Metformin exerts a protective effect on articular cartilage in osteoarthritis rats by inhibiting the PI3K/AKT/mTOR pathway

doi:10.12307/2025.282

Abstract

Abstract: BACKGROUND: Studies have shown that metformin has anti-inflammatory, anti-tumor, anti-aging and vasoprotective effects, and can inhibit the progression of osteoarthritis, but its specific mechanism of action remains unclear.
OBJECTIVE: To investigate the mechanism of metformin on cartilage protection in a rat model of osteoarthritis.
METHODS: Forty male Sprague-Dawley rats were randomly divided into four groups (n=10 per group): blank, control, sham-operated, and metformin groups. The blank group did not undergo any surgery. In the sham-operated group, the joint cavity was exposed. In the model group and the metformin group, the modified Hulth method was used to establish the osteoarthritis model. At 1 day after modeling, the rats in the metformin group were given 200 mg/kg/d
metformin by gavage, and the model, blank, and sham-operated groups were given normal saline by gavage. Administration in each group was given for 4 weeks consecutively. Hematoxylin-eosin staining, toluidine blue staining, and safranin O-fast green staining were used to observe the morphological structure of rat knee joints. Immunohistochemical staining and western blot were used to detect the protein expression of SOX9, type II collagen, a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), Beclin1, P62, phosphatidylinositol 3-kinase (PI3K), p-PI3K, protein kinase B (AKT), p-AKT, mammalian target of rapamycin (mTOR), and p-mTOR in rat cartilage tissue.
RESULTS AND CONCLUSION: The results of hematoxylin-eosin, toluidine blue and safranin O-fast green staining showed smooth cartilage surface of the knee joints and normal histomorphology in the blank group and the sham-operated group, while in the model group, there was irregular cartilage surface of the knee joint and cartilage damage, with a decrease in the number of chondrocytes and the content of proteoglycans in the cartilage matrix. In the metformin group, there was a significant improvement in the damage to the structure of the cartilage in the knee joints of the rats, and the cartilage surface tended to be smooth, with an increase in the number of chondrocytes and the content of proteoglycans in the cartilage matrix. Immunohistochemistry staining and western blot results showed that compared with the control and sham-operated groups, the expression of SOX9, type II collagen, and Beclin1 proteins in the cartilage tissue of rats in the model group was significantly decreased (P < 0.05). Conversely, the expression of ADAMTS5, P62, as well as p-PI3K, p-AKT, and p-mTOR proteins was significantly increased (P < 0.05). Furthermore, compared with the model group, the expression of SOX9, type II collagen, and Beclin1 proteins in the cartilage tissue of rats in the metformin group was significantly increased (P < 0.05), while the expression of ADAMTS5, P62, as well as p-PI3K, p-AKT, and p-mTOR proteins was significantly decreased (P < 0.05). To conclude, Metformin can improve the autophagy activity of chondrocytes and reduce the degradation of cartilage matrix in osteoarthritis rats by inhibiting the activation of PI3K/AKT/mTOR signaling pathway, thus exerting a protective effect on articular cartilage.

Key words: osteoarthritis, articular cartilage, autophagy, metformin, PI3K/AKT/mTOR signaling pathway

CLC Number:

Xu Tianjie, Fan Jiaxin, Guo Xiaoling, Jia Xiang, Zhao Xingwang, Liu kainan, Wang Qian. Metformin exerts a protective effect on articular cartilage in osteoarthritis rats by inhibiting the PI3K/AKT/mTOR pathway [J]. Chinese Journal of Tissue Engineering Research, 2025, 29(5): 1003-1012.

Figures/Tables 12

2.1 实验动物数量分析 40只SD大鼠全部进入结果分析。 2.2 各组大鼠膝关节软骨病理观形态察结果见图1。苏木精-伊红染色结果显示，空白组软骨表面光滑，组织形态正常，软骨细胞单个排列，假手术组软骨形态与空白组无明显差别；模型组软骨表面不规则，软骨组织出现缺损，软骨层变薄，软骨细胞呈簇状分布且细胞数量减少；二甲双胍组软骨表面光滑，软骨层明显增厚，软骨细胞数量增多且排列整齐。甲苯胺蓝染色结果显示，空白组和假手术组软骨细胞可见蓝紫色的细胞核，软骨基质中的蛋白多糖染色均匀，呈蓝色或蓝紫色，潮线完整；模型组软骨细胞数量减少，软骨基质中的蛋白多糖染色较浅，潮线不完整；相较于模型组，二甲双胍组软骨细胞数量增多，软骨基质染色深染，但略浅于空白组和假手术组。番红O-固绿染色结果显示，空白组和假手术组软骨细胞可见蓝色的细胞核，软骨基质染色均匀，呈红色，潮线完整；模型组软骨细胞数量减少，部分软骨组织番红染色较浅，潮线不完整；二甲双胍组软骨细胞数量增多，潮线完整，软骨基质较模型组深染，但仍浅于空白组和假手术组。空白组、假手术组、模型组与二甲双胍组大鼠膝关节软骨Mankin评分分别为0，0.33±0.50，5.67±1.12，1.56±0.73，空白组与假手术组Mankin评分比较差异无显著性意义(P > 0.05)，二甲双胍组、模型组Mankin评分高于空白组和假手术组(P < 0.05)，二甲双胍组Mankin评分低于模型组(P < 0.05)。 2.3 各组大鼠膝关节软骨组织中ADAMTS5表达免疫组化染色结果显示，各组大鼠膝关节软骨组织软骨细胞内均有不同程度ADAMTD5蛋白表达，可见棕黄色颗粒分布于细胞质内。与空白组比较，假手术组ADAMTS5阳性细胞数量与染色程度无明显差异；与空白组和假手术组比较，模型组ADAMTS5阳性细胞数量明显增多且染色较深；与模型组比较，二甲双胍组ADAMTS5阳性细胞数量减少且染色稍浅，见图2A。免疫组化染色平均吸光度值分析结果显示，假手术组大鼠软骨组织中ADAMTS5蛋白阳性表达与空白组比较无明显差异(P > 0.05)；与空白组和假手术组比较，模型组大鼠软骨组织中ADAMTD5蛋白阳性表达增多(P < 0.01)；与模型组比较，二甲双胍组大鼠软骨组织中ADAMTS5蛋白阳性表达降低(P < 0.01)，见图2B。 Western blotting检测结果显示，与空白组比较，假手术组大鼠软骨组织中ADAMTS5蛋白表达无明显差异(P > 0.05)；与空白组和假手术组比较，模型组大鼠软骨组织中ADAMTD5表达升高(P < 0.001，P < 0.05)；与模型组比较，二甲双胍组大鼠软骨组织中ADAMTS5蛋白表达降低(P < 0.001)，见图3。Western blotting检测结果与免疫组化学染色结果一致。 2.4 各组骨关节炎大鼠软骨组织中SOX9和Ⅱ型胶原表达免疫组化染色结果显示，各组软骨组织软骨细胞内均有不同程度SOX9和Ⅱ型胶原蛋白表达，SOX9阳性表达为棕黄色颗粒分布于细胞核，Ⅱ型胶原阳性表达为棕黄色颗粒分布于细胞质。与空白组比较，假手术组SOX9和Ⅱ型胶原阳性细胞数量与染色程度无明显差异；与对照组和假手术组比较，模型组SOX9与Ⅱ型胶原阳性细胞数量减少且染色较浅；与模型组比较，二甲双胍组SOX9与Ⅱ型胶原阳性细胞数量增多，染色较深，见图4A。免疫组化染色平均吸光度值分析结果显示，假手术组软骨组织中SOX9和Ⅱ型胶原蛋白表达与空白组无明显差异(P > 0.05)；与空白组和假手术组比较，模型组软骨组织中SOX9和Ⅱ型胶原蛋白表达降低(P < 0.01或P < 0.001)；与模型组比较，二甲双胍组软骨组织中SOX9和Ⅱ型胶原蛋白表达升高 (P < 0.05，P < 0.001)，见图4B。 Western blotting检测结果显示，假手术组与空白组软骨组织中SOX9和Ⅱ型胶原蛋白表达无明显差异(P > 0.05)；与空白组和假手术组比较，模型组软骨组织中SOX9和Ⅱ型胶原蛋白表达降低(P < 0.01或P < 0.001)；与模型组比较，二甲双胍组软骨组织中SOX9和Ⅱ型胶原蛋白升高(P < 0.05，P < 0.01)，但仍低于空白组(P < 0.05)，见图5。Western blotting检测结果与免疫组化染色结果一致。 2.5 各组大鼠膝关节软骨组织中P62、Beclin1表达免疫组化染色结果显示，各组软骨组织软骨细胞内均有不同程度的P62和Beclin1蛋白表达，P62与Beclin1阳性表达均呈棕黄色分布于细胞质内。与空白组比较，假手术组P62与Beclin1阳性细胞数量与染色程度无明显差异；与空白组和假手术组比较，模型组P62阳性细胞数量增多且染色较深，Beclin1阳性细胞数量减少且染色较浅；与模型组比较，二甲双胍组P62阳性细胞数量减少且染色较浅，Beclin1阳性细胞数量增多且染色较染，见图6A。免疫组化染色图平均吸光度值分析分析显示，空白组软骨组织中P62、Beclin1蛋白表达与假手术组无明显差异(P > 0.05)；与空白组和假手术组比较，模型组软骨组织中P62蛋白表达升高(P < 0.01)，Beclin1蛋白表达降低(P < 0.001)；与模型组比较，二甲双胍组软骨组织中P62蛋白表达降低 (P < 0.05)，Beclin1蛋白表达升高(P < 0.05)，见图6B。 Western blotting检测结果显示，空白组软骨组织中P62、Beclin1蛋白表达与假手术组无明显差异(P > 0.05)；与空白组和假手术组比较，模型组软骨组织中P62蛋白表达升高(P < 0.05)，Beclin1蛋白表达降低(P < 0.001)；与模型组比较，二甲双胍组软骨组织中P62蛋白表达降低(P < 0.05)，Beclin1蛋白表达升高(P < 0.05)，见图7。Western blotting检测结果与免疫组化染色结果一致。 2.6 各组大鼠膝关节软骨组织中PI3K/AKT/mTOR信号通路蛋白表达免疫组化染色结果显示，各组软骨组织软骨细胞内均有不同程度的PI3K、p-PI3K、AKT、p-AKT、mTOR、p-mTOR蛋白表达，各蛋白阳性表达可见棕黄色颗粒分布于细胞质。各组PI3K、AKT、mTOR阳性细胞数量与染色程度无明显差异；与空白组比较，假手术组p-PI3K、p-AKT、p-mTOR阳性细胞数量与染色程度无明显差异；与空白组和假手术组比较，模型组p-PI3K、p-AKT、p-mTOR阳性细胞数量增多且染色较深；与模型组比较，二甲双胍组p-PI3K、p-AKT、p-mTOR阳性细胞数量减少且染色较浅，见图8A。免疫组化染色染色平均吸光度值定量分析结果显示，假手术组p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR蛋白平均吸光度比值与空白组比较差异无显著性意义(P > 0.05)；与空白组和假手术组比较，模型组p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR蛋白平均吸光度比值升高(P < 0.01或P < 0.001)；与模型组比较，二甲双胍组p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR蛋白平均吸光度比值降低(P < 0.05)，见图8B。 Western blotting检测结果显示，各组PI3K、AKT、mTOR蛋白表达比较差异无显著性意义(P > 0.05)；假手术组p-PI3K、p-AKT、p-mTOR蛋白表达与空白组比较差异无显著性意义(P > 0.05)；与空白组和假手术组比较，模型组p-PI3K、p-AKT、p-mTOR蛋白表达升高(P < 0.05，或P < 0.01或P < 0.001)；与模型组比较，二甲双胍组p-PI3K、p-AKT、p-mTOR蛋白表达降低(P < 0.05)，见图9。 "

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