二甲双胍通过AMPK信号通路对小鼠骨关节炎保护作用的研究

Metformin protects osteoarthritis in mice by regulating the AMPK signaling pathways

目的研究二甲双胍对小鼠骨关节炎及软骨细胞分化的影响。方法切除小鼠右膝关节的内侧半月板和前交叉韧带造模骨关节炎,造模成功后,随机分为2组,实验组给予二甲双胍(2mg/kg/d)而对照组予等量的生理盐水灌胃干预治疗8周,通过HE、番红O-快速绿切片染色和Micro-CT观察关节结构变化,进行Mankin评分评估关节炎严重程度,通过Western Blot检测各组关节软骨AMPK、mTOR的活性及自噬程度;取新生小鼠肋软骨和膝关节软骨进行细胞培养,随机分为实验组(2mmol/L的二甲双胍)和空白对照组,观察软骨细胞形态、数量,MTT法检测细胞活性,Western Blot检测软骨细胞AMPK、mTOR的活性及自噬程度。结果 (1)成功构建了小鼠骨关节炎模型,HE、番红O-快速绿染色、MicroCT结果及Mankin评分证明二甲双胍能保护骨关节炎;(2)关节软骨Western Blot结果显示二甲双胍能激活AMPK,增强自噬保护骨关节炎;(3)MTT法显示二甲双胍抑制软骨细胞增殖分化,Western Blot结果显示二甲双胍促进软骨细胞自噬。结论二甲双胍通过激活AMPK信号通路,抑制mTOR信号通路,促进软骨细胞自噬,发挥保护骨关节炎作用。

Objective Study the effect of metformin on osteoarthritis and chondrocyte differentiation in mice. Methods Mice were divided into a control group and an experimental group whose anterior cruciate ligament and medial meniscus excision were done in the right knee of both groups for arthritis model; Aftersuccess of model establishment, the experimental group was given metformin （2mg/kg/d） and the control group was given saline as intervention treatment for 8 weeks; Then through the HE, sarranine O-fast green slice staining and Micro-CT structural change was observed and grading was performed according to Mankin ratings; The AMPK, mTOR activity and degree of autophagy of articular cartilage in two groups were tested by Western Blot; Chondrocytes were cultivated in vitro and stimulated by metformin to observe the cell morphology, and amount. The cell activity was determined by MTT method .The cell activity of AMPK, mTOR and autophagy were detected by Western Blot. Results （1）We succeeded in establishing the model of osteoarthritis in mice and found metformin could protect the osteoarthritis by Micro - CT, HE and sarranine O-fast green slice staining and Mankin ratings. （2） Western Blot results showed metformin activated AMPK and enhanced autophagy to protect osteoarthritis. （3） MTT method showed metformin inhibited Chondrocytes differentiation,and Western Blot results showed metformin promoted chondrocytes autophagy. Conclusions Metformin activates AMPK signaling pathways, inhibits mTOR signaling pathways, protecting chondrocyte autophagy,thus protects osteoarthritis in mice.