二妙散通过α7nAChR-JAK2/STAT3途径改善胶原诱导性关节炎大鼠炎症的免疫学机制

Immunological mechanism of Ermiao Powder improving inflammation in rats with collagen-induced arthritis throughα7nAChR-JAK2/STAT3 pathway

探讨二妙散通过α7烟碱型乙酰胆碱受体(α7nAchR)-Janus激酶2(JAK2)/信号转导及转录激活因子3(STAT3)途径改善类风湿关节炎大鼠炎症的免疫学机制。56只雌性Wistar大鼠随机分为正常(HG)组、胶原诱导性关节炎模型(CM)组、迷走神经切断(VA)组、假手术(SH)组、二妙散治疗模型(EM)组、二妙散治疗迷走神经切断(EV)组、二妙散治疗假手术(ES)组,每组8只。除HG组外,其他组均构建胶原诱导性关节炎(CIA)大鼠模型,造模成功后进行颈部单侧迷走神经切断术及假手术(仅分离迷走神经),术后7 d开始给药治疗,持续治疗35 d。记录大鼠体质量及关节情况,观察大鼠脾脏病理学变化,ELISA检测大鼠血清中白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)含量,qRT-PCR、Western blot和免疫组化检测脾脏中α7nAchR-JAK2/STAT3通路核心基因mRNA及蛋白表达。结果显示,CM组对比HG组、VA组对比CM组和SH组,大鼠体质量显著降低(P<0.05,P<0.01),关节炎评分显著升高(P<0.05,P<0.01),踝关节明显肿胀伴畸形,脾脏淋巴小结显著增大,血清中IL-6、IL-1β、TNF-α含量显著增加(P<0.05,P<0.01),脾脏中JAK2、STAT3 mRNA水平显著升高(P<0.05,P<0.01),磷酸化JAK2(p-JAK2)/JAK2、磷酸化STAT3(p-STAT3)/STAT3水平显著升高(P<0.05,P<0.01),JAK2、p-JAK2、STAT3、p-STAT3细胞数增加(P<0.05,P<0.01)。EM组对比CM组、ES组对比SH组,大鼠体质量显著升高(P<0.01),关节炎评分显著下降(P<0.05,P<0.01),踝关节肿胀度明显减轻,脾脏淋巴小结体积减小,炎症减轻,血清中IL-6、IL-1β、TNF-α含量显著下降(P<0.05,P<0.01),脾脏JAK2、STAT3 mRNA表达显著降低(P<0.05,P<0.01),p-JAK2/JAK2、p-STAT3/STAT3水平显著降低(P<0.05,P<0.01),JAK2、p-JAK2、STAT3、p-STAT3细胞数量显著减少(P<0.05,P<0.01),α7nAChR mRNA及蛋白表达显著上升(P<0.01)。EV组对比VA组,大鼠体质量上升及关节炎评分无明显差异,脾脏淋巴小结数量减少不显著,体积仍较大,炎症无明显改善,血清IL-6、IL-1β、TNF-α含量表达不显著,脾脏中α7nAChR、JAK2、STAT3 mRNA无明显差异,脾脏中p-JAK2/JAK2、α7nAChR蛋白表达显著降低(P<0.05,P<0.01),p-STAT3/STAT3蛋白表达不显著,JAK2、p-JAK2、STAT3、p-STAT3细胞数量无明显差异。以上实验结果显示,单侧迷走神经切断促进磷酸化的JAK2和STAT3表达增加,加剧炎症状态,二妙散通过迷走神经激活α7nAChR介导的JAK2/STAT3途径改善CIA大鼠炎症,提示α7nAchR-JAK2/STAT3途径可能是治疗类风湿关节炎的潜在靶标。

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 ofα7nAChR-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-1βand 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-αdecreased(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 ofα7nAChR 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-1βand TNF-αwere not significantly different,and there were no significant differences inα7nAChR,JAK2,and STAT3 mRNA expression in the spleen.The protein expression levels of p-JAK2/JAK2 andα7nAChR 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 theα7nAChR-mediated JAK2/STAT3 pathway through the vagus nerve,suggesting that theα7nAchR-JAK2/STAT3 pathway may be a potential target for the treatment of rheumatoid arthritis.