二甲双胍通过Wnt/β-catenin信号通路对U251细胞氧糖剥夺/复糖复氧损伤的作用机制研究

Mechanism of Metformin on Oxygen-glucose Deprivation/reoxygenation-induced U251 Cells Injury through Wnt/β-catenin Signaling Pathway

目的探讨二甲双胍对U251细胞氧糖剥夺/复糖复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)损伤的作用机制。方法用OGD/R后的U251细胞模型来模拟人脑缺血再灌注损伤。将U251细胞分为6组,即空白对照组、模型组、二甲双胍中剂量组、二甲双胍高剂量组、激动剂组(Wnt3a,Wnt/β-catenin信号通路激动剂)、抑制剂组(二甲双胍+XAV939,Wnt/β-catenin信号通路抑制剂)。除空白对照组外,其余各组细胞均予以氧糖剥夺/复糖复氧2h后再灌注24h处理,建立OGD/R模型,造模前24h动物均予以二甲双胍、Wnt3a和XAV939处理。采用CCK-8法和乳酸脱氢酶(lactate dehydrogenase,LDH)实验检测细胞活力和毒性,DHE染色检测细胞ROS形成情况,采用酶联免疫吸附试验(enzyme-linked immunoadsordent assay,ELISA)检测细胞中谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)、白细胞介素-6(interleukin-6,IL-6)、诱生型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平,Western blot法检测细胞β-catenin、cyclin D1、p-GSK-3β(Ser9)及GSK-3β蛋白表达水平。结果与空白对照组比较,模型组、二甲双胍组、激动剂组及抑制剂组的LDH、ROS、MDA、IL-6、iNOS和TNF-α水平明显升高,SOD、GSH-Px、β-catenin、cyclin D1、p-GSK-3β(Ser9)蛋白相对表达量和细胞活力明显下降。与模型组比较,二甲双胍组、激动剂组的LDH、ROS、MDA、IL-6、iNOS和TNF-α水平明显下降,SOD、GSH-Px、β-catenin、cyclin D1、p-GSK-3β(Ser9)蛋白相对表达量和细胞活力明显升高。与二甲双胍组比较,抑制剂组的LDH、ROS、MDA、IL-6、iNOS和TNF-α水平明显升高,SOD、GSH-Px、β-catenin、cyclin D1、p-GSK-3β(Ser9)蛋白相对表达量和细胞活力明显下降。结论二甲双胍通过Wnt/β-catenin信号通路对OGD/R的U251细胞炎症和氧化应激,从而发挥神经保护作用。

Objective To investigate the mechanism of metformin on oxygen-glucose deprivation/reoxygenation(OGD/R)injury in U251 cells.Methods Human glioma cell line U251 cells were cultured and divided into 6groups:blank control group,model group,metformin medium dose group,metformin high dose group,agonist group(Wnt3a,Wnt/β-catenin signaling pathway agonist),inhibitor group(metformin+XAV939,Wnt/β-catenin signaling pathway inhibitor).Except for the blank control group,the cells in the other groups were subjected to OGD/R for 2h and then reperfusion for 24h to establish the OGD/R model.The animals were treated with metformin,Wnt3a and XAV93924h before modeling.Cell viability and toxicity were detected by CCK-8method and lactate dehydrogenase(LDH)assay.ROS formation was detected by DHE staining.Glutathione peroxidase(GSH-Px),superoxide dismutase(SOD)malondialdehyde(MDA),interleukin-6(IL-6),inducible nitric oxide synthase(iNOS)and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunoadsordent assay(ELISA).The protein expression levels ofβ-catenin,cyclin D1,p-GSK-3β(Ser9)and GSK-3βwere detected by Western blot.Results Compared with blank control group,LDH,ROS,MDA,IL-6,iNOS and TNF-αin model group,metformin group,agonist group and inhibitor group were significantly increase.The relative expression levels of SOD,GSH-Px,β-catenin,cyclin D1,p-GSK-3β(Ser9)and cell viability were significantly decreased.Compared with model group,the levels of LDH,ROS,MDA,IL-6,iNOS and TNF-αin metformin group and agonist group were significantly decreased,while the relative expression levels of SOD,GSH-Px,β-catenin,cyclin D1,p-GSK-3β(Ser9)and the cell viability were significantly increased.Compared with metformin group,LDH,ROS,MDA,IL-6,iNOS and TNF-αin metformin group and inhibitor group were significantly increase,the relative expression levels of SOD,GSH-Px,β-catenin,cyclin D1,p-GSK-3β(Ser9)and the cell viability were significantly decreased.Conclusion Metformin may play a protective role in OGD/R of U251 cells through Wnt/β-catenin signaling pathway.