二甲双胍通过ERK/p38 MAPK信号通路抑制软骨细胞损伤

Metformin inhibits chondrocyte injury via ERK/p38 MAPK signaling pathway

目的探讨二甲双胍(metformin,Met)能否通过ERK/p38 MAPK信号通路抑制白介素-1β(interleukin-1β,IL-1β)诱导的大鼠软骨细胞损伤。方法胰酶消化法分离出原代大鼠软骨细胞,贴壁培养至第3代备用。以浓度为10 ng/ml IL-1β刺激软骨细胞构建软骨细胞损伤模型,并以软骨细胞活力下降及凋亡增加作为模型构建成功的评判标准。用不同浓度的Met(0.01,0.1,1.0,10 mmol/L)分别对软骨细胞损伤模型进行干预,以CCK-8法筛选出Met的最适干预浓度,并以此浓度进行后续实验。取第3代软骨细胞按干预方式分为3组:control组(培养基作用24 h)、IL-1β组(IL-1β作用24 h)、IL-1β+Met组(先后用IL-1β和Met各作用24 h)。用流式细胞术检测细胞凋亡率,用Western blot检测细胞中IL-1β、COX-2、MMP-13、p-p38MAPK、p-ERK的蛋白表达。结果与control组比较,IL-1β组软骨细胞活力降低(P<0.001)、凋亡增加(P<0.001),说明造模成功。1.0 mmol/L的Met对软骨细胞损伤模型的干预效果最佳,故以1.0 mmol/L为最适干预浓度。与IL-1β组比较,IL-1β+Met组软骨细胞活力增高(P<0.01)、凋亡减少(P<0.05)。与control组比较,IL-1β组IL-1β、COX-2、MMP-13、p-p38MAPK和p-ERK蛋白表达增加(P<0.01);与IL-1β组比较,IL-1β+Met组IL-1β、COX-2、MMP-13、p-p38MAPK和p-ERK蛋白表达降低(P<0.05)。结论二甲双胍(Met)可抑制IL-1β诱导的软骨细胞损伤,其机制可能与ERK/p38 MAPK信号通路下调有关。

Objective To explore whether metformin can inhibit IL-1β-induced chondrocyte injury through the ERK/p38 MAPK signaling pathway.Methods Primary rat chondrocytes isolated by trypsin digestion were cultured to the third-generation by adherent culture method.The third generation chondrocytes were stimulated with IL-1βat the concentration of 10 ng/ml to establish the model of chondrocyte injury.The decrease of chondrocyte viability and the increase of chondrocyte apoptosis were used as the criteria of the successful model.The established chondrocyte injury models were interfered with different concentrations of metformin(0.01,0.1,1.0,10 mmol/L).The optimal concentration of metformin was selected by CCK-8 method for the following experiments.The third generation chondrocytes were divided into three groups according to the intervention mode.Chondrocytes in control group were exposed to medium for 24 h.Chondrocytes in IL-1βgroup were exposed to IL-1βsolution for 24 h.Chondrocytes in IL-1β+Met group were exposed to IL-1βsolution for 24 h and then metformin solution for another 24 h.The apoptosis rates were estimated by flow cytometry and the protein levels of IL-1β,COX-2,MMP-13,p-p38MAPK and p-ERK were examined by Western blot.Results Compared to control group,the chondrocyte vitality was reduced in IL-1βgroup(P<0.001),while the chondrocyte apoptosis was increased(P<0.001),indicating that the chondrocyte injury model was successfully built.The efficacy was best after intervence with 1.0 mmol/L Met,so 1.0 mmol/L metformin was selected as the optimal concentration for the following experiments.Compared to IL-1βgroup,chondrocyte vitality was increased in IL-1β+Met group(P<0.01),while chondrocyte apoptosis was reduced(P<0.05).Compared to control group,the protein levels of IL-1β,COX-2,MMP-13,p-p38MAPK and p-ERK in IL-1βgroup were increased(P<0.01).Compared to IL-1βgroup,the protein levels of IL-1β,COX-2,MMP-13,p-p38MAPK and p-ERK in IL-1β+Met group were reduced(P<0.05).Conclusion Metformin can inhibit the IL-1β-induced chondrocyte injury through the downregulation of ERK/p38 MAPK signaling pathway.