VEGF-B通过ERK1/2信号通路对RGC-5细胞缺氧损伤的保护作用

Vascular endothelial growth factor B alleviates hypoxia injury in RGC-5 cell line,via ERK1/2 signaling pathway

目的探讨血管内皮生长因子B(vascular endothelial growth factor-B,VEGF-B)对氯化钴(cobalt chloride hexahydrate,Co Cl2)诱导的大鼠视网膜神经节细胞株(retinal ganglion cells-5,RGC-5)缺氧损伤的保护作用及其机制。方法 Co Cl2孵育RGC-5细胞24h诱导缺氧模型。将RGC-5细胞分为正常组、缺氧组、VEGF-B+缺氧组、PD98059(ERK1/2阻滞剂,PD)+VEGF-B+缺氧组。倒置显微镜观察细胞的形态变化;CCK-8法检测细胞存活率;流式细胞仪Annexin V-APC/7-AAD双染色法检测细胞凋亡率;Western Blot检测蛋白Bcl-2、Bax及ERK1/2、pERK1/2表达水平。结果 1缺氧使细胞皱缩变小变圆,正常组及药物组细胞体积大有突起。2与正常组相比,缺氧使细胞存活率减低;与缺氧组相比,药物组细胞存活率均升高。3与正常组相比,缺氧组正常细胞率下降、细胞凋亡率升高;与缺氧组相比,药物组正常细胞率升高、细胞凋亡率下降。4与正常组相比,缺氧组Bcl-2、Bcl-2/Bax比值降低,Bax增多;与缺氧组相比,药物组Bcl-2、Bcl-2/Bax比值增高,Bax减少。四组总ERK1/2表达量不变,pERK1/2表达量缺氧组较正常组增多,VEGF-B+缺氧组较缺氧组表达量高,PD+VEGF-B+缺氧组较VEGF-B+缺氧组减少。结论 VEGF-B部分通过ERK1/2信号通路增加Bcl-2表达,降低Bax表达从而抑制Co Cl2诱导的RGC-5细胞凋亡。

Objective：To expore the protective effects and the underlying mechanism of vascular endothelial growth fac-tor-B（VEGF-B）in retinal ganglion cells line（RGC-5）in rats with hypoxia injury induced by cobalt chloride hexahydrate （ CoCl2 ）. Methods：RGC-5 were incubated with CoCl2 to induce hypoxia injury as the apoptosis model. RGC-5 were divided into four groups：normal group,hypoxia group,VEGF-B ＋Hypoxia group and PD98059（ inhibitor of ERK1/2,PD）＋VEGF-B＋Hypoxia group. The morphology of cells was observed by inverted microscope. The cell viability rate was detec-ted by CCK-8 assay. Annexin-V APC/7-AAD double staining flow cytometry was used to test the normal cells rate and ap-optotic cells rate. The levels of Bcl-2,Bax,EKK1/2 and p-ERK1/2 protein expression were detected by using Western Blot. Results：① The morphology of cells in the normal group and VEGF-B ＋Hypoxia group were big and with many processes. The cells of the hypoxia group shrinked to be round and smaller. ② The cell viability was decreased by CoCl2 treatment compared with the normal group. VEGF-B treatment significantly increased cell viability compared with the hy-poxia group. ③ The normal cells rate decreased and the apoptotic cells rate increased in the hypoxia group compared with the normal group. VEGF-B treatment significantly increased normal cells rate and decreased apoptotic cells rate compared with the hypoxia group. ④ Compared with the normal group,Bcl-2 protein expression and Bcl-2/Bax ratio decreased and Bax protein expression increased in the hypoxia group. However,VEGF-B treatment up-regulated Bcl-2 protein expression and Bcl-2/Bax ratio and down-regulated Bax protein expression compared with the hypoxia group. Moreover,hypoxia treat-ment up-regulated level of p-ERK1/2 protein compared with Normal group. Level of p-ERK1/2 protein was higher in VEGF-B＋Hypoxia group compared with the hypoxia group but there was no change of total ERK1/2 protein expression in the four groups. Conclusion：VEGF-B can inhibit apoptosis induced by CoCl2 in RGC-5 cell line through up-regulating Bcl-2 protein expression and down-regulating Bax protein expression,partially via ERK1/2 signaling pathway.