基于Wnt/β-catenin信号途径探讨健骨方对骨关节炎动物模型软骨保护效应研究

Exploration on Protective Effect of Jiangu Formula(健骨方)on Cartilage of Osteoarthritis Animal Model Based on Wnt/β-catenin Signal Pathway

目的探究健骨方通过调节Wntβ-catenin信号途径在骨关节炎(osteoarthritis,OA)动物模型的作用机制。方法36只大鼠随机分为Sham组、OA组和OA+健骨方组(n=12)。OA组和OA+健骨方组大鼠关节腔内注射碘乙酸钠制作大鼠OV模型,OA+健骨方组使用健骨方灌胃。比较各组大鼠膝关节水肿和承重情况,分别通过甲苯胺蓝染色和番红O染色检测骨关节病变和软骨损伤。利用酶联免疫吸附法检测血清炎性细胞因子。分别通过qPCR和Western Blot检测Wntβ-catenin信号通路。结果3组的各项指标差异显著(P<0.05)。其中OA组的膝盖水肿[(3.82±0.45)mm)]和膝关节承不对称性[(22.63±3.06)g]显著高于Sham组(P<0.05)。OA+健骨方组的膝盖水肿[(2.97±0.40)mm]和膝关节承不对称性[(15.32±2.55)g]显著低于OA组(P<0.05)]。OA模型大鼠骨关节表面不光滑,并且出现严重的炎性浸润。与OA组比较,OA+健骨方组的炎性浸润和损伤情况均有所缓解。对照组关节表面光滑,番红素O均匀分布,软骨细胞排列规则。OA组番红蛋白O染色明显减少,细胞间基质丢失以及软骨层变薄或破坏。OA+健骨方组的组番红蛋白O染色比OA组多,可观察到软骨表面粗糙,基质染色强度降低但高于OA组。OA组的白细胞介素(interleukin,IL)-1β[(28.62±3.75)pg/mL]、IL-6[(16.54±3.21)pg/mL]和肿瘤坏死因子(tumor necrosis factor,TNF)-α[(21.44±3.54)pg/mL]显著高于Sham组(P<0.05)。OA+健骨方组的IL-1β[(11.67±3.01)pg/mL]、IL-6[(10.89±2.45)pg/mL]和TNF-α[(14.87±3.12)pg/mL]显著低于OA组(P<0.05)。OA组的Wnt和β-catenin mRNA和蛋白的水平显著高于Sham组(P<0.05),OA+健骨方组的Wnt和β-catenin mRNA和蛋白的水平显著低于OA组(P<0.05)。结论健骨方可以通过抑制Wnt/β-catenin通路抑制炎性反应,从而缓解软骨损伤,减轻OA大鼠模型的症状。

Objective To explore the mechanism of Jiangu Formula(健骨方)in osteoarthritis(OA)animal models by regulating Wntβ-catenin signaling pathway.Methods Thirty-six rats were randomly divided into Sham group,OA group and OA+Jiangu Formula group(n=12).Rats in OA group and OA+Jiangu Formula group were injected with sodium iodoacetate into the joint cavity to make rat OV model.In the OA+Jiangu Formula group,the Jiangu Formula was given by gavage.The edema and weight bearing of the knee joint of each group were compared.Toluidine blue staining and Safranin O staining were used to detect bone and joint disease and cartilage damage.Enzyme-linked immunosorbent assay was used to detect serum inflammatory cytokines.The Wntβ-catenin signaling pathway was detected by qPCR and Western blot respectively.Results The indicators of the three groups were significantly different(P<0.05).The knee edema[(3.82±0.45)mm]and knee joint bearing asymmetry[(22.63±3.06)g]in the OA group were significantly higher than those in the Sham group(P<0.05).The knee edema[(2.97±0.40)mm]and knee joint bearing asymmetry[(15.32±2.55)g]in the OA+Jiangu Formula group were significantly lower than those in the OA group(P<0.05).The surface of bone joints in OA model rats was not smooth,and severe inflammatory infiltration appeared.Compared with those of the OA group,the inflammatory infiltration and injury of the OA+Jiangu Formula group were all relieved.The joint surface of the control group was smooth,lysoene O was evenly distributed,and chondrocytes were arranged regularly.In the OA group,the staining of saffron O was significantly reduced,the intercellular matrix was lost,and the cartilage layer was thinned or destroyed.The OA+Jiangu Formula group had more safranine O staining than the OA group.The cartilage surface was rough and the matrix staining intensity was decreased but higher than that of the OA group.Interleukin(IL)-1β[(28.62±3.75)pg/mL],IL-6[(16.54±3.21)pg/mL]and tumor necrosis factor(TNF)-α[(21.44±3.54)pg/mL]in OA group was significantly higher than that in the Sham group(P<0.05).IL-1β[(11.67±3.01)pg/mL],IL-6[(10.89±2.45)pg/mL]and TNF-α[(14.87±3.12)pg/mL]in the OA+Jiangu Formula group were significantly lower than those in the OA group(P<0.05).The levels of Wnt andβ-catenin mRNA and protein in the OA group were significantly higher than those in the Sham group(P<0.05).The levels of Wnt andβ-catenin mRNA and protein in the OA+Jiangu Formula group were significantly lower than those in the OA group(P<0.05).Conclusion Jiangu Formula can inhibit the inflammatory response by inhibiting the Wnt/β-catenin pathway,thereby alleviating cartilage damage and reducing the symptoms of OA rat models.