Abstract：
This study investigated the immunological mechanisms of Ermiao powder in the treatment of rheumatoid arthritis rats through the alpha 7 nicotinic acetylcholine receptor(α7nAChR)-Janus kinases 2(JAK2)/signal transducer and activator of transcription 3(STAT3) signaling pathway. A total of 56 female Wistar rats were randomly divided into the normal group(HG, n=8), collagen-induced arthritis(CIA) model group(CM, n=8), vagotomy group(VA, n=8), sham group(SH, n=8), Ermiao Powder treatment model group(EM, n=8), Ermiao Powder treatment for vagotomy group(EV, n=8) and Ermiao Powder treatment for sham group(ES, n=8). Following the establishment of CIA models in all groups except the HG group, the rats underwent unilateral vagotomy and sham operation(only the vagus nerve was separated). Drug treatment was started 7 days after surgery and continued for 35 days. The body weight and joints of rats were recorded, the pathological changes of the spleen of rats were observed, the contents of interleukin-6(IL-6), interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) in serum were detected by enzyme-linked immunosorbent assay(ELISA), and the mRNA and protein expression of α7nAChR-JAK2/STAT3 pathway core genes in spleen were detected by qRT-PCR, Western blot and immunohistochemistry. RESULTS:: showed that CM group(compared with HG group) and VA group(compared with CM group and SH group) had significantly decreased body weight(P<0.05, P<0.01), increased arthritis score(P<0.05, P<0.01), swollen ankle joints with deformity, and increased and enlarged lymph nodes in the spleen. There were also notable increases in the serum levels of IL-6, IL-1β and TNF-α(P<0.05, P<0.01), and in the mRNA expressions of JAK2 and STAT3 in the spleen(P<0.05, P<0.01). The protein levels of phosphorylated JAK2(p-JAK2)/JAK2 and phospho-STAT3(p-STAT3)/STAT3 were significantly increased(P<0.05, P<0.01), and the number of JAK2, p-JAK2, STAT3 and p-STAT3 cells increased(P<0.05, P<0.01). EM group(compared with CM group) and ES group(compared with SH group) exhibited significantly increased body weight(P<0.01), decreased arthritis scores(P<0.05, P<0.01), reduced swelling of ankle joint, and decreased number and volume of lymph nodes in the spleen. Furthermore, serum levels of IL-6, IL-1β, and TNF-α decreased(P<0.05, P<0.01), the mRNA expression of JAK2 and STAT3 in spleen decreased(P<0.05, P<0.01), the protein levels of p-JAK2/JAK2 and p-STAT3/STAT3 decreased(P<0.05, P<0.01), and the number of JAK2, p-JAK2, STAT3 and p-STAT3 cells decreased(P<0.05, P<0.01), whereas the mRNA and protein expressions of α7nAChR were significantly increased(P<0.01). Compared with the VA group, there was no significant differences in weight gain and arthritis scores in the EV group. The number of lymph nodes in the spleen was not significantly reduced and the volume was still large, suggesting the inflammation was not significantly improved. The serum levels of IL-6, IL-1β and TNF-α were not significantly different, and there were no significant differences in α7nAChR, JAK2, and STAT3 mRNA expression in the spleen. The protein expression levels of p-JAK2/JAK2 and α7nAChR in spleen were lower(P<0.05, P<0.01), while p-STAT3/STAT3 protein expression was not significantly different. Besides, the two groups had no significant difference in the number of JAK2, p-JAK2, STAT3, and p-STAT3 cells. The results suggested that unilateral vagotomy promoted the increase of phosphorylated JAK2 and STAT3 expressions and exacerbated inflammation. In contrast, Ermiao Powder alleviated the inflammation in rheumatoid arthritis rats by activating the α7nAChR-mediated JAK2/STAT3 pathway through the vagus nerve, suggesting that the α7nAchR-JAK2/STAT3 pathway may be a potential target for the treatment of rheumatoid arthritis.
摘要：
本研究通过α7烟碱型乙酰胆碱受体(α7nAChR)-Janus激酶2(JAK2)/信号转导和转录激活因子3(STAT3)信号通路,探讨二妙散治疗类风湿关节炎大鼠的免疫学机制。将56只雌性Wistar大鼠随机分为正常组(HG，n=8),胶原诱导性关节炎（CIA）模型组（CM，n=8),迷走神经切断术组(VA，n=8),假手术组(SH，n=8),二妙散治疗模型组(EM，n=8),二妙散治疗迷走神经切断术组（EV，n=8）和假手术组（ES，n=8)。在除HG组外的所有组建立CIA模型后，大鼠行单侧迷走神经切断术和假手术(仅分离迷走神经)。手术后7天开始药物治疗并持续35天。记录大鼠体重和关节,观察大鼠脾脏的病理变化,白细胞介素-6（IL-6）的含量，酶联免疫吸附试验(ELISA)检测血清中白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α),qRT-PCR检测脾脏中α7nAChR-JAK2/STAT3通路核心基因的mRNA和蛋白表达,Westernblot和免疫组织化学。结果：显示CM组（与HG组相比）和VA组（与CM组和SH组相比）体重明显下降（P＆lt;0.05，P＆lt;0.01），关节炎评分增加（P＆lt;0.05，P＆lt;0.01），踝关节肿胀，畸形，脾脏淋巴结增多和肿大。血清IL-6、IL-1β和TNF-α水平也显著升高(P<0.05,P<0.01),脾脏中JAK2和STAT3的mRNA表达（P＆lt;0.05，P＆lt;0.01）。磷酸化JAK2(p-JAK2)/JAK2和磷酸化STAT3(p-STAT3)/STAT3蛋白水平显著升高(P<0.05,P<0.01),JAK2，p-JAK2，STAT3和p-STAT3细胞数量增加（P＆lt;0.05，P＆lt;0.01）。EM组（与CM组相比）和ES组（与SH组相比）均表现出明显的体重增加（P＆lt;0.01），关节炎评分降低（P＆lt;0.05，P＆lt;0.01），踝关节肿胀减少，脾脏中淋巴结的数量和体积减少。此外，血清IL-6，IL-1β，TNF-α降低（P＆lt;0.05，P＆lt;0.01），脾脏中JAK2和STAT3的mRNA表达降低（P＆lt;0.05，P＆lt;0.01），p-JAK2/JAK2和p-STAT3/STAT3蛋白水平降低(P<0.05,P<0.01),JAK2，p-JAK2，STAT3和p-STAT3细胞数量减少（P＆lt;0.05，P＆lt;0.01），而α7nAChR的mRNA和蛋白表达明显升高（P＆lt;0.01）。与VA组相比，EV组的体重增加和关节炎评分没有显着差异。脾脏内的淋巴结数目没有明显减少,体积仍然很大,提示炎症没有明显改善。血清IL-6、IL-1β、TNF-α水平差异无统计学意义,α7nAChR无显著差异,JAK2和STAT3mRNA在脾脏中表达。脾脏中p-JAK2/JAK2和α7nAChR的蛋白表达水平较低（P＆lt;0.05，P＆lt;0.01），而p-STAT3/STAT3蛋白表达无显著差异。此外,两组的JAK2,p-JAK2,STAT3和p-STAT3细胞数量差异无统计学意义.结果表明，单侧迷走神经切断术促进了磷酸化JAK2和STAT3表达的增加，并加剧了炎症反应。相比之下,二妙散通过迷走神经激活α7nAChR介导的JAK2/STAT3通路减轻类风湿关节炎大鼠炎症,提示α7nAchR-JAK2/STAT3通路可能是治疗类风湿关节炎的潜在靶点。
