文献类型：期刊文献

中文题名：热补针法对类风湿关节炎寒证家兔模型膝关节滑膜组织TLR4-MyD88-ERK1/2信号通路的影响

英文题名：Effect of heat-reinforcing needling on the TLR4-MyD88-ERK1/2 signaling pathway in the synovial tissue of the knee joint of rabbits with rheumatoid arthritis with cold syndrome

作者：苏成红[1];杜小正[1];方晓丽[1];刘强[1];刘莉梅[1];袁博[2];井维尧[1];张小娜[1];宋亚文[2]

第一作者：苏成红

机构：[1]甘肃中医药大学针灸推拿学院,兰州730000;[2]甘肃中医药大学附属医院郑氏针灸科

第一机构：甘肃中医药大学针灸推拿学院

年份：2022

卷号：45

期号：7

起止页码：745

中文期刊名：北京中医药大学学报

外文期刊名：Journal of Beijing University of Traditional Chinese Medicine

收录：CSTPCD;;北大核心:【北大核心2020】；CSCD:【CSCD2021_2022】；

基金：国家自然科学基金项目(No.82060891);甘肃省自然科学基金项目(No.21JR7RA568);国家中医药管理局甘肃郑氏针法学术流派传承工作室项目(No.2305135901)。

语种：中文

中文关键词：类风湿关节炎;热补针法;Toll样受体4;髓样分化因子88;丝裂原活化蛋白激酶1/2;家兔

外文关键词：rheumatoid arthritis;heat-reinforcing needling;TLR4;MyD88;ERK1/2;rabbit

摘要：目的 探讨热补针法治疗类风湿关节炎(RA)寒证的抗炎机制。方法 采用卵蛋白干粉、弗氏完全佐剂联合低温冷冻法制备RA寒证家兔模型。按随机数字表法将50只家兔(雌雄各半)随机分为正常组、模型组、瑞沙托维(TAK-242)组、热补针法组及脂多糖(LPS)联合热补针法组,每组10只。TAK-242组腹腔注射Toll样受体4(TLR4)抑制剂TAK-242(0.75 mg/kg),热补针法组于“足三里”行热补针法针刺,LPS联合热补针法组行热补针法针刺合腹腔注射TLR4激动剂LPS(0.25 mg/kg)。连续治疗14 d后,比较各组家兔膝关节周径、皮肤温度;HE染色法观察家兔膝关节滑膜组织形态学;免疫组化法检测各组家兔膝关节滑膜组织中的肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)表达水平;Western blotting法检测各组家兔膝关节滑膜组织TLR4、髓样分化因子88(MyD88)、丝裂原活化蛋白激酶1/2(ERK1/2)的蛋白表达水平;实时荧光PCR检测各组家兔哺乳动物雷帕霉素靶蛋白(mTOR)、核糖体蛋白S6激酶(p70S6K)mRNA的表达。结果 与模型组比较,各干预组家兔膝关节周径缩小、皮肤温度升高(P<0.01);HE染色法示,TAK-242组及热补针法组家兔膝关节关节囊内侧黏膜基本平整,血管翳增殖减轻,存在少量炎性细胞浸润,而LPS联合热补针法组关节囊内侧黏膜粗糙,血管翳增殖,炎性细胞浸润聚集明显;滑膜组织中TNF-α、IL-1β、IL-6平均光密度均降低(P<0.01);TLR4、MyD88、ERK1/2蛋白表达水平及mTOR、p70S6K mRNA表达水平均降低(P<0.01)。与LPS联合热补针法组比较,热补针法组及TAK-242组家兔膝关节周径缩小、皮肤温度升高(P<0.01);滑膜组织中TNF-α、IL-1β、IL-6平均光密度均降低(P<0.01);TLR4、MyD88、ERK1/2蛋白表达水平及mTOR、p70S6K mRNA表达水平均降低(P<0.01)。结论 热补针法可以有效改善RA寒证家兔模型膝关节肿胀,缓解炎症反应,其机制可能与抑制滑膜组织TLR4-MyD88-ERK1/2通路,拮抗细胞增殖活性,减少炎性因子TNF-α、IL-1β、IL-6的分泌有关。
Objective We aimed to investigate the anti-inflammatory mechanism of heat-reinforcing needling on rheumatoid arthritis(RA) with cold syndrome.Methods The RA with cold syndrome model was established by injecting ovalbumin dry powder and Freund’s complete adjuvant combined with cold freezing.Fifty rabbits(50% male and 50% female) were randomly divided into the normal group, the model group, the resatorvid(TAK-242) group, the heat-reinforcing needling group, and the lipopolysaccharide(LPS) combined with heat-reinforcing needling group, 10 rabbits in each group.Rabbits in the TAK-242 group were intraperitoneally injected with the TLR4 inhibitor TAK-242(0.75 mg/kg);rabbits in the heat-reinforcing needling group were treated with “Zusanli”(ST 36) with heat-reinforcing needling;and rabbits in the LPS combined with heat-reinforcing needling group were injected intraperitoneally with the TLR4 agonist LPS(0.25 mg/kg) on the basis of heat-reinforcing needling. Each group received continuous treatment for 14 days. The circumference and skin temperature of the knee joint of the rabbits were recorded;the histomorphology of the synovial membrane of the rabbit knee joints was observed by HE staining;the expression levels of TNF-α, IL-1β, and IL-6 in synovial tissues were detected by immunohistochemistry;the protein expression levels of TLR4, MyD88, and ERK1/2 in synovial tissues were detected by Western blotting;and the mRNA levels of mTOR and p70 S6 K were detected by real-time PCR.Results The circumference of the knee joint was lower and the skin temperature of the knee joint was higher(P<0.01) in all intervention groups compared with the model group. The HE staining of the TAK-242 group and the heat-reinforcing needling group showed that the inner mucosa of the joint capsule was basically flat, the proliferation of pannus was reduced, and there was a small amount of inflammatory cell infiltration, while the LPS combined with the heat-reinforcing needling group showed that the inner mucosa of the joint capsule was rough, the pannus proliferated, and the inflammatory cell infiltration and aggregation were obvious. The expression of TNF-α, IL-1β, and IL-6(P<0.01), the protein levels of TLR4, MyD88, and ERK1/2, and the mRNA levels of mTOR and p70 S6 K were lower(P<0.01) in all intervention groups compared with the model group.The circumference of the knee joint was lower and the skin temperature of the knee joint was higher(P<0.01) in the heat-reinforcing needling group and the TAK-242 group compared with the LPS combined with heat-reinforcing needling group;the levels of TNF-α, IL-1β, and IL-6(P<0.01), the protein levels of TLR4, MyD88, and ERK1/2, and the mRNA levels of mTOR and p70 S6 K were lower(P<0.01) in the heat-reinforcing needling group and the TAK-242 group compared with the LPS combined with the heat-reinforcing needling group.Conclusion Heat-reinforcing needling can effectively reduce knee swelling and inflammatory infiltration in RA with cold syndrome model rabbits.The possible mechanism is related to the inhibition of the TLR4-MyD88-ERK1/2 pathway, antagonism of cell proliferation activity, and a decrease of TNF-α, IL-1β, and IL-6 secretion by heat-reinforcing needling.