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

我们之前报道过，类风湿性关节炎（RA）患者自身抗体识别的靶自身抗原之一是钙依赖性半胱氨酸蛋白酶（calpain）的内源性抑制剂calpastatin， RA患者血清中的IgG部分抑制calpastatin的功能并增加calpain的蛋白水解活性。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，并可能参与软骨降解。本研究旨在通过calpain-calpastatin系统探讨RA的发病机制及新的治疗策略。calpain抑制剂E-64-d对小鼠关节炎模型的改善作用腹腔注射calpain抑制剂E-64-d能明显抑制Balb/c小鼠单克隆抗II型胶原抗体诱导的关节炎。定量RT-PCR结果显示，e -64-d处理小鼠关节组织中IL-1和IL-6 mRNA水平明显受到抑制。当添加E-64-d时，RA患者培养的滑膜细胞中IL-6和IL-1的产生也呈剂量依赖性地受到抑制。calpastatin过表达对成纤维细胞和T细胞活化的影响Th1细胞中calpastatin的自然表达明显低于Th2细胞，尤其是活化的Th1细胞。用人钙pastatin cdna -重组逆转录病毒载体转染Balb/c小鼠脾CD4+T细胞和3T3成纤维细胞后，IL-6和干扰素-γ的产生明显受到抑制。因此，在RA患者中，钙蛋白酶活性的增加可能通过CD4+T细胞从Th2分化到Th1，以及滑膜细胞中IL-6的过度产生参与关节炎的恶化。

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.