抑制转位蛋白在氧糖剥夺/复糖复氧模型中对BV-2细胞损伤和自噬的影响

Effects of knocking down TSPO on proliferation and autophagy of BV2 cells in OGD/R models

目的观察转位蛋白TSPO对OGD/R致BV2小胶质细胞损伤的作用,并初步探究TSPO在该模型中的自噬机制。方法离体培养BV2小胶质细胞,将实验分为正常组、OGD/R组、OGD/R+小发夹RNA阴性对照组(OGD/R+NCshRNA)、OGD/R+TSPO小发夹RNA组(OGD/R+TSPOshRNA),细胞中TSPO mRNA和TSPO蛋白的表达量分别采用qRT-PCR、Western blot方法检测。为探讨TSPO在OGD/R中致BV2小胶质细胞损伤和自噬作用,采用CCK-8实验检测细胞活力、自由氧(ROS)检测细胞毒性,qRT-PCR检测各组自噬相关mRNA(p62 mRNA、LC3B mRNA、Beclin-1 mRNA)表达情况、Western blot检测各组中自噬相关蛋白(p62、LC3Ⅱ/LC3Ⅰ、Beclin-1)表达情况。结果与正常组对比,OGD/R组中细胞TSPO mRNA和蛋白表达明显增高,细胞生存率明显降低,细胞毒性明显升高,LC3B mRNA、Beclin-1 mRNA水平升高,p62 mRNA表达量明显下降,LC3Ⅱ/LC3Ⅰ、Beclin-1蛋白水平升高,p62蛋白表达明显下降,自噬水平被激活;与OGD/R组比较,OGD/R+NCshRNA组细胞活力、细胞毒性和自噬水平差异无统计学意义,而OGD/R+TSPOshRNA组细胞生存率明显增高,细胞毒性和自噬水平明显降低。结论抑制TSPO在OGD/R模型中对BV2小胶质细胞损伤有明显的保护作用,该机制可能与抑制自噬相关。

Aim To investigate the effects of proliferation and autophagy of BV2 cells in OGD/R models when the 18 ku translocator protein(TSPO)was inhibited.Methods BV2 microglia were cultured in vitro and the model established by oxygen-glucose deprivation/reperfusion(OGD/R),the cells were divided into control group and OGD/R group,OGD/R+small hairpin RNA negative control group(OGD/R+NCshRNA),OGD/R+TSPO small hairpin RNA group(OGD/R+TSPOshRNA).The expression of TSPO mRNA and TSPO protein were detected by qRT-PCR and Western blot,respectively.In order to study the effect of TSPO on BV2 microglial cells in OGD/R injury and autophagy,the cell viability was tested by CCK-8 assey,the cytotoxicity was detected by reactive oxygen species(ROS),autophagy-related mRNA(p62 mRNA,LC3B mRNA,Beclin-1 mRNA)expressions were detected by qRT-PCR,and the expression levels of autophagy-related proteins(p62,LC3Ⅱ/LC3Ⅰ,Beclin-1)were detected by Western blot in each group.Result The expression of TSPO mRNA and protein increased significantly in OGD/R group while compared to control group,the cell death and cytotoxicity increased significantly,the expression levels of LC3B mRNA and Beclin-1 mRNA increased,while the p62 mRNA decreased significantly,the levels of LC3Ⅱ/LC3Ⅰand Beclin-1 protein increased,the expression of p62 protein decreased significantly in OGD/R group,and the autophagy was activated;compared with OGD/R group,the different levels of cell viability,cytotoxicity and autophagy in OGD/R+NCshRNA group were not statistically significant.But the survival rate of cells in OGD/R+TSPOshRNA group significantly increased,the levels of cytotoxicity and autophagy were significantly reduced.Conclusions The inhibition of TSPO has a significant protective effect on OGD/R injury model in BV2 microglial cells,which may be related to the inhibition of autophagy.