基于Bcl-2与mTOR蛋白“crosstalk”探究活血定眩胶囊对血管内皮细胞自噬的作用机制

Mechanism of Huoxue Dingxuan Capsules on autophagy in vascular endothelial cells based on"crosstalk"of Bcl-2 and mTOR protein

基于B淋巴细胞瘤-2(Bcl-2)与雷帕霉素靶蛋白(mTOR)间的蛋白“crosstalk”探究活血定眩胶囊(HXDX)对血管内皮细胞自噬的作用机制。将bEnd.3细胞分为空白对照组、模型组、活血定眩胶囊组,免疫共沉淀(CO-IP)实验后蛋白免疫印迹法(WB)检测Bcl-2与mTOR的结合情况,染色后激光共聚焦显微镜观察Bcl-2与mTOR蛋白的共定位情况;构建bEnd.3细胞内Bcl-2 siRNA模型,将bEnd.3细胞分为5组,空白对照组、氧糖剥夺(OGD)组、转染Bcl-2 siRNA组、阴性对照(NC)组、HXDX组;构建bEnd.3细胞内mTOR siRNA模型,将bEnd.3细胞分为5组,空白对照组、OGD组、转染mTOR siRNA组、NC组、HXDX组,WB检测自噬相关蛋白的表达。CO-IP实验结果显示Bcl-2与mTOR蛋白在bEnd.3细胞可共定位表达,活血定眩胶囊含药血清干预后Bcl-2、mTOR蛋白的表达显著上升。筛选并转染Bcl-2-mus-384后诱导bEnd.3细胞自噬,Bcl-2 siRNA组中Bcl-2表达较空白对照组和模型组显著降低,mTOR蛋白的表达较OGD组显著降低、人微管相关蛋白轻链3Ⅱ/Ⅰ(LC3Ⅱ/LC3Ⅰ)表达较模型组显著升高,活血定眩胶囊含药血清干预后,HXDX组中Bcl-2、mTOR表达较Bcl-2 siRNA组显著升高,LC3Ⅱ/LC3Ⅰ、重组人自噬效应蛋白1(Beclin1)表达较Bcl-2 siRNA组显著降低;转染mTOR-mus-7061诱导bEnd.3细胞自噬后,在mTOR siRNA组中,mTOR蛋白表达较空白对照组和模型组显著降低,LC3Ⅱ/LC3Ⅰ、Beclin1蛋白表达较模型组明显升高;经活血定眩胶囊含药血清干预后HXDX组中Bcl-2、mTOR表达较mTOR siRNA组明显升高,LC3Ⅱ/LC3Ⅰ、Beclin1蛋白表达较mTOR siRNA组明显降低。该研究从细胞水平探讨了bEnd.3细胞自噬过程中Bcl-2与mTOR“crosstalk”,并说明了活血定眩胶囊含药血清对其的调节作用,为椎动脉型颈椎病的治疗提供基础。

This paper investigated the mechanism of Huoxue Dingxuan Capsules(HXDX)on autophagy in vascular endothelial cells based on the"crosstalk"of Bcl-2 and mTOR protein.bEnd.3 cells were divided into a blank control group,a model group,and an HXDX group.CO-IP experiments were conducted,and then Western blot(WB)was used to detect the binding of Bcl-2 and mTOR.Co-localization of Bcl-2 with mTOR protein was observed by laser confocal microscopy after staining.The model of Bcl-2 siRNA in bEnd.3 cells was constructed,and the bEnd.3 cells were divided into five groups,including blank control group,oxygen-glucose deprivation(OGD)group,transfected Bcl-2 siRNA group,negative control(NC)group,and HXDX group.The model of mTOR siRNA in bEnd.3 cells was constructed,and the bEnd.3 cells were divided into five groups,including blank control group,OGD group,transfected mTOR siRNA group,NC group,and HXDX group.The expression of autophagy-related proteins was detected by WB.The results of CO-IP experiments showed that Bcl-2 and mTOR proteins could be co-localized and expressed in bEnd.3 cells,and the expression of Bcl-2 and mTOR proteins increased after the intervention of the HXDX-containing serum.After screening and transfection with Bcl-2-mus-384,autophagy of bEnd.3 cells was induced.The expression of Bcl-2 in the Bcl-2 siRNA group was significantly decreased compared with the blank control group and model group.The expression of mTOR protein was significantly lower than that of the OGD group,and the expression of human microtubule-associated protein light chain 3Ⅱ/Ⅰwas significantly higher than that of the model group.After the intervention of the HXDX-containing serum,the expression of Bcl-2 and mTOR was increased in the HXDX group compared with that in the Bcl-2 siRNA group,and the expression of LC3Ⅱ/LC3Ⅰand Beclin1 was decreased in the HXDX group compared with that in the Bcl-2 siRNA group.After transfecting mTOR-mus-7061 and inducing autophagy of bEnd.3 cells,in the mTOR siRNA group,mTOR protein expression was decreased compared with the blank control and model groups,and LC3Ⅱ/LC3Ⅰand Beclin1 protein expression was increased compared with the model group.After the intervention of the HXDX-containing serum,the expression of Bcl-2 and mTOR was increased in the HXDX group compared with that in the mTOR siRNA group,and the expression of LC3Ⅱ/LC3Ⅰand Beclin1 proteins was decreased compared with the mTOR siRNA group.This study explored the"crosstalk"of Bcl-2 and mTOR during autophagy in bEnd.3 cells at the cellular level and illustrated the modulating effect of the HXDX-containing serum,which provided a basis for the treatment of cervical spondylosis of vertebral artery type.