基于Wnt-Notch信号探讨苗药五藤膏治疗膝骨关节炎的分子机制

The pathogenesis of knee osteoarthritis(KOA) is not entirely clear. The latest domestic and foreign study have confirmed that Wnt and Notch signal play an important role in KOA chondrocyte differentiation, proliferation and apoptosis, and also have important significance in the maintenance of chondrocyte phenotype and substrate metabolism,the abnormal activation of signal pathway may be the key factor lead to the imbalance of chondrocyte metabolism and the formation of KOA. This topic based on the Miao medicine theory of external therapy“choking for disease,dredging for health”,put forward the therapy of “dredging sinew and scattering blood”,have composed the Wuteng Plaster which mainly includes local Miao medicine,targeted for Notch-Wnt signal to research the effect of Wuteng plaster for the expression level of Wnt signal upstream and downstream gene,Notch receptors and corresponding gametes in KOA model chondrocyte,to discuss the molecular mechanism of Miao medicine Wuteng plaster in regulating the related genes of Wnt-Notch signal in KOA chondrocyte.for this, We based on the previous studies,propose the scientific hypothesis that Wnt and Notch signal may be the key regulatory factors in the KOA chondrocyte lesion,Miao medicine Wuteng plaster maybe maintain the balance of chondrocyte differentiation, proliferation and apoptosis by regulating and controlling the expression level of related genes and protein in Wnt and Notch signal in KOA chondrocyte,this hypothesis has solid theoretical basis and sufficient research foundation.

膝骨关节炎（KOA）的发病机制尚不完全清楚。据最新的国内外研究证实，Wnt和Notch信号在KOA软骨细胞分化、增殖和凋亡、维持软骨细胞表型及基质代谢等方面发挥重要角色，其信号通路的异常激活可能是软骨细胞代谢失衡和KOA形成的关键因素。本课题根据苗医学“壅塞为病，通达为康”的外治理论，提出“通筋散血”的治疗方法，以贵州苗药为主组成五藤膏，以Notch-Wnt信号为靶向研究五藤膏对KOA模型软骨细胞Wnt上下游基因和Notch受体与相应配子的表达水平，探讨苗药五藤膏调节KOA软骨细胞Wnt-Notch信号相关基因的分子机制。为此，我们在前期研究的基础上，提出Wnt和Notch信号为KOA软骨细胞病变的关键调节因子，苗药五藤膏可能通过调控KOA软骨细胞Wnt和Notch信号相关基因和蛋白的表达水平，从而维持KOA软骨细胞分化、增殖与凋亡平衡的科学假说，具有坚实的理论依据和充分的研究基础。

膝骨关节炎（KOA）发病机制尚未明确，大量研究证实Wnt-Notch信号与KOA发病密切相关。通过前期临床试验，我们发现中医药外治KOA疗效确切，本研究拟通过Wnt-Notch信号探讨中医药治疗KOA的分子机制。实验以兔KOA模型为研究对象，分为空白组、模型组、巴布膏组和五藤膏低、中、高剂量组，于用药1、2、3、4w分批处死。病理切片示空白组软骨表面平整，细胞量较多，分布均匀，排列整齐，潮线完整；模型组软骨表面不规则，潮线识别度差，细胞量少，排列紊乱；五藤膏高剂量组软骨细胞量较多，排列整齐，染色情况较好，潮线识别度较高；五藤膏低、中剂量组及巴布膏组层次界限较模糊，细胞量较少，局部存在聚集，分布散乱，个别潮线不完整。PCR法测Wnt3a、Wnt5a、Wnt7a、OPG、RANK、RANKL、MMP-3、MMP-13、c-myc mRNA、Notch1、Notch4表达量均降低，其表达量空白组<五藤膏高剂量组<巴布膏组<五藤膏中、低剂量组<模型组。WB法测β-catenin、GSK-3β、Notch1、Notch4、SMAD1、SMAD5蛋白表达均升高，其表达量空白组>巴布膏组>五藤膏高、中、低剂量组>模型组。ELISA法测Jagged1、Jagged2、DLL1表达量均降低，其表达量空白组<巴布膏组<五藤膏高剂量组、中剂量、低剂量组<模型组。以上研究结果表明苗药五藤膏可能通过调控Wnt-Notch信号，促进KOA模型兔软骨细胞β-catenin、GSK-3β、Notch1、Notch4、SMAD1、SMAD5蛋白表达，抑制Wnt3a、Wnt5a、Wnt7a、OPG、RANK、RANKL、MMP-3、MMP-13、c-myc mRNA、Notch1、Notch4、Jagged1、Jagged2、DLL1表达，延缓KOA的病程进展，从而达到改善及治疗作用。