电针通过激活腺苷A2A受体抑制p38 MAPK通路减轻胶原诱导性关节炎大鼠的关节骨侵蚀  

Inhibiting p38 MAPK signaling pathway to reduce joint bone erosion in rats with collagen-induced arthritis by activating A2A adenosine receptor after treating with electroacupuncture

作　　者：唐可婧 郑琪琪 宋芷葳 杜忠衡 叶天申[2] TANG Ke-jing;ZHENG Qi-qi;SONG Zhi-wei;DU Zhong-heng;YE Tian-shen(School of First Clinical Medicine,School of Information and Engineering,Wenzhou Medical University,Wenzhou 325035,Zhejiang,China;Department of Acupuncture,Tuina and Physiotherapy,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325000,Zhejiang,China)

机构地区：[1]温州医科大学第一临床医学院、信息与工程学院,温州325035 [2]温州医科大学附属第一医院针推理疗科,温州325000

出　　处：《第二军医大学学报》2021年第7期740-748,共9页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金面上项目(81674053);浙江省自然科学基金探索项目(LY20H270015).

摘　　要：目的基于p38 MAPK信号通路,探究电针刺激足三里、三阴交激活腺苷A2A受体(A2AR)减轻胶原诱导性关节炎(CIA)大鼠关节炎症及骨侵蚀的作用机制。方法将32只SD大鼠随机分为对照组、模型组、药物组和电针组,每组8只。模型组、药物组和电针组大鼠均通过胎牛Ⅱ型胶原与弗氏佐剂混合液2次注射免疫复制CIA大鼠模型,药物组于造模成功后每隔7 d腹腔注射甲氨蝶呤2 mg/kg、连续14 d,电针组于造模成功后予大鼠双侧足三里、三阴交每天电针(2/100 Hz疏密波,输出电流2 mA)刺激20 min、连续14 d。通过CT观察大鼠后足骨质破坏情况,H-E、番红O-固绿及抗酒石酸酸性磷酸酶(TRAP)染色分别观察膝关节滑膜、软骨及破骨细胞病理组织学变化,蛋白质印迹法测定膝关节滑膜组织中A2AR、p38、磷酸化p38(P-p38)、NF-κB、T细胞核因子c1(NFATc1)蛋白表达,ELISA法测定外周血清IL-1β含量。结果与对照组相比,模型组大鼠后足骨密度下降,空间结构紊乱;膝关节软骨明显受损,滑膜组织过度增生;TRAP阳性的破骨细胞数目增加(P<0.01);膝关节滑膜组织中A2AR、p38、P-p38、NF-κB、NFATc1表达均上调(P均<0.01);外周血清中IL-1β含量升高(P<0.01)。与模型组比较,电针组和药物组大鼠后足整体结构、膝关节滑膜及软骨破坏明显改善;TRAP阳性破骨细胞数目减少(P<0.01);A2AR表达上调(P<0.01),p38、P-p38、NF-κB、NFATc1表达下调(P均<0.01);外周血清中IL-1β含量降低(P<0.01)。与药物组比较,电针组的TRAP阳性破骨细胞数目增加(P<0.05);A2AR蛋白表达下调(P<0.01),NF-κB、NFATc1蛋白表达上调(P均<0.05),p38表达变化不明显(P>0.05),但P-p38表达上调(P<0.05)。结论电针刺激足三里、三阴交可减轻CIA大鼠的关节骨侵蚀损害,其关节保护作用机制可能与电针激活A2AR后抑制p38 MAPK通路进而减少破骨细胞生成有关。Objective To explore the mechanism of electroacupuncture at“Zusanli”(ST36)and“Sanyinjiao”(SP06)to activate A2A adenosine receptor(A2AR)for reducing joint inflammation and bone erosion in rats with collagen-induced arthritis(CIA)based on p38 mitogen-activated protein kinase(MAPK)signaling pathway.Methods A total of 32 male SD rats were evenly randomized into control,CIA model,methotrexate(MTX)-treated and electroacupuncture-treated groups.The rats in model,MTX-treated and electroacupuncture-treated groups were immunized to replicate the CIA model by twice injection of fetal bovine type Ⅱ collagen and Freund’s adjuvant mixture.After the model was successfully established,the rats in the MTX-treated group were injected with MTX 2 mg/kg every 7 days for 14 days,and those in the electroacupuncturetreated group were treated with electroacupuncture(2/100 Hz density wave and output current of 2 mA)bilaterally at ST36 and SP06 for 20 min every day for 14 days.The pathological changes of hind foot bone were observed by computed tomography(CT)imaging;the histopathological changes of synovium,cartilage and osteoclasts of the knee joint were observed by hematoxylineosin(H-E),safranin O-fast green and tartrate-resistant acid phosphatase(TRAP)staining;the expression of A2AR,p38,phosphorylated p38(P-p38),nuclear factorκB(NF-κB)and nuclear factor-activated T cell 1(NFATc1)proteins in the knee synovial tissue was determined by Western blotting;and the peripheral serum interleukin 1β(IL-1β)content was measured by enzyme-linked immunosorbent assay(ELISA).Results Compared with the control group,the bone density of hind foot in the model group was decreased and the spatial structure was disordered;the cartilage of knee joint was obviously damaged and the synovial tissue was hyperplastic;the number of TRAP positive osteoclasts was increased significantly(P<0.01),while the expression of A2AR,p38,P-p38,NF-κB and NFATc1 in the knee synovial tissue was significantly up-regulated(P<0.01);and the peripheral serum IL-1βconte

关 键 词：电针 甲氨蝶呤 胶原诱导性关节炎 腺苷A2A受体 p38丝裂原活化蛋白激酶信号通路 

分 类 号：R593.22[医药卫生—内科学]