染料木素通过激活PI3K/AKT/mTOR信号通路抑制骨性关节炎兔软骨细胞退变

Genistein inhibits osteoarthritis rabbit chondrocyte degeneration by activating PI3K/AKT/mTOR signaling pathway

目的:探究染料木素(Gen)通过调节磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对骨性关节炎(OA)兔软骨细胞退变的影响。方法:将新西兰大白兔分为对照组和模型组,通过前交叉韧带离断术构建OA兔模型;HE染色及改良的Mankin评分法观察兔软骨组织病理学情况,分离培养软骨细胞,将其分为对照组、OA组、Gen-L组、Gen-H组、Gen-H+LY294002组,免疫组化法检测软骨细胞基质金属蛋白酶-13(MMP-13)及Ⅱ型胶原(COL2)表达;流式细胞仪检测细胞凋亡情况;Western Blot检测软骨组织MMP-13、COL2、B淋巴细胞瘤-2基因(Bcl-2)、Bax、Beclin-1、PI3K/AKT/mTOR通路蛋白表达。结果:对照组兔软骨组织未发现病理现象;模型组兔软骨组织表面染色不均、软骨细胞明显减少;与对照组相比,模型组兔软骨组织Mankin总评分、基质分布、纤维化、软骨细胞集落、软骨细胞缺失评分显著增加(P<0.05);与对照组相比,OA组软骨细胞COL2、Bcl-2、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR表达显著降低,细胞凋亡率、MMP-13、Bax表达显著增加(P<0.05);与OA组相比,Gen-L组、Gen-H组COL2、Bcl-2、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR表达显著增加,细胞凋亡率、MMP-13、Bax表达显著降低(P<0.05);与Gen-H组相比,Gen-H+LY294002组逆转上述指标变化。结论:Gen通过激活PI3K/AKT/mTOR通路来抑制OA兔软骨细胞退变。

Objective:To explore the effects of genistein(Gen)on the degeneration of chondrocytes in osteoarthritis(OA)rabbits by regulating the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signaling pathway.Methods:The New Zealand white rabbits were divided into a control group and a model group.Anterior cruciate ligament dissection was used to construct the OA rabbit model;HE staining and the modified Mankin scoring method were used to observe the pathology of rabbit cartilage tissue;the chondrocytes were isolated,cultured and divided into control group,OA group,Gen-L group,Gen-H group,and Gen-H+LY294002 group according to the treatment;immunohistochemical method was used to detect the expression of matrix metalloproteinase-13(MMP-13)and typeⅡcollagen(COL2)in chondrocytes;flow cytometry was used to detect cell apoptosis;Western Blot was used to detect the expression of MMP-13,COL2,B lymphocyte tumor-2 gene(Bcl-2),Bax,Beclin-1,and PI3K/AKT/mTOR pathway proteins in cartilage tissue.Results:No pathological changes were found in the cartilage tissue of the rabbits in the control group,while in the model group,the surface of cartilage tissue in rabbits was stained unevenly and chondrocytes were significantly reduced.Compared with those in the control group,the cartilage tissue Mankin total score,matrix distribution,fibrosis,chondrocyte colony,and chondrocyte loss score in the model group were significantly increased(P<0.05).Compared with those in the control group,the expression of chondrocytes COL2,Bcl-2,p-PI3K/PI3K,p-AKT/AKT,and p-mTOR/mTOR in the OA group was significantly reduced,and the apoptosis rate,expression of MMP-13 and Bax were significantly increased(P<0.05).Compared with those in the OA group,the expression of COL2,Bcl-2,p-PI3K/PI3K,p-AKT/AKT,and p-mTOR/mTOR in the Gen-L group and Gen-H group increased significantly,cell apoptosis rate,and expression of MMP-13 and Bax decreased significantly(P<0.05).Compared with Gen-H group,Gen-H+LY294002 group reversed the above indexes’changes.Conclusion:Gen can inhibit the degeneration of chondrocytes in OA rabbits by activating the PI3K/AKT/mTOR pathway.