【摘要】 目的：观察黄芪甲苷对视网膜神经节细胞（retinal ganglion cell, RGC）损伤的保护作用，并基于腺苷酸活化蛋白激酶（adenosine monophosphate activated kinase, AMPK）/哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）/Unc-51-样激酶（Unc-51-like kinase, ULK）自噬通路探讨相关作用机制。方法：取KM乳小鼠，制备原代RGC。采用Brn3a染色检测RGC纯度，台盼蓝染色检测RGC活力。采用CCK-8法检测谷氨酸钠的最佳造模浓度和黄芪甲苷的最佳药物浓度。将RCC分为空白组和谷氨酸钠组(20 mmol·L^-1),透射电镜检测自噬溶酶体数量。将RGC分为对照组、模型组(20 mmol·L^-1谷氨酸钠)、低剂量黄芪甲苷组(25μmol·L^-1)、中剂量黄芪甲苷组(50μmol·L^-1)及高剂量黄芪甲苷组(100μmol·L^-1),流式细胞术检测细胞凋亡率。采用Western blot法检测RGC的微管相关蛋白轻链3-Ⅱ（microtubule-associated protein light chain 3-Ⅱ,LC3-Ⅱ）、AMPK、p-AMPK、mTOR、p-mTOR、ULK、p-ULK表达水平。结果：RGC纯度为95.03%,细胞活率为93.3%。谷氨酸钠损伤RGC的半数抑制浓度（half maximal inhibitory concentration, IC50）为20 mmol·L^-1（P<0.05）。透射电镜显示，与空白组比较，谷氨酸钠组RGC同时期的自噬溶酶体数量增加。流式细胞术显示，与对照组比较，模型组RGC凋亡率升高；与模型组比较，黄芪甲苷低剂量组、中剂量组、高剂量组RGC凋亡率降低（P<0.001）。Western blot显示，与对照组比较，模型组RGC的AMPK、p-AMPK、mTOR、p-mTOR、ULK表达水平升高，LC3-Ⅱ表达水平降低（P<0.05）。与模型组比较，高剂量黄芪甲苷组RGC的AMPK、mTOR、ULK表达水平降低，LC3-Ⅱ、p-AMPK、p-ULK表达水平升高（P<0.05）。结论：黄芪甲苷可发挥对谷氨酸钠损伤RGC的保护作用，其机制可能与调控AMPK/mTOR/ULK信号通路，从而促进自噬有关。更多还原

【Abstract】 Objective: To observe the protective effect of astragaloside on retinal ganglion cell（RGC） damage.The mechanism of action was explored based on the adenosine monophosphate activated kinase（AMPK）/mammalian target of rapamycin（mTOR）/Unc-51-like kinase（ULK） autophagy pathway.Methods: Primary RGCs were prepared from KM lactating mice, and the purity of RGCs was detected with Brn3a staining, and the viability of RGCs was detected with Taipan blue staining.The CCK-8 method was used to detect the optimal modeling concentration of monosodium glutamate and the optimal drug concentration of astragaloside.The RCC was divided into blank group and sodium glutamate group(20 mmol·L^-1),and the number of autophagic lysosomes was detected with transmission electron microscopy.The RGC was divided into control group, model group(20 mmol·L^-1 sodium glutamate),low-dose astragaloside group(25 μmol·L^-1),medium-dose astragaloside group(50 μmol·L^-1) and high-dose astragaloside group(100 μmol·L^-1),and the apoptosis rate was detected by flow cytometry.The expression levels of microtubule-associated protein light chain type 3-Ⅱ（LC3-Ⅱ）,AMPK,p-AMPK,mTOR,p-mTOR,ULK,and p-ULK of RGC were detected with Western blot.Results: The purity of RGC was 95.03% and the cell viability was 93.3%.The half maximal inhibitory concentration（IC50） of RGC damaged by monosodium glutamate was 20 mmol·L^-1（P<0.05）.With transmission electron microscopy it was showed an increase in the number of autophagic lysosomes in RGCs in the monosodium glutamate group compared with the blank group（P<0.05）.With flow cytometry it was showed that the apoptosis rate of RGCs in the model group was elevated compared with that of the control group; the apoptosis rate of RGCs in the low, medium, and high doses of astragaloside group was decreased compared with that of the model group（P<0.05）.With Western blot it was showed that the expression levels of AMPK,p-AMPK,mTOR,p-mTOR,and ULK of RGCs in the model group were elevated compared with that of the control group, and the LC3-Ⅱ expression levels were decreased（P<0.05）.Compared with that of the model group, the expression levels of AMPK,mTOR,and ULK in RGC in the high-dose astragaloside group were reduced, and the expression levels of LC3-Ⅱ,p-AMPK,and p-ULK were increased（P<0.05）.Conclusion: Astragaloside can exert a protective effect on sodium glutamate injured RGCs, which may be related to the modulation of the AMPK/mTOR/ULK signaling pathway, thereby promoting autophagy.更多还原