背景：临床研究表明，针刀治疗能够改善类风湿关节炎患者的症状，减轻关节肿胀，改善受损关节的活动功能，但尚无其具体作用机制的实验研究报道。

目的：观察针刀治疗对胶原诱导性关节炎大鼠滑膜组织Bcl-2、Bax表达的影响。

方法：将40只大鼠随机分为正常对照组10只，造模组30只。造模组大鼠采用二次免疫法制备胶原诱导性关节炎模型，随机选取20只造模成功大鼠随机分为模型组和针刀组。正常组和模型组不予处理；针刀组大鼠于左膝关节周围触及髌内外支持带、膝内外侧副韧带、髌底上缘中点及髌韧带的中点，每次选取上述2点予以针刀治疗，1次/周，共治疗3次。实验过程中监测大鼠一般情况，测量大鼠左后足趾厚度，进行关节炎指数评分；苏木精-伊红染色法观察关节滑膜病理形态；应用实时定量PCR法、免疫荧光技术分别检测滑膜组织Bcl-2、Bax的基因表达水平及免疫荧光强度。实验方案经湖北中医药大学动物实验伦理委员会批准(批准号为00)。

结果与结论：①与正常组相比，模型组左后足趾厚度及关节炎指数评分明显增加(P＜ 0.01，P＜ 0.01)；与模型组相比，针刀组左后足趾厚度及关节炎指数评分减小(P＜ 0.05，P＜ 0.05)；②病理形态上，模型组较正常组滑膜层数增厚，大量炎症细胞浸润，针刀组较模型组滑膜层数减少，炎症细胞浸润减轻；③各组间滑膜组织Bax mRNA表达水平比较，差异无显著性意义(P＞ 0.05)；针刀组Bcl-2 mRNA、Bcl-2/Bax mRNA表达较模型组明显降低(P＜ 0.01，P＜ 0.01)；④与模型组比较，针刀组Bcl-2/Bax荧光强度明显降低(P＜ 0.01)；⑤结果表明，针刀治疗类风湿关节炎的机制可能与下调滑膜组织中Bcl-2表达，降低Bcl-2/Bax比值有关。

BACKGROUND:It has been shown that acupotomy therapy can improve the symptoms of rheumatoid arthritis, reduce the swelling of the joints, and improve the function of the damaged joints in clinical practice. However, the specific mechanism has not been reported in animal experiments.

OBJECTIVE:To observe the effect of acupotomy therapy on the expression of Bcl-2 and Bax in synovial tissue of collagen-induced arthritis rats.

METHODS:Forty rats were prepared for the experiment. Ten of them were randomly selected as normal controls, and the other 30 rats were used to manufacture collagen-induced arthritis models via secondary immunization. After the modeling, 20 successful models were randomly selected for subsequent experiments and randomly divided into a model group and an acupotomy group, with 10 in each group. Normal group and model group remain untreated. The medial and lateral patellofemoral ligament, the tibial and fibular collateral ligament, the midpoint of the upper margin of the patella and the midpoint of the patellar ligament around the left knee joint were selected as points of acupotomy treatment, two of which were selected for each acupotomy session. Rats in the acupotomy group were treated once a week for 3 continuous weeks. The general condition of experimental rats was monitored daily, thickness of the leftposterior toe was measured and arthritis index was evaluated. Hematoxylin-eosin staining was executed to observe the pathological morphology of the synovialtissue. Real-time quantitative PCR and immunofluorescence staining were implemented to measure gene expression and immunofluorescence intensity of Bcl-2 and Bax in synovial tissue, respectively. The study protocol was approved by the Animal Ethic Committee of Hubei University of Traditional Chinese Medicine with an approval No. 00.

RESULTS AND CONCLUSION:Compared with the normal group, the thickness of the left posterior toe and arthritis index score of the model group were significantly increased (P＜ 0.01,P＜ 0.01). Compared with the model group, the left hind toe thickness and arthritis index score of the acupotomy group were decreased (P＜ 0.05,P＜ 0.05). Hematoxylin-eosin staining showed that: pathologically, the synovial layers of the model group were thicker, with infiltration of a large number of inflammatory cells. However, the synovial layers of the acupotomy group were fewer with less infiltration of inflammatory cells. RT-PCR showed that there was no significant difference in the expression of Bax mRNA in the synovial tissue between all the groups (P＞ 0.05). The expressions of Bcl-2 mRNA and Bcl-2/Bax mRNA were significantly reduced in the acupotomy group compared with the model group (P＜ 0.01,P＜ 0.01). Immunofluorescence detection indicated that the fluorescence intensity of Bcl-2/Bax in the acupotomy group was significantly reduced compared with the model group (P＜ 0.01). To conclude, the down-regulation of Bcl-2 expression and Bcl-2/Bax ratio in the synovial tissue may be one of its mechanisms for improving rheumatoid arthritis.

0 引言 Introduction

类风湿关节炎是一种以关节滑膜增生、慢性炎症为最主要病理表现的自身免疫性疾病[1]，目前在国内外仍属难治之症。近年来，作者导师运用针刀治疗类风湿关节炎取得了较好的临床疗效，且在研究中发现，针刀治疗通过调节类风湿关节炎患者关节滑液中肿瘤坏死因子α、白细胞介素6和白细胞介素10炎症细胞因子的水平，减轻了关节炎症反应；通过调节与类风湿关节炎发病密切相关的关节滑液基质金属蛋白酶的表达，有效调控了关节软骨细胞外基质的降解；且针刀治疗后类风湿关节炎患者血清中类风湿因子、C-反应蛋白、血沉含量降低，针刀治疗通过活化血清中自由基清除剂超氧化物歧化酶，有效清除多余自由基，从而在一定程度上保护受损的滑膜及骨组织[2-3]。随着针刀治疗类风湿关节炎的临床观察日益增多[4-5]，其疗效也逐渐受到认可，但其具体作用机制却未见实验研究报道。