Wang Hongxing, Wu Hui, Fang Kehua, Chang Xiaotian
Medical Research Center of the Affiliated Hospital of Qingdao University, Qingdao, China.
Department of Clinical Laboratory, Qilu Hospital, Shandong University, Jinan, China.
Front Pharmacol. 2021 Apr 19;12:658511. doi: 10.3389/fphar.2021.658511. eCollection 2021.
Uridine diphosphate (UDP) is an extracellular nucleotide signaling molecule implicated in diverse biological processes via specific activation of pyrimidinergic receptor P2Y, G Protein-Coupled, 6 (P2Y6). There is very little knowledge about the function and mechanism of UDP in rheumatoid arthritis (RA). This study used a quasi-targeted liquid chromatography-mass spectrometry (LC-MS) approach to investigate the unique expression of metabolites in RA synovial fluids (SF) ( = 10) with samples from osteoarthritis (OA) as controls ( = 10). RA fibroblast-like synoviocytes (FLSs) were collected from synovial tissues ( = 5) and cultured with UDP or MRS2578, a P2Y6 antagonist, and FLSs from OA were used as controls ( = 5). Rats with collagen-induced arthritis (CIA) were injected with UDP, MRS2578 or both ( = 9 for each group). P2Y6 expression was examined using real-time PCR, Western blotting and immunohistochemistry. Cell proliferation, apoptosis and migration of RA FLSs were measured using CCK-8 assay, real-time cell analysis, flow cytometry, wound healing assay and Transwell assay, respectively. The UDP levels in the culture medium, synovial fluid ( = 36) and peripheral blood ( = 36) of RA and CIA rats were measured using a Transcreener UDP Assay. Levels of proinflammatory cytokines were measured using a flow assay. Interleukin-6 (IL-6) levels were measured using ELISA and flow. LC-MS analysis detected significantly increased UDP levels in RA SF compared with OA SF, and the level was positively correlated with anticyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF)levels in RA. The increased UDP concentration was verified in the blood and synovial fluids of RA patients compared with samples from OA patients and healthy volunteers, respectively. UDP stimulated cell proliferation, migration and IL-6 secretion in RA FLSs and inhibited their apoptosis in culture, and MRS2578 inhibited these effects of UDP. UDP injection accelerated CIA and stimulated IL-6 production rather than other proinflammatory cytokines in the rat model, but simultaneous injection of MRS2578 suppressed these effects and alleviated CIA. P2Y6 expression was increased in RA and CIA synovial tissues. These results suggest that UDP is highly expressed in RA and stimulates RA pathogenesis by promoting P2Y6 activities to increase IL-6 production.
尿苷二磷酸（UDP）是一种细胞外核苷酸信号分子，通过嘧啶能受体P2Y、G蛋白偶联6（P2Y6）的特异性激活参与多种生物学过程。关于UDP在类风湿关节炎（RA）中的功能和机制知之甚少。本研究采用准靶向液相色谱-质谱（LC-MS）方法，以骨关节炎（OA）样本（n = 10）为对照，研究RA滑膜液（SF）（n = 10）中代谢物的独特表达。从滑膜组织（n = 5）中收集RA成纤维样滑膜细胞（FLS），用UDP或P2Y6拮抗剂MRS2578培养，以OA的FLS作为对照（n = 5）。给胶原诱导性关节炎（CIA）大鼠注射UDP、MRS2578或两者（每组n = 9）。使用实时PCR、蛋白质印迹和免疫组织化学检测P2Y6表达。分别使用CCK-8测定、实时细胞分析、流式细胞术、伤口愈合测定和Transwell测定来测量RA FLS的细胞增殖、凋亡和迁移。使用Transcreener UDP测定法测量RA和CIA大鼠的培养基、滑膜液（n = 36）和外周血（n = 36）中的UDP水平。使用流式测定法测量促炎细胞因子水平。使用ELISA和流式法测量白细胞介素-6（IL-6）水平。LC-MS分析检测到与OA SF相比，RA SF中的UDP水平显著升高，且该水平与RA中的抗环瓜氨酸肽（anti-CCP）和类风湿因子（RF）水平呈正相关。与OA患者和健康志愿者的样本相比，分别在RA患者的血液和滑膜液中验证了UDP浓度的升高。UDP刺激RA FLS的细胞增殖、迁移和IL-6分泌，并在培养中抑制其凋亡，而MRS2578抑制UDP的这些作用。在大鼠模型中，注射UDP加速了CIA并刺激了IL-6的产生，而不是其他促炎细胞因子，但同时注射MRS2578可抑制这些作用并减轻CIA。RA和CIA滑膜组织中P2Y6表达增加。这些结果表明，UDP在RA中高表达，并通过促进P2Y6活性以增加IL-6产生来刺激RA发病机制。
