黄芪甲苷对谷氨酸钠损伤视网膜神经节细胞的保护作用  

Protective Effect of Astragaloside on Retinal Ganglion Cells Injured by Sodium Glutamate

作　　者：税小丁 尚孟秋 罗钰 陈涛[1] 王妍[1] 丁琨 韦企平[1,3] 廖良[1,3] SHUI Xiaoding;SHANG Mengqiu;LUO Yu;CHEN Tao;WANG Yan;DING Kun;WEI Qiping;LIAO Liang(Beijing University of Chinese Medicine,Beijing China 100029;Eye Hospital,China Academy of Chinese Medical Sciences,Beijing China 100040;Dongfang Hospital,Beijing University of Chinese Medicine,Beijing China 100078)

机构地区：[1]北京中医药大学,北京100029 [2]中国中医科学院眼科医院,北京100040 [3]北京中医药大学东方医院,北京100078

出　　处：《中医学报》2024年第12期2632-2639,共8页Acta Chinese Medicine

基　　金：国家自然科学基金面上项目(81973909)。

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。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(25μmol·L^(-1)),medium-dose astragaloside group(50μmol·L^(-1))and high-dose astragaloside group(100μmol·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

关 键 词：黄芪甲苷 视网膜神经节细胞 AMPK/mTOR/ULK信号通路 外伤性视神经病变 自噬 谷氨酸钠 

分 类 号：R285.5[医药卫生—中药学] R276.7[医药卫生—中医学]