Analysis of pathogenesis and control of arthritis in rheumatoid arthritis by calpain-calpastatin system

我们先前报道，类风湿关节炎(RA)患者自身抗体识别的靶抗原之一是钙化半胱氨酸蛋白酶(Calain)的内源性抑制物calastatin，而RA患者血清中的免疫球蛋白组份抑制calastatin的功能并增强calain的蛋白分解活性。钙蛋白酶被认为是一种中性蛋白酶，调节各种炎症因子的激活，可能参与软骨的降解。本研究旨在探讨Calain-calastatin系统治疗类风湿关节炎的机制及新的治疗策略。1.Calain抑制剂对小鼠关节炎模型的改善作用Calain抑制剂E--d可显著抑制抗II型胶原抗体诱导的小鼠关节炎。定量RT-PCR结果显示，E--d组小鼠关节组织中IL-1和IL-6mRNA的表达水平明显受到抑制。加入E--d后，RA患者滑膜细胞中IL-6和IL-1的产生也受到抑制，且呈剂量依赖关系。2.Calastatin过表达对成纤维细胞和T细胞的激活作用Th1细胞，尤其是活化的Th1细胞，其自然表达明显低于Th2细胞。重组逆转录病毒载体转染BALB/c小鼠和3T3成纤维细胞后，IL-6和干扰素-γ的产生受到明显抑制，提示类风湿关节炎患者可能通过向Th2和Th1细胞分化而增加Calain活性，并在滑膜细胞中过度产生IL-6。

We have previously reported that one of target autoantigens recognized by autoantibodies in rheumatoid arthritis (RA) patients is calpastatin, an endogenous inhibitor of calcium-dependent cysteine proteinase (calpain), and that IgG fraction of RA patient sera inhibits the function of calpastatin and increases the proteolytic activity of calpain. Calpain is thought to be a neutral proteinase that regulates activation of various inflammation factors and may be involved in cartilage degradation. Our study aims to investigate the pathogenesis and new therapeutic strategy of RA by calpain-calpastatin system.1.Amelioration of mouse model arthritis by calpain inhibitorMonoclonal anti-type II collagen antibodies-induced arthritis in Balb/c mice was significantly inhibited by intraperitoneal injection of calpain inhibitor E-64-d. Quantitative RT-PCR demonstrated that mRNA levels of IL-1 and IL-6 were significantly suppressed in the joint tissues of E-64-d-treated mice. Production of IL-6 and IL-1 was also suppressed in dose-dependent manner in cultured synoviocytes from RA patients when E-64-d was added.2.Effect of over expression of calpastatin in activation of fibroblasts and T cellsNatural expression of calpastatin was significantly decreased in Th1 cells, especially in activated Th1 cells, than in Th2 cells. When splenic CD4+T cells from Balb/c mice and 3T3 fibroblasts were transfected by human calpastatin cDNA-recombinant retrovirus vector, production of IL-6 and interferon-γ was significantly suppressed.Thus, in RA patients, increased calpain activity may be involved in exacerbation of arthritis by differentiating CD4+T cells from Th2 to Th1, as well as over production of IL-6 in synovial cells.