电针抑制CIA小鼠破骨细胞形成的腺苷A2AR途径机制

Acupuncture is frequently employed in diseases with a local inflammatory component, such as rheumatoid arthritis (RA), but some of the latest trials assessing the benefits of acupuncture in RA found that acupuncture was not better than sham acupuncture implying that the analgesic effects observed are related to a strong placebo response(Amezaga Urruela M, Suarez-Almazor ME. Curr Rheumatol Rep. 2012 December ; 14(6): 589–597). And the mechanism of inhibition of joint damage in acupuncture treatment of RA deserves further study..Now days, a growing number of researchers set out to explore the mechanism of adenosine and its receptors mediated inflamtory analgesic effects of acupuncture. Goldman found adenosine A1 receptors are involved in acupuncture analgesia arthritis model (Goldman N, Chen M, Fujita T, Xu Q, Peng W, Liu W, Jensen TK, Pei Y, Wang F, Han X, Chen JF, Schnermann J, Takano T, Bekar L, Tieu K, Nedergaard M. Nat Neurosci, 2013, 13(7):883-8); meanwhile, many studies have shown that adenosine A2A recopters (A2AR) involved in collagen-induced arthritis (CIA) of anti-inflammatory and jiont damage reduction; Our preliminary study also suggests that electroacupuncture (EA) treatment can persistently elevates adenosine in peripheral blood and ameliorates CIA(Tian-shen Ye, Zhong-heng Du, Zhi-hui Li, Wen-xia Xie, Ka-te Huang, Yong Chen, Zhou-yang Chen, Huan Hu, Jun-lu Wang and Jian-qiao Fang.. eCAM, 2016, Article ID 3632168, 10 pages.) and exerts an anti-inflammatory effect resulting in significant joint damage reduction role by activation of A2AR in the synovial tissue of CIA (Qi-hui Li, Wen-xia Xie, Xiao-pei Li, Ka-te Huang, Zhong-heng Du, Wen-jie Cong, Long-hua Zhou, Tian-shen Ye, and Jiang-Fan Chen. eCAM, 2015, Article ID 809560, 11 pages). On the other hand, studies have found that activation of A2AR reduces osteoclast formation via protein kinase A (PKA) and ERK1/2 mediated suppression of NF-κB nuclear translocation, suggesting a mechanism by which adenosine could target local bone erosion in RA (Aránzazu Mediero, Miguel Perez-Aso and Bruce N. Cronstein. British Journal of Pharmacology (2013) 169 1372–1388 ). Therefore, we put forward the scientific hypothesis: EA exerts a joint damage reduction effect on CIA mice probably is associated with inhibiting osteoclast formation by A2AR—AC—cAMP—PKA—ERK1/2—NF-κB puthway..We made the CIA model from C57BL/B6 mice including wild type mice and A2A knockout mice respectively, each treatment group received EA at “ zusanli and sanyinjiao” aupoints equally, we uitlized the micro dialysis, cell culture technology，RT-PCR, WB, ELISA and mmunohistochemistry, to detect the concentrations of adenosine, A2AR, TNF-a, IL-1β, RANK, RANKL et al. in joint of each mice before and after EA, then the joint organization will take out using tartrateresistant acid phosphatase (TRAP) staining to identify osteoclasts and to determine the expression of PKA, ERK1/2 and NF-κB et al. Further study in vitro is to research the A2AR mediate inhibition pathway through synovial fibroblasts induce osteoclast formation from mononuclear cell by co-culture technology. This study is aim to elucidate the inner associations among joint damage reduction effects of EA, and A2AR mediated inhibition of osteoclast formation from whole body, tissue, cell, molecular and genetic aspects, the results may offer a scientific clue for the mechanism study of acupuncture’s joint damage reduction effects.

如何优选安全的疗法抑制关节不可逆损害是类风湿关节炎（RA）中医药治疗领域的重大课题。研究证实激活腺苷A2A受体（A2AR）对RA有抗炎作用；Goldman发现的腺苷途径介导针刺抗炎镇痛开辟了RA针刺机理研究的新方向。结合RA骨侵蚀治疗研究新进展（激活A2AR通过调节PKA和ERK1/2减少NF-κB核易位而抑制破骨细胞形成）及本课题组前期研究发现电针激活A2AR可减轻RA模型（胶原诱导关节炎，CIA）的关节损害，提出科学假说：电针减轻CIA小鼠关节损害机制可能与A2AR途径抑制破骨细胞形成有关。项目拟采用A2A基因敲除小鼠CIA模型，电针足三里、三阴交，运用微透析、细胞共培养等技术，证实电针可增加关节局部腺苷浓度；并运用体内、体外实验方法阐明A2AR介导电针抑制破骨细胞形成及其细胞内机制。研究从整体、细胞、基因层面揭示电针抑制CIA关节损害规律有重要意义，将为电针临床治疗RA提供理论依据。

电针激活腺苷A2A受体（A2AR）抑制类风湿关节炎（RA）不可逆损害研究是中医药领域的重要方向。本项目研究电针抑制CIA小鼠破骨细胞形成的腺苷A2AR途径机制，主要内容为：1）通过构建C57BL/6小鼠WT及A2A KO的CIA模型，运用微透析、HPLC等技术，从动物组织形态、细胞和基因等水平，检测电针对小鼠膝关节腔内腺苷浓度及TNF-a、IL-1β、RANKL的表达和分布的影响，并明确其对破骨细胞分化的相关因子的调节作用；2）通过14天电针治疗后取炎症关节组织检测破骨细胞的数量及活性；并进一步采用细胞共培养、“针灸血清”及“工具药”等体内外研究技术，从细胞、分子和基因等水平阐明A2AR可介导电针抑制破骨细胞形成，并明确其细胞内信号转导途径。.研究重要结果及关键数据：1）已证明连续电针干预可以使CIA模型血清腺苷浓度保持高位，激活炎症关节滑膜细胞A2AR，发挥抗炎和减轻关节损害作用。2）从细胞水平证实了电针激活A2AR后可通过本项目预计的AC—cAMP—PKA—ERK1/2—NF-κB途径抑制破骨细胞形成（已发表SCI论文，IF:2.482）；还发现P38—NF-κB—NFATc1也是重要途径，获得省自然资助，并待发表SCI论文1篇；3）已完成A2A基因敲除小鼠CIA造模及电针治疗和体外破骨细胞培养，预计2021年内再发表SCI2-3篇。2） 关键数据：培养1名硕士研究生，发表SCI1篇，国内核心期刊论文3篇，举办会议一次，讲座2次，获得省自然面上项目资助。.科学意义：本研究从整体、细胞、分子基因层面揭示电针抑制CIA关节损害规律，建立了针刺治疗类风湿关节炎的腺苷信号转导途径抗炎保护关节效应研究方向，研究成果将为针灸临床治疗RA的提供更加完善的科学理论依据。本研究具有鲜明的引领性或开创性特征，旨在通过独辟蹊径取得开拓性成果，必将引领或拓展科学前沿，具有重要的科学意义和良好的应用前景。