黄芪皂苷通过调控蛋白激酶R样内质网激酶途径抑制高糖诱导的人视网膜色素上皮细胞自噬的研究

目的:探讨黄芪皂苷通过调控蛋白激酶R样内质网激酶（PERK）途径影响高糖诱导的人视网膜色素上皮细胞（ARPE-19细胞）自噬水平。方法:体外培养人视网膜色素上皮细胞，实验分为正常对照组（5.5 mmol/L葡萄糖），高糖组（30 mmol/L葡萄糖），黄芪皂苷组（30 mmol/L葡萄糖+2.5、5、10 μg/mL黄芪皂苷）。噻唑蓝比色（MTT）法检测ARPE-19细胞活性，流式细胞仪检测细胞凋亡率，蛋白免疫印迹（Western blot）法检测自噬相关蛋白及PERK途径关键因子表达水平。添加PERK特异性抑制剂GSK2606414，Western blot检测对细胞自噬水平的影响。结果:与正常对照组比较，高糖组ARPE-19细胞活性降低，凋亡率升高，自噬相关蛋白LC3表达水平明显升高，另一自噬相关蛋白p62蛋白表达水平明显降低。PERK途径关键因子葡萄糖调节蛋白78（GRP78）、Perk和增强子结合蛋白的同源蛋白（CHOP）表达水平明显上调，差异具有统计学意义（P＜0.05）。与高糖组比较，黄芪皂苷组ARPE-19细胞活性升高，凋亡率降低，LC3蛋白表达水平明显下调，p62蛋白表达水平明显上调，GRP78、Perk和CHOP蛋白水平明显降低，差异具有统计学意义（P＜0.05）。PERK特异性抑制剂GSK2606414能够进一步抑制黄芪皂苷组ARPE-19细胞LC3蛋白表达水平，上调p62蛋白表达水平，差异具有统计学意义（P＜0.05）。结论:黄芪皂苷能够抑制高糖诱导的人视网膜色素上皮细胞自噬水平，其作用机制可能与抑制蛋白激酶R样内质网激酶途径有关。

Astragalus saponins inhibit high glucose-induced autophagy in human retinal pigment epithelial cells by regulating protein kinase R-like endoplasmic reticulum kinase pathway

AIM: To investigate the effects of astragalus saponins on the regulation of PERK pathway in high glucose-induced autophagy in human retinal pigment epithelial cells. METHODS: Human retinal pigment epithelial cells were cultured in vitro. The experiment was divided into NC group (5.5 mmol/L glucose), HG group (30 mmol/L glucose), and Ast group (30 mmol/L glucose+2.5, 5, 10 μg/mL astragalus saponins). The activity of ARPE-19 cells was detected by MTT. The apoptotic rate was detected by flow cytometry. The expression levels of autophagy-related proteins and key factors of PERK pathway were detected by Western blot. The effect on the level of autophagy was determined by Western blot analysis with the addition of PERK-specific inhibitor GSK2606414. RESULTS:Compared with the normal control group, the activity of ARPE-19 cells in the high glucose group was decreased. The apoptotic rate was increased, the expression level of autophagy-related protein LC3 was significantly increased. The expression level of p62 protein was significantly decreased. The protein levels of GRP78, Perk and CHOP, the key factors of PERK pathways, were significantly up-regulated, and the difference was statistically significant (P＜0.05). Compared with the high glucose group, the activity of ARPE-19 cells in the astragalus saponins group was increased. The apoptotic rate was decreased, the expression level of LC3 protein was down-regulated, the expression level of p62 protein was up-regulated, and the protein levels of GRP78, Perk and CHOP were significantly decreased. The concentration dependence was statistically significant (P＜0.05). PERK-specific inhibitor GSK2606414 was able to further inhibit the expression of LC3 protein in the ARPE-19 cells of the astragalus saponins group and up-regulate the expression of p62 protein, the difference was statistically significant (P＜0.05). CONCLUSION: Astragalus saponins can inhibit the autophagy of human retinal pigment epithelial cells induced by high glucose, and its mechanism is related to the inhibition of protein kinase R-like endoplasmic reticulum kinase pathway.