千草方熏洗对膝骨关节炎大鼠软骨的保护作用及相关机制

Protective Effects of Qiancao Fang Fumigation on Cartilage in Rats with Knee Osteoarthritis and Related Mechanism

【目的】观察千草方熏洗对膝骨关节炎大鼠软骨蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路表达的影响,探讨千草方熏洗对膝骨关节炎大鼠软骨的保护作用及相关机制。【方法】将32只SD大鼠随机分成4组,即正常组、模型组、千草方组和透明质酸组,每组8只。除正常组,其他3组大鼠采用改良Hulth法构建膝骨关节炎模型。制模后,千草方组大鼠给予千草方熏洗,透明质酸组大鼠给予关节腔内注射透明质酸,模型组不给予任何治疗。治疗30 d。给药结束后,采用苏木素-伊红(HE)染色法和Masson染色法观察大鼠膝关节软骨组织,行Mankin’s评分,酶联免疫吸附分析(ELISA)检测血清白细胞介素(IL)-6、IL-10和肿瘤坏死因子α(TNF-α)的含量,免疫组织化学法检测软骨组织Ki67的表达,蛋白免疫印迹(Western Blot)法检测软骨组织Akt、磷酸化Akt(p-Akt)、mTOR、磷酸化mTOR(p-mTOR)、糖原合成酶激酶3β(GS3Kβ)、磷酸化GS3Kβ(p-GS3Kβ)和细胞周期蛋白D1(CyclinD1)的表达。【结果】与正常组比较,模型组大鼠膝关节软骨细胞排列紊乱,结构不清晰,Mankin’s评分升高(P <0.05),血清IL-10含量下降,IL-6和TNF-α含量升高(P <0.05),软骨组织Ki67表达量,p-Akt/Akt、p-mTOR/mTOR、p-GSK3β/GSK3β和CyclinD1的相对表达量均显著减少(P <0.05)。与模型组比较,千草方组大鼠膝关节软骨表面更加光滑,结构较清晰,软骨基质着色较均匀,Mankin’s评分降低(P <0.05),血清IL-10含量升高,IL-6和TNF-α含量降低(P <0.05),软骨组织p-Akt/Akt、p-mTOR/mTOR、p-GSK3β/GSK3β和CyclinD1的相对表达量显著增加(P <0.05)。【结论】千草方熏洗能够通过促进Akt/mTOR信号通路活化及软骨细胞增殖来缓解膝骨关节炎。

Objective To observe the effects of Qiancao Fang fumigation on protein kinase B(Akt)/mammalian target of rapamycin(mTOR)pathway in cartilage from rats with knee osteoarthritis(KOA),so as to explore the protective effects of Qiancao Fang fumigation on cartilage of knee osteoarthritis rats and the related mechanisms.Methods Thirty-two SD rats were randomly divided into 4 groups,namely normal group,model group,Qiancao Fang group and hyaluronic acid(HA)group,8 rats in each group. Apart from the normal group,the rats in the other groups were induced into KOA model by the improved Hulth method. After modeling,Qiancao Fang group was given fumigation of Qiancao Fang,and HA group was given intra-articular injection of HA,the model group had no treatment. The treatment lasted 30 days. After medication,the pathological features of cartilage tissue were observed by haematoxylin-eosin(HE)and Masson staining methods,and Mankin’s scoring was carried out,the serum levels of interleukin-6(IL)-6,IL-10 and tumor necrosis factor α(TNF-α)were detected by enzyme linked immunosorbent assay(ELISA),the expression level of Ki67 in cartilage tissue was detected by immunohistochemistry,and the expression levels of Akt,phosphorylated Akt(p-Akt),mTOR,phosphorylated mTOR(p-mTOR),glycogen synthase kinase-3 beta(GSK3β),phosphorylated GSK3β(p-GSK3β)and CyclinD1 in cartilage tissue were detected by Western blotting assay. Results Compared with the normal group,the cartilage cells in knee joint were disordered and cartilage structure were not clear in the model group,the Mankin’s scores were enhanced(P <0.05),serum IL-10 content was decreased,serum IL-6 and TNF-α contents were increased(P < 0.05),and the expression level of Ki67 and relative expression levels of p-Akt/Akt,p-mTOR/mTOR,p-GSK3β/GSK3β and CyclinD1 in cartilage tissue were lowered(P < 0.05). Compared with the model group,the cartilage surface of knee joint was smoother,the cartilage structure was clearer,and the cartilage matrix was evenly colored in the Qiancao Fang group,the Mankin’s scores were decreased,the serum IL-10 content was increased,the serum IL-6 and TNF-α contents were significantly decreased(P < 0.05),and the expression level of Ki67 and relative expression levels of p-Akt/Akt, p-mTOR/mTOR, p-GSK3β/GSK3β and CyclinD1 in cartilage tissue were raised(P < 0.05). Conclusion Qiancao Fang has effects on relieving KOA through promoting activation of Akt/mTOR pathway and proliferation of cartilage cells.