二甲双胍对27-羟基胆固醇诱导的HT22细胞损伤的保护作用

The protective effects of metformin on 27-hydroxycholesterol induced injury in HT22 cells

目的:探究二甲双胍(Met)对27-羟基胆固醇(27-HC)诱导的HT22细胞损伤的保护作用。方法:将HT22细胞分为对照组(control)、27-HC处理组(27-HC)和27-HC联合Met处理组(27-HC+Met)。采用MTT法确定27-HC诱导HT22细胞损伤模型的最佳浓度及不同浓度的Met对细胞活性的影响,确定Met的最佳预保护浓度;利用Hoechst 33342染色观察细胞凋亡;利用商品化的试剂盒检测HT22细胞中超氧化物歧化酶(SOD)和乳酸脱氢酶(LDH)的活性;利用Western Blot法检测HT22细胞中NMDA受体亚单位2B(NR2B)和AMP活化蛋白激酶(AMPK)蛋白表达及AMPK蛋白的磷酸化水平。结果:与对照组相比,27-HC诱导后的HT22细胞活性显著下降(P <0.05),细胞形态异常且凋亡细胞增加,LDH活性升高,SOD活性降低,NR2B的表达和p-AMPK水平降低;Met预处理后,细胞活性提高,细胞形态趋于正常且凋亡细胞减少,LDH活性降低,SOD活性升高,NR2B的表达和p-AMPK水平增加。结论:Met通过AMPK/NR2B信号通路调节细胞氧化应激水平,发挥对27-HC诱导的HT22细胞损伤的保护作用。

Objective: To investigate the protective effects of metformin(Met) on 27-hydroxycholesterol(27-HC) induced injury in HT22 cells.Methods: HT22 cells were divided into control group,27-HC group,and 27-HC + Met group.The optimal concentration of 27-HC for constructing HT22 cell damage model were selected by performing MTT assay.Besides,the influence of Met at different concentrations on cell viability were assessed to determine the optimal pre-protective concentration.Furthermore,Hoechst33342 staining was applied to determine the apoptotic cells.Activities of superoxide dismutase(SOD) and lactate dehydrogenase(LDH) in HT22 cells following the designed treatment were detected by corresponding commercial kits.In addition,Western Blot was used to detect NMDA receptor 2B subunit(NR2 B) and AMP-activated protein kinase(AMPK) protein expression and AMPK protein phosphorylation level in HT22 cells.Results: In comparison with the control group,27-HC treatment significantly reduced HT22 cell viability(P <0.05),caused abnormal cell morphology and induced cell apoptosis,elevated the activity of LDH,reduced the activity of SOD,and decreased the expression of NR2 B and the level of p-AMPK.Moreover,Met pretreatment increased cell viability,restored cell morphology to be normal and decreased cell apoptosis,reduced the activity of LDH,elevated the activity of SOD,and increased the expression of NR2B and the level of p-AMPK.Conclusion: Met exerts its protective effects against 27-HC induced injury in HT22 cells by modulating the oxidative stress level via AMPK/NR2B signaling pathway.