Effect of acupuncture on arthritic pathological injury in rats with type Ⅱ collagen-induced rheumatoid arthritis.

To observe the effect of acupuncture stimulation of "Yanglingquan"(GB34), "Zusanli"(ST36) and "Xuanzhong" (GB39) on arthritis index (AI), joint synovial membrane pathology, serum-related immunoinflammatory factors, and expressions of tumor suppressor gene mt-p53, nuclear factor kappa B (NF-κB) and peroxisome proliferator activated receptor gamma (PPARγ) in knee joint synovial tissue of rats with type Ⅱ collagen-induced arthritis (CIA), so as to explore its possible mechanisms underlying improvement of rheumatoid arthritis (RA).Male SD rats were used in the present study. The CIA model was established by subcutaneous injection of collagen emulsion (200 μL/rat) in the tail root region on the first day and repeat (100 μL/rat) once on the 9th day. Eighteen successful CIA rats were randomized into model, medication and acupuncture groups, with 6 rats in each group. Other 6 normal rats were used as the normal control group. For rats of the medication group, leflunomide (1.9 mg/kg) was administrated by gavage, once a day, and for rats of the acupuncture group, manual acupuncture stimulation was applied to bilateral GB34, ST36, GB39 for 30 min, once a day, for 12 weeks. The arthritis index (AI) score (0-4 points) was evaluated once every week. The contents of IL-6, IL-17 and TNF-α in the serum were determined by ELISA. Histopathological changes of the ankle joint were observed by H.E. staining. The protein and mRNA expression levels of mt-p53, NF-κB p65, and PPARγ in the knee joint synovial tissue were determined by Western blot and quantitative real time PCR, separately.Compared with the normal control group, the AI scores at different time-points after modeling, contents of serum TNF-α, IL-6 and IL-17, expression levels of mt-p53, NF-κB p65, PPARγ proteins and mRNAs were significantly increased in the model group (P<0.01, P<0.05). In comparison with the model group, the AI scores at the 10th week in the medication group and at the 3rd, 9th and 10th week in the acupuncture group, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of mt-p53 and NF-κB p65 proteins in both medication and acupuncture groups, as well as mt-p53 and NF-κB p65 mRNAs in the medication group were apparently decreased (P<0.01, P<0.05), while the expression levels of PPARγ protein in both medication and acupuncture group and PPARγ mRNA in the medication group were significantly up-regulated (P<0.05, P<0.01). No significant differences were found between the acupuncture and medication groups in down-regulating the AI score and serum TNF-α, IL-6 and IL-17 contents. The effect of acupuncture was weaker than that of medication in down-regulating the expression of mt-p53 and NF-κB p65 proteins and mRNAs and in up-regulating PPARγ mRNA (P<0.01). H.E. results showed ankle cartilage hyperplasia, reduced joint cavity, mild fibroproliferation and inflammatory cell infiltration in the surrounding soft tissue of the ankle joint in rats of the model group, which was milder in both medication and acupuncture groups.Acupuncture stimulation can improve the degree of joint inflammation and swelling in CIA rats, which may be related to its effects in inhibiting the overexpression of immunoinflammatory factors in serum and regulating expression of mt-p53, NF-κB p65, PPARγ mRNAs and proteins in the synovial tissue.目的: 观察针刺“阳陵泉”“足三里”“悬钟”对Ⅱ型胶原蛋白诱导性关节炎（CIA）大鼠关节炎指数（AI）、关节滑膜病理形态、血清相关免疫炎性因子及关节滑膜组织突变型p53（mt-p53）、核转录因子-κB（NF-κB）p65、滑膜过氧化物酶体增殖物激活受体-γ（PPARγ）蛋白表达的影响，探讨针刺治疗类风湿性关节炎（RA）的可能机制。方法: 随机选取6只雄性SD大鼠作为正常组，其余大鼠采用牛源性Ⅱ型胶原蛋白和完全弗氏佐剂建立CIA模型。将造模成功的18只大鼠随机分为模型组、来氟米特组、针刺组，每组6只。来氟米特组予来氟米特1.9 mg/kg灌胃，每日1次；针刺组针刺“阳陵泉”“足三里”“悬钟”，留针30 min，每日1次。以上均干预12周。每周进行关节炎指数（AI）评分；ELISA法检测血清中肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6、IL-17含量；HE染色法观察大鼠踝关节的病理学变化；Western blot法检测膝关节滑膜组织中mt-p53、NF-κB p65、PPARγ蛋白表达水平；荧光定量PCR法检测膝关节滑膜组织中mt-p53、NF-κB p65、PPARγ mRNA表达水平。结果: 与正常组比较，模型组大鼠AI评分升高（P<0.01，P<0.05），血清中TNF-α、IL-6、IL-17含量升高（P<0.01），膝关节滑膜组织中mt-p53、NF-κB p65、PPARγ蛋白及mRNA表达水平均升高（P<0.01，P<0.05）。与模型组比较，针刺组和来氟米特组大鼠AI评分降低（P<0.01，P<0.05），血清TNF-α、IL-6及IL-17含量降低（P<0.01），关节滑膜组织中NF-κB p65蛋白及mRNA表达水平均降低（P<0.01），PPARγ蛋白表达水平升高（P<0.05）；来氟米特组mt-p53蛋白和mRNA表达水平降低（P<0.01），PPARγ mRNA表达水平升高（P<0.01）。与来氟米特组比较，针刺组大鼠滑膜组织中mt-p53、NF-κB p65蛋白及mRNA表达水平升高（P<0.01），PPARγ mRNA表达水平下降（P<0.01）。HE染色结果显示，模型组大鼠踝关节软骨增生，关节腔缩小，周围软组织见轻度纤维增生和炎性细胞浸润；针刺组和来氟米特组大鼠踝关节增生和炎性细胞浸润状态明显改善。结论: 针刺“阳陵泉”“足三里”“悬钟”能够改善CIA大鼠关节炎性反应和肿胀程度，其机制可能与抑制大鼠血清中炎性因子的过度表达及调节滑膜组织中mt-p53、NF-κB p65、PPARγ mRNA转录水平和蛋白表达水平有关。.