Objective: To observe the effect of Miao medicinal acupuncture therapy on transient receptor potential vanilloid (TRPV) channel in knee joint synovial tissue of the rabbits with knee osteoarthritis (KOA) model and to explore the mechanism of Miao medicinal acupuncture therapy in treatment of KOA.
Methods: Of 34 New Zealand male rabbits, 6 rabbits were selected randomly as the normal group. KOA model was established in the rest rabbits by injecting a mixture of papain and L-cysteine in right knee joints. The 24 successfully modeled rabbits were randomized into a model group, a Miao medicinal acupuncture therapy group, a dermal needle group and a smearing group, 6 rabbits in each one. In the Miao medicinal acupuncture therapy group, Miao medicinal acupuncture therapy was adopted, in which, the roller type of dermal needle was used on the surface of right knee joint [a rectangle shape formed by "Xuehai" (SP 10), "Liangqiu" (ST 34), "Yanglingquan" (GB 34) and "Yinlingquan" (SP 9)], rolling in a "" shape, on which, Miao medicinal solution was smeared in advance. In the dermal needle group, the rolling stimulation was exerted on the right the right knee joint surface with the roller type of dermal needle. In the smearing group, Miao medicinal solution was smeared on the right knee joint surface. The intervention was given once every two days, 3 times weekly and the intervention was exerted consecutively for 4 weeks. Successively, on day 1, 21, 28, 35, 42 and 49 of experiment, paw withdrawal threshold (von Frey threshold) after mechanical stimulation was detected in the rabbits. HE staining was adopted to observe the histomorphological changes of the right knee joint cartilage in the rabbits. ELISA was used to determine the contents of interleukin-1 (IL-1β) and tumor necrosis factor-α (TNF-α) in the right knee synovial fluid. Western blot method and real-time PCR were used to determine the relative expressions of protein and mRNA of TRPV1 and TRPV4 in knee synovial tissue of the rabbits.
Results: Compared with the normal group, on day 49 of experiment, von Frey threshold was reduced significantly in the rabbits of the model group (P<0.01), the integrety of cartilage surface of knee joint was seriously damaged, the structural layers were disordered, the chondrocytes were clustered, the tide lines were distorted and the matrix staining disappeared. The contents of IL-1β and TNF-α of the right knee synovial fluid were increased significantly (P<0.01), and the relative expressions of protein and mRNA of TRPV1 and TRPV4 in the synovial tissue of the right knee were increased significantly (P<0.01). Compared with the model group, on day 49 of experiment, von Frey threshold was increased significantly in each of the Miao medicinal acupuncture therapy group, the dermal needle group and the smearing group (P<0.01). The right knee joint cartilage was complete in morphology, the structure clear was in layer, the cells were arranged in order and the matrix staining was uniform. The contents of IL-1β and TNF-α of the right knee synovial fluid were reduced significantly (P<0.01, P<0.05), and the relative expressions of protein and mRNA of TRPV1 and TRPV4 in the synovial tissue of the right knee were reduced significantly (P<0.01). Compared with either the dermal needle group or the smearing group, on day 49 of experiment, von Frey threshold was increased significantly in the Miao medicinal acupuncture therapy group (P<0.01). The right knee joint cartilage surface was even, the structure layers were clear. The contents of IL-1β and TNF-α of the right knee synovial fluid were reduced significantly (P<0.05), and the relative expressions of protein of TRPV1 and TRPV4 in the synovial tissue were reduced (P<0.01, P<0.05). Compared with the smearing group, the relative expression of TRPV1 mRNA in the synovial tissue was reduced significantly in the Miao medicinal acupuncture therapy group (P<0.05).
Conclusion: Miao medicinal acupuncture therapy plays a role in treatment of KOA probably through inhibiting the expressions of IL-1β and TNF-α of knee synovial fluid and down-regulating the expressions of protein and mRNA of TRPV1 and TRPV4 in knee synovial tissue.
目的：观察苗医弩药针疗法对膝骨性关节炎（KOA）模型家兔膝关节滑膜组织瞬时感受器电位香草酸受体(TRPV)通道的影响，探讨苗医弩药针疗法治疗KOA的作用机制。方法：从34只新西兰雄性家兔中随机选取6只作为正常组，其余家兔采用右膝关节腔注射木瓜蛋白酶和L-半胱氨酸混合液的方法建立KOA模型，将造模成功的24只家兔随机分为模型组、弩药针组、皮肤针组和涂抹组，各6只。弩药针组家兔采用苗医弩药针疗法于右膝关节面（以血海、梁丘、阳陵泉、阴陵泉为4点的长方形内)上呈“米”字型滚刺；皮肤针组采用皮肤滚针于右膝关节面进行滚刺；涂抹组以弩药液涂抹右膝关节面。隔日1次，每周3次，连续干预4周。于实验第1、21、28、35、42、49天检测家兔机械刺激的缩足阈值（von Frey阈值）。干预结束后，HE染色法观察家兔右膝关节软骨组织形态学改变；ELISA法检测家兔右膝关节液中白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）的含量；Western blot法和real-time PCR检测家兔右膝关节滑膜组织中TRPV1、TRPV4蛋白和mRNA的相对表达量。结果：与正常组比较，模型组家兔实验第49天von Frey阈值显著降低（P<0.01）；右膝关节软骨表面完整性严重破坏，结构层次紊乱，软骨细胞成簇，潮线扭曲，基质染色消失；右膝关节液中IL-1β、TNF-α含量显著升高（P<0.01）；右膝关节滑膜组织中TRPV1、TRPV4蛋白和mRNA相对表达水平显著升高（P<0.01）。与模型组比较，弩药针组、皮肤针组和涂抹组家兔实验第49天von Frey阈值显著升高（P<0.01）；右膝关节软骨形态较完整，结构层次较清晰，细胞排列有序，基质染色较均匀；右膝关节液中IL-1β、TNF-α含量显著降低（P<0.01，P<0.05）；右膝关节滑膜组织中TRPV1、TRPV4蛋白和mRNA的相对表达水平均显著降低（P<0.01）。与皮肤针组和涂抹组比较，弩药针组家兔实验第49天von Frey阈值显著升高（P<0.01）；右膝关节软骨表面平整，结构层次清晰；右膝关节液中IL-1β、TNF-α含量降低（P<0.05）；右膝关节滑膜组织中TRPV1、TRPV4蛋白相对表达量降低（P<0.01，P<0.05）；与涂抹组比较，弩药针组家兔右膝关节滑膜组织TRPV1 mRNA相对表达量显著降低（P<0.05）。结论：苗医弩药针疗法可能通过抑制膝关节液中IL-1β、TNF-α的表达，下调膝关节滑膜组织中TRPV1、TRPV4表达，发挥治疗KOA作用。.
Keywords: Miao medicinal acupuncture therapya; knee osteoarthritisa; transient receptor potential vanilloid 1 (TRPV1)a; transient receptor potential vanilloid 4 (TRPV4).