盘龙七片对膝骨关节炎大鼠关节软骨的保护作用及对Wnt通路的调控机制

Protective effect of Panlongqi Tablet on articular cartilage of knee osteoarthritis rats and its regulatory mechanism on Wnt pathway

目的探讨盘龙七片对膝骨关节炎(KOA)大鼠关节软骨的保护作用及对Wnt通路的调控机制。方法选用96只SPF级8周龄雄性SD大鼠构建膝骨关节炎大鼠模型,观察大鼠活动状态,并经CT验证。实验分为假手术组、模型组、塞来昔布组和低、中、高剂量盘龙七片组6组,每组16只。按照实验分组给药,观察各组大鼠软骨形态并进行大体评分,行HE染色,酶联免疫吸附(ELISA)法测定大鼠滑膜中TNF-α、IL-1β及MMP-13水平,蛋白印迹(WB)法测定软骨中Wnt-4、β-catenin水平。结果手术后,大鼠出现膝骨关节炎症状并经CT验证。与假手术组相比,模型组大鼠膝骨关节可见裂纹,表面呈毛刺状、暗黄色,软骨可见增生、赘生物;HE染色可见软骨细胞数减少,骨赘、血管增生,结构混乱不清,表面凹陷,深层结缔组织可见明显裂隙,潮线消失;大体评分、TNF-α、IL-1β、MMP-13、Wnt-4及β-catenin水平显著升高(均P<0.05)。与模型组相比,经低、中、高剂量盘龙七片治疗后,大鼠膝骨关节软骨表面趋向光滑,色泽逐渐明亮,增生、赘生物减少;HE染色可见软骨细胞数逐渐增多,结构趋向分明,裂隙减少,伴有少量炎症细胞浸润;大体评分、TNF-α、IL-1β、MMP-13、Wnt-4及β-catenin水平明显降低(均P<0.05)。结论盘龙七片可缓解KOA大鼠症状,具有一定的临床应用价值,这提示盘龙七片可能成为治疗KOA的候选药物。

Objective To study the protective effect of Panlongqi Tablet on articular cartilage of knee osteoarthritis(KOA)rats and its regulatory mechanism of Wnt pathway.Methods The rat model of knee osteoarthritis was established.The activity of rats was observed and verified by CT.The experiment was divided into sham operation group,model group,celecoxib group and low,medium and high dose Panlongqi Tablet groups.The drug was administered according to the experimental group.The cartilage morphology of rats in each group was observed and scored.HE staining was performed.The levels of TNF-α,IL-1β and MMP-13 in synovium of rats were measured by enzyme-linked immunosorbent assay(ELISA),and Wnt-4 andβ-Catenin in cartilage were measured by Western blot.Results The symptoms of knee osteoarthritis appeared in the rats after operation and were verified by CT.Compared with the sham operation group,the model group had cracks on the knee joints,with burr like and dark yellow surface,hyperplasia and vegetations on the cartilage.HE staining showed that the number of chondrocytes decreased,osteophytes and blood vessels proliferated,the structure was confused and unclear,the surface was sunken,the deep connective tissue showed obvious cracks,and the tide line disappeared.The general score,TNF-α level,IL-1β level,MMP-13 level,Wnt-4 level and β-catenin level was significantly increased(P<0.05).Compared with the model group,after the treatment of low,medium and high doses of Panlongqi Tablets,the cartilage surface of the knee joint of the rats tended to be smooth,bright,with less proliferation and vegetation.HE staining showed that the number of chondrocytes increased gradually,the structure tended to be clear,the fissures decreased.Accompanied by a small number of inflammatory cell infiltration,the general score,TNF-α level,IL-1β level,MMP-13 level,Wnt-4 level and β-catenin level decreased significantly(P<0.05).Conclusion Panlongqi Tablet may alleviate the symptoms of knee osteoarthritis rats by inhibiting Wnt pathway activity,so as to protect articular cartilage.