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

使用蛋白酶抑制剂改善肝纤维化/肝硬化和肝再生受损

基本信息

批准号：
13670579
负责人：
KOJIMA Soichi
金额：
$ 2.24万
依托单位：
The Institute of Physical and Chemical Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670579/
关键词：
Hepatic Fibrosis Cirrhosis Impaired Liver Regeneration TGF-β Activation Plasmin Plasma Kallikrein Protease Inhibitor Hepatic Stellate Cells Cutting Edge Antibody 肝硬変

项目摘要

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.

转化生长因子-β（transforminggrowthfactor-β，TGF-β）是一种强有力的纤维化和生长抑制细胞因子，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。潜伏性TGF-β通过用蛋白酶如纤溶酶（PLN）或血浆激肽释放酶（PLK）特异性切割潜伏性相关肽（LTP）部分而生理活化。在目前的研究中，使用动物模型，我们已经确定TGF-β在肝纤维化/肝硬化中由PLN分泌和激活，或在受损的肝再生中由PLK分泌和激活，因此用蛋白酶抑制剂包括FOY 305（甲磺酸卡莫司他）抑制激活可预防疾病的发展。然后，我们确定了不同的切割位点的PLN和PLK的切割部分，并提出了抗体在兔免疫的序列肽开始或结束于这些切割位点。纯化的抗体对N-末端的PLK切割位点特异性识别PLK切割的β，但不是未切割的β，和弱识别PLN切割的β在Western印迹。将来自LPS预处理和部分肝切除小鼠的肝切片用该抗体以及抗活性TGF-β抗体特异性免疫染色。与免疫前抗体相比，抗切割的TGF-β抗体还对来自暴发性肝炎患者的肝切片进行了微弱但显著的染色，表明可能发生PLK依赖性活化反应以产生活性TGF-β，其抑制患者肝脏的再生。此外，我们发现了一种化合物CXZ，其干扰TGF-β信号传导，从而在抑制肝星状细胞活化方面与蛋白酶抑制剂发挥协同作用，这表明CXZ和蛋白酶抑制剂对肝脏疾病的有希望的联合治疗。