黄芪甲苷通过JAK2/STAT3通路减轻MPP+诱导的帕金森病SK-N-SH细胞模型损伤

Astragaloside IV attenuates the injury of SK-N-SH cell model of Parkinson's disease induced by MPP+through JAK2/STAT3 pathway

目的探讨黄芪甲苷对1-甲基-4-苯基吡啶离子(MPP+)诱导的帕金森病(PD)细胞模型损伤的影响及分子机制。方法将对数期的SK-N-SH细胞按照随机数字表法分为Mock组、MPP+组、Ast组和AG490组。Mock组采用常规培养,MPP+组采用0.6 mmol/L的MPP+处理,Ast组采用40 nmol/L黄芪甲苷预处理2 h后再用0.6 mmol/L的MPP+处理,AG490组采用1μmol/L酪氨酸蛋白激酶2(JAK2)/信号转导子和转录激活子3(STAT3)通路抑制剂AG490和40 nmol/L的黄芪甲苷预处理2 h后再用0.6 mmol/L的MPP+处理。四甲基偶氮唑盐比色法(MTT)检测细胞存活率,流式细胞术检测细胞凋亡,DCFH-DA探针法检测活性氧(ROS)水平,试剂盒测定超氧化物歧化酶(SOD)和乳酸脱氢酶(LDH)的活性,蛋白质印迹法检测活化的半胱氨酸天冬氨酸蛋白酶-3/-9(Clevaed Caspase-3/-9)、磷酸化酪氨酸蛋白激酶2(p-JAK2)和磷酸化信号转导子和转录激活子3(p-STAT3)蛋白表达。结果与Mock组比较,MPP+处理的SK-N-SH细胞存活率和SOD活性明显降低,p-JAK2和p-STAT3蛋白表达明显下调,凋亡率、LDH活性、ROS水平、Clevaed Caspase-3和Clevaed Caspase-9的表达水平明显升高,差异均有统计学意义(P<0.05);黄芪甲苷处理能够逆转MPP+诱导的SK-N-SH细胞损伤;而JAK2/STAT3通路抑制剂AG490逆转了黄芪甲苷对SK-N-SH细胞损伤保护作用。结论黄芪甲苷通过激活JAK2/STAT3信号通路减轻MPP+诱导的SK-N-SH细胞损伤。

Objective To investigate the effect of astragaloside IV on 1-methyl-4-phenylpyridinium(MPP+)-induced injury of Parkinson's disease(PD)cell model and its molecular mechanism.Methods SK-N-SH cells in log phase were divided into Mock group,MPP+group,Ast group and AG490 group according to the random number table method.The Mock group was cultured routinely,the MPP+group was treated with 0.6 mmol/L MPP+,the Ast group was pretreated with 40 nmol/L astragaloside IV for 2 h and then treated with 0.6 mmol/L MPP+,and the AG490 group was treated with 1μmol/L Janus Kinase2(JAK2)/signal transducer and activator of transcription 3(STAT3)pathway inhibitor AG490 and 40 nmol/L astragaloside IV were pretreated for 2 h and then treated with 0.6 mmol/L MPP+.Tetramethylazolium salt colorimetric method(MTT)was used to detect cell viability,flow cytometry was used to detect cell apoptosis,DCFH-DA probe method was used to detect reactive oxygen species(ROS)levels,and the kit was used to detect superoxide dismutase(SOD)and lactate dehydrogenase(LDH)activity,Western blotting test to detect activated caspase-3/-9(Clevaed Caspase-3/-9),activated caspase-9(Clevaed Caspase-9),phosphorylated JAK2(p-JAK2)and phosphorylated STAT3(p-STAT3)protein expression.Results Compared with the Mock group,the survival rate and SOD activity of SK-N-SH cells treated with MPP+were significantly reduced,the expression of p-JAK2 and p-STAT3 protein was significantly down-regulated,apoptosis rate,LDH activity,ROS level,the expression level of Clevaed Caspase-3 and Clevaed Caspase-9 was significantly increased,and the differences were statistically significant(P<0.05).Astragaloside IV treatment can reverse the injury of SK-N-SH cells induced by MPP+;the JAK2/STAT3 pathway inhibitor AG490 reversed the protective effect of Astragaloside IV on SK-N-SH cells.Conclusion Astragaloside IV can alleviate MPP+-induced damage to SK-N-SH cells by activating the JAK2/STAT3 signaling pathway.