Improvement of Hepatic fibrosis/cirrhosis and impaired liver regeneration using protease inhibitors

转化生长因子-β是一种强有力的促纤维化和抑制生长的细胞因子，由两种不同的基因产物组成，并以潜伏期的形式分泌。在与受体结合并发挥其活性之前，它必须被激活。潜伏的转化生长因子-β通过潜伏期相关肽(LAP)部分与纤溶酶(PLN)或血浆激肽释放酶(PLK)等酶的特异性切割而在生理上被激活。在目前的研究中，我们利用动物模型证实，转化生长因子-β在肝纤维化/肝硬变时由磷脂酰肌醇分泌和激活，或在肝再生受损时由磷脂酰肌醇激活，因此用包括FOY305(卡莫司坦甲磺酸)在内的蛋白酶抑制剂抑制其激活可阻止疾病的发展。然后，我们用LAP部分的PLN和PLK来确定不同的裂解部位，并通过免疫从这些裂解部位开始或结束的LAP序列多肽来提高兔的抗体水平。在Western Blot中，针对PLK裂解位点N端的纯化抗体特异性地识别PLK裂解的LAP，而不能识别未裂解的LAP，弱识别PLN裂解的LAP。用该抗体和抗活性转化生长因子-β抗体对脂多糖预处理小鼠和部分肝切除小鼠的肝切片进行了特异性免疫染色。与免疫前抗体相比，抗切割的LAP抗体对重型肝炎患者的肝脏切片的染色较弱，但明显染色，提示可能发生PLK依赖的激活反应，产生活性的转化生长因子-β，从而抑制患者肝脏的再生。此外，我们还发现了一种化合物CXZ，它可以干扰转化生长因子-β信号转导，从而在抑制蛋白水解酶抑制剂抑制肝星状细胞活化方面发挥协同作用，提示CXZ与蛋白水解酶抑制剂联合治疗肝病具有很好的前景。

Transforming growth factor-β(TGF-β), a potent fibrogenic and growth-suppressing cytokine, is composed of two different gene products, and secreted as latent form. It must be activated before binding to receptors and exerting its activities. Latent TGF-β is physiologically activated by specific cleavage of latency-associated peptide (LAP) portion with proteases such as plasmin (PLN) or plasma kallikrein (PLK). In the current study, using animal models, we have established that TGF-β is secreted and activated either by PLN in liver fibrosis/cirrhosis or by PLK in impaired liver regeneration, and therefore that inhibition of the activation with protease inhibitors including FOY305 (camostat mesilate) prevents the development of the diseases. We then determined different cleavage sites by PLN and PLK in LAP portion and raised antibodies in rabbits by immunizing LAP sequence peptides beginning from or ending at these cleavage sites. Purified antibody against N-terminus of PLK-cleaved site specifically recognized PLK-cleaved LAP, but not uncleaved LAP, and weakly recognized PLN-cleaved LAP in Western Blot. Liver sections from LPS-pretreated and partial hepatectomized mice were specifically immunostained with this antibody as well as anti-active TGF-β antibody. The anti-cleaved LAP antibody also weakly but significantly stained liver sections from patients with fulminant hepatitis compared to pre-immune antibody, suggesting that PLK-dependent activation reaction may occur to produce active TGF-β, which suppresses regeneration of the patient liver. In addition, we discovered a compound CXZ, which interferes with TGF-β signaling and thus exerts a synergism in suppressing the activation of hepatic stellate cells with the protease inhibitor, suggesting a promising combination therapy of CXZ and protease inhibitors against the liver diseases.