摘要
目的探讨白藜芦醇(RES)通过激活核因子E2相关因子2(Nrf2)/NAD(P)H:醌氧化还原酶1(NQO1)信号通路在调控小鼠海马神经元HT22细胞铁死亡过程中的保护机制。方法采用Erastin诱导HT22细胞模型模拟铁死亡, 设置对照组、Erastin组、40、80 μmol/L RES处理组, 以及Erastin+RES+ML385组。通过细胞计数试剂盒(CCK-8)检测细胞活力, 流式细胞术和原位缺口末端标记法(TUNEL)法评估凋亡, 试剂盒测定细胞内活性铁和活性氧(ROS)含量, 荧光定量聚合酶链式反应(qPCR)和蛋白质印迹法(Western blot)分析Nrf2/NQO1信号通路。组间差异通过ANOVA和Tukey’s post hoc测试确定。结果 0.6 μmol/L的Erastin组细胞活力显著低于40 μmol/L的RES组(51.00±2.90比68.12±2.24, t=2.902, P<0.05);RES显著降低细胞凋亡率, 80 μmol/L RES处理的HT22细胞凋亡率显著低于Erastin组(6.12±0.93比18.21±2.05, t=3.858, P<0.05), 效果最佳。80 μmol/L的RES组细胞内活性铁和ROS含量显著低于Erastin组(活性铁:43.14±3.13比65.11±4.07, t=3.821, P<0.05;ROS:8.04±0.83比25.02±1.57, t=5.852, P<0.05)。RES预处理后Nrf2、NQO1和HO-1 mRNA及蛋白表达显著高于Erastin组(NQO1 mRNA:0.32±0.04比1.01±0.07, t=3.928, P<0.05;Nrf2 mRNA:0.25±0.03比1.04±0.06, t=3.785, P<0.05;HO-1 mRNA:0.26±0.05比1.02±0.08, t=3.957, P<0.05;NQO1蛋白:0.96±0.04比0.45±0.07, t=3.792, P<0.05;Nrf2蛋白:0.91±0.07比0.32±0.05, t=3.957, P<0.05;HO-1蛋白:1.21±0.04比0.25±0.03, t=4.535, P<0.05), 而ML385逆转了RES的保护效果, 导致RES+Erastin+ML385组的NQO1、Nrf2、HO-1蛋白表达显著少于RES+Erastin组(NQO1:0.67±0.05比0.92±0.08, t=4.276, P<0.05;Nrf2:0.71±0.05比0.94±0.07, t=4.121, P<0.05;HO-1:0.64±0.04比1.16±0.04, t=3.978, P<0.05)。结论 RES可能通过激活Nrf2/NQO1信号通路, 从而抑制Erastin诱导的神经元HT22细胞铁死亡。
ObjectiveTo investigate the potential mechanism of resveratrol regulating iron death in HT22 cells of mouse hippocampal neurons by quantitative polymerase chain reaction(qPCR)and Western blotting.Differences between groups were determined by ANOVA and Tukey’s post hoc tests.MethodsHT22 cell ferroptosis model was induced by Erastin.The groups of control,Erastin,40 and 80μmol/L RES,and Erastin+RES+ML385 were set up.Cell viability was tested by cell counting kit-8(CCK-8)assay,apoptosis was detected by flow cytometry and terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL),intracellular iron and ROS determined by kits,and Nrf2/NQO1 detected by quantitative polymerase chain reaction(qPCR)and Western blotting.Differences between groups were determined by ANOVA and Tukey’s post hoc tests.ResultsThe cell viability of 0.6μmol/L Erastin group was significantly lower than that of 40μmol/L RES group(51.00±2.90 vs.68.12±2.24,t=2.902,P<0.05).The apoptosis rate of HT22 cells treated with 80μmol/L RES was significantly lower than that of Erastin group(6.12±0.93 vs.18.21±2.05,t=3.858,P<0.05).The contents of intracellular active iron and ROS in 80μmol/L RES group were significantly lower than those in Erastin group(active iron:43.14±3.13 vs.65.11±4.07,t=3.821,P<0.05;ROS:8.04±0.83 vs.25.02±1.57,t=5.852,P<0.05).The mRNA and protein expressions of Nrf2,NQO1 and HO-1 after RES pretreatment were significantly higher than those in Erastin group(NQO1 mRNA:0.32±0.04 vs.1.01±0.07,t=3.928,P<0.05;Nrf2 mRNA:0.25±0.03 vs.1.04±0.06,t=3.785,P<0.05;HO-1 mRNA:0.26±0.05 vs.1.02±0.08,t=3.957,P<0.05;NQO1 protein:0.96±0.04 vs.0.45±0.07,t=3.792,P<0.05;Nrf2 protein:0.91±0.07 vs.0.32±0.05,t=3.957,P<0.05;HO-1 protein:1.21±0.04 vs.0.25±0.03,t=4.535,P<0.05),while ML385 reversed the protective effect of RES,resulting in significantly lower expression of NQO1,Nrf2 and HO-1 protein in RES+Erastin+ML385 group than in RES+Erastin group(NQO1:0.67±0.05 vs.0.92±0.08,t=4.276,P<0.05;Nrf2:0.71±0.05 vs.0.94±0.07,t=4.1
作者
张林
郭田田
王纵
李力
杨扬
Zhang Lin;Guo Tiantian;Wang Zong;Li Li;Yang Yang(Department of Vascular Neurosurgery,Zhumadian Central Hospital,Zhumadian 463000,China;Department of Infection Management,Zhumadian Central Hospital,Zhumadian 463000,China;Department of Neurosurgery,Zhumadian Central Hospital,Zhumadian 463000,China;Department of Research Management,Zhumadian Central Hospital,Zhumadian 463000,China)
出处
《中华实验外科杂志》
CAS
2024年第8期1751-1754,共4页
Chinese Journal of Experimental Surgery
基金
河南医学科技攻关计划联合共建立项项目(LHGJ20200701)。
