Detection and regulation of hepatic diseases targeting TGF-β activation reaction.

转化生长因子（Transforming growth factor，TGF）-β是一种分子量为25 kD的二聚体多肽，在肝脏疾病的发病机制中起着关键作用，在肝脏疾病的发病过程中，TGF-β以高分子量的潜伏形式产生，并被纤溶酶（plasma protein，PLN）和血浆激肽释放酶（plasma kallikrein，PLK）激活。用低分子量蛋白酶抑制剂阻断活化反应可防止动物模型中疾病的发展。基于这些发现，我们发现（1）PLN和PLK分别在人潜伏性TGF-β1的K56-L57和R58-L59之间切割。我们生产了特异性识别由蛋白酶降解形成的新表位的抗体，即每个切割位点的切割末端。抗R58抗体强烈染色的肝脏切片与暴发性肝炎患者。(2)用抗L59抗体建立ELISA检测小鼠肝再生障碍模型、大鼠四氯化碳肝纤维化模型和胆管结扎肝纤维化模型血清中L59降解片段，与血清转氨酶水平和肝羟脯氨酸水平具有良好的相关性。(3)含有切割位点的诱饵肽阻断了肝星状细胞中TGF-β的活化，并改善了小鼠模型中的肝再生。(4)在硬皮病中，TGF-β2是主要的同种型，MMP而不是PLN和PLK似乎参与其活化反应。(5)CXZ可抑制Smad的磷酸化，成功地改善了小鼠模型的肝再生。这些结果表明，在肝星状细胞周围发生PLN/PLK依赖的活化反应，产生活性TGF-β，这可能诱导纤维化并抑制肝细胞增殖，从而解决了诱饵肽在肝炎治疗中的潜在用途。这些结果还表明，一个潜在的用途，作为一种新的生物标志物的肝纤维化的一种降解产物。

Transforming growth factor (TGF)-β, a 25 kD dimeric polypeptide playing a pivotal role in the pathogenesis of liver diseases, is produced as a high molecular weight latent form, and activated by plasmin (PLN) and plasma kallikrein (PLK) during pathogenesis of liver diseases. Blockage of the activation reaction with low molecular weight protease inhibitors prevented the development of the diseases in animal models. Based upon these findings, here, we found that(1) PLN and PLK cleaved between K56-L57 and R58-L59 in LAP portion of human latent TGF-β1, respectively. We produced antibodies that specifically recognize the neo-epitopes formed by protease degradation, namely the cut ends of each cleavage site. The anti-R58 antibodies strongly stained liver sections from patients with fulminant hepatitis.(2) The anti-L59 antibodies were successively used to establish ELISA to detect LAP degradation fragments in serum of mouse impaired liver regeneration model as well as rat CCl4 and bile duct ligation hepatic fibrosis models, showing a good correlation with serum transamidase levels and hepatic hydroxyproline levels.(3) Decoy peptides containing the cleavage sites blocked the activation of TGF-β in the hepatic stellate cells and improved hepatic regeneration in the mouse model.(4) In scleroderma TGF-β2 is the main isoform and MMP, but not PLN and PLK, appeared to be involved in its activation reaction.(5) CXZ, which has been shown to suppress phosphorylation of Smad, successfully improved hepatic regeneration in the mouse model.These results suggest that a PLN/PLK-dependent activation reaction occurs around hepatic stellate cells to produce active TGF-β, which may induce fibrosis and inhibit the proliferation of hepatocytes, thereby addressing a potential use for decoy peptides in hepatitis therapy. These results also suggest a potential usage of a LAP degradate as a novel biomarker for liver fibrosis.