,apoptosis rate,ROS level and the expression of IL-1β,TNF-α,p53 and p-STAT3 were increased significantly in hypoxia group(P<0.05);compared with hypoxia group, apoptosis rate,ROS level and the expression of IL-1β,TNF-α,p53 and p-STAT3 were significantly decreased in hypoxia+pcDNA3.1-GRK5 group,apoptosis rate,ROS level and the expression of IL-1β,TNF-α,p53 and p-STAT3 were significantly higher in hypoxia group+GRK5-siRNA group(P<0.05).Conclusion:Over expression of GRK5 can reduce the astrocytes apoptosis and ROS level,down regulation IL-1β,TNF-α,p53 expression and STAT3 signaling pathway,and the Inhibition of GRK5 expression is the opposite.