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

我们以前曾报道，在类风湿关节炎（RA）患者的自身抗体识别的靶抗原之一是钙蛋白酶抑制剂，钙依赖性半胱氨酸蛋白酶（calpain）的内源性抑制剂，和RA患者血清中的IgG组分抑制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。本研究旨在通过钙蛋白酶-钙蛋白酶抑制素系统探讨RA的发病机制和新的治疗策略。1.钙蛋白酶抑制剂对小鼠关节炎模型的改善腹腔注射钙蛋白酶抑制剂E-64-d可明显抑制单克隆抗II型胶原抗体诱导的Balb/c小鼠关节炎。定量RT-PCR结果显示，E-64-d处理的小鼠关节组织中IL-1和IL-6的mRNA水平明显受到抑制。E-64-d对RA滑膜细胞IL-6和IL-1的产生也有抑制作用，且呈剂量依赖性。2.钙蛋白酶抑制蛋白过表达对成纤维细胞和T细胞活化的影响Th 1细胞中钙蛋白酶抑制蛋白的天然表达明显低于Th 2细胞，尤其是活化的Th 1细胞。将人钙蛋白酶抑制蛋白cDNA重组逆转录病毒载体转染Balb/c小鼠脾CD 4 +T细胞和3 T3成纤维细胞后，IL-6和IFN-γ的产生明显受到抑制，提示RA患者钙蛋白酶活性的增加可能通过诱导CD 4 +T细胞由Th 2向Th 1分化，以及滑膜细胞IL-6的过度产生而参与关节炎的加重。

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.