微小RNA-146b-5p在小胶质细胞糖氧剥夺/复氧损伤中的作用与机制

The Role and Mechanism of MicroRNA-146b-5p in Glucose and Oxygen Deprivation/Reoxygenation Injury of Microglia

目的:探究微小RNA-146b-5p(miR-146b-5p)在小胶质细胞糖氧剥夺/复氧损伤(OGD/R)中的作用及机制。方法:建立小鼠小胶质细胞系EOC 13.31的OGD/R模型。将miR-146b-5p模拟物、miR-146b-5p抑制剂及相应的阴性对照,分别转染至EOC 13.31细胞中,然后再分别进行OGD/R处理,分别命名为OGD/R+miR-146b-5p mimic组、OGD/R+miR-146b-5p inhibitor组、OGD/R+NC mimic组和OGD/R+NC inhibitor组,以常规培养的细胞为对照组(Control组),只行OGD/R处理的细胞为OGD/R组,48h后检测相关指标。实时定量反转录聚合酶链反应(qRT-PCR)检测miR-146b-5p表达;采用细胞计数试剂盒-8(CCK-8)检测细胞存活率;原位末端标记法(TUNEL)检测细胞凋亡率;酶联免疫吸附法(ELISA)测定超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)含量;蛋白免疫印迹法(Western blotting)检测TRAF6蛋白表达;生物信息学和双荧光素酶报告基因实验分析miR-146b-5p和TRAF6之间的关系。结果:与Control组相比,OGD/R组中miR-146b-5p表达显著下调(P<0.01),TRAF6蛋白表达显著上调(P<0.01)。与OGD/R+NC mimic组比较,OGD/R+miR-146b-5p mimic组细胞存活率显著增高,凋亡率显著降低,MDA含量显著降低,SOD、CAT含量显著增高,TNF-α,IL-1β和IL-6含量显著降低,含TRAF6-WT的荧光素酶活性显著降低,TRAF6蛋白表达显著降低(P<0.05或P<0.01)。与OGD/R+NC inhibitor组比较,OGD/R+miR-146b-5p inhibitor组细胞的存活率显著降低,凋亡率显著升高,MDA含量显著增高,SOD、CAT含量显著降低,TNF-α,IL-1β和IL-6含量显著升高,TRAF6蛋白表达显著增加(P<0.05或P<0.01)。结论:miR-146b-5p通过靶向TRAF6促进小胶质细胞OGD/R模型细胞存活,抑制细胞凋亡,减轻氧化应激和炎症反应。

Objective:To explore the mechanism of microRNA-146b-5p(miR-146b-5p)in microglia glucose deprivation/reoxygenation injury(OGD/R).Method:An OGD/R model of mouse microglia cell line EOC 13.31 was established,and miR-146b-5p mimic,miR-146b-5p inhibitor and corresponding controls were transfected into EOC 13.31 cells and then treated with OGD/R.The expression of miR-146b-5p was detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR);cell counting kit-8(CCK-8)was used to detect cell viability;in situ end labeling(TUNEL)was used to detect cell apoptosis rate;enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of superoxide substance dismutase(SOD),catalase(CAT),malondialdehyde(MDA),tumor necrosis factor-α(TNF-α),interleukin 1β(IL-1β)and interleukin 6(IL-6);Western blotting method was used to detect the expression of TRAF6 protein;bioinformatics and dual luciferase reporter gene experiments was used to analyze the relationship between miR-146b-5p and TRAF6.Results:Compared with the control group,the expression of miR-146b-5p in OGD/R was down-regulated(P<0.01),and the expression of TRAF6 protein was up-regulated(P<0.01).Compared with the OGD/R+NC mimic group,the survival rate of the cells in the OGD/R+miR-146b-5p mimic group was significantly increased(P<0.01),the apoptosis rate was significantly reduced(P<0.01),and the content of MDA was significantly reduced(P<0.05),the content of SOD and CAT were significantly increased(P<0.01),and the content of TNF-α,IL-1βand IL-6 were significantly decreased(P<0.05,P<0.01),the luciferase activity of TRAF6-WT was significantly reduced(P<0.01),and the expression of TRAF6 protein was significantly reduced(P<0.01).Compared with the OGD/R+NC inhibitor group,the survival rate of the cells in the OGD/R+miR-146b-5p inhibitor group was significantly reduced(P<0.01),the apoptosis rate was significantly increased(P<0.05),and the content of MDA was significantly increased(P<0.01),the content of SOD and CAT were significantly reduced(P<0.01,P<0.05),the content of TNF-α,IL-1βand IL-6 were significantly increased(P<0.01,P<0.05),the expression of TRAF6 protein was significantly increased(P<0.01),and the differences were statistically significant.Conclusion:miR-146b-5p can promote the survival rate of ODG model of microglial cells,inhibit cell apoptosis,and reduce oxidative stress and inflammatory response by targeting TRAF6.