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

转化生长因子-β(TGF-β)是一种有效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，以潜伏形式分泌。它必须在与受体结合并发挥其活性之前被激活。潜伏TGF-β是通过潜伏相关肽（LAP）部分与蛋白酶（如纤溶酶（PLN）或血浆钾激肽（PLK））的特异性裂解而被生理激活的。在目前的研究中，通过动物模型，我们已经确定TGF-β在肝纤维化/肝硬化中由PLN分泌和激活，在肝再生受损时由PLK分泌和激活，因此用蛋白酶抑制剂（包括FOY305（甲磺酸卡莫司他））抑制其激活可以防止疾病的发展。然后，我们通过LAP部分的PLN和PLK确定了不同的裂解位点，并通过免疫从这些裂解位点开始或结束的LAP序列肽在家兔中产生抗体。纯化的抗PLK-cleaved位点n端抗体在Western Blot中特异性识别PLK-cleaved LAP，但不能识别non -cleaved LAP，弱识别PLN-cleaved LAP。lps预处理和部分肝切除小鼠的肝切片用该抗体和抗活性TGF-β抗体特异性免疫染色。与免疫前抗体相比，抗裂解LAP抗体对重型肝炎患者肝脏切片也有微弱但显著的染色，提示可能发生plk依赖性激活反应，产生活性TGF-β，抑制患者肝脏再生。此外，我们还发现了一种化合物CXZ，它可以干扰TGF-β信号，从而与蛋白酶抑制剂协同抑制肝星状细胞的活化，提示CXZ与蛋白酶抑制剂联合治疗肝脏疾病是一种很有前景的治疗方法。

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.