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

转化生长因子(TGF)-β是一种分子量为25kd的二聚体多肽，在肝脏疾病的发病过程中起着关键作用。TGF -β是一种高分子量的潜伏形式，在肝脏疾病的发病过程中被纤溶蛋白（PLN）和血浆钾激肽（PLK）激活。在动物模型中，用低分子量蛋白酶抑制剂阻断激活反应可以阻止疾病的发展。基于这些发现，我们发现(1)人潜伏TGF-β1的LAP部分，PLN和PLK分别在K56-L57和R58-L59之间分裂。我们制造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端。抗r58抗体强烈染色暴发性肝炎患者的肝切片。(2)利用抗l59抗体建立ELISA法检测小鼠受损肝再生模型、大鼠CCl4和胆管结扎肝纤维化模型血清中LAP降解片段，与血清转氨酶水平和肝脏羟脯氨酸水平有良好的相关性。(3)含有裂解位点的诱饵肽阻断了肝星状细胞TGF-β的活化，促进了小鼠肝再生。(4)硬皮病中TGF-β2是其主要亚型，MMP参与其活化反应，PLN和PLK不参与。(5) CXZ已被证明可以抑制Smad的磷酸化，在小鼠模型中成功地改善了肝脏再生。这些结果表明，肝星状细胞周围发生PLN/ plk依赖性激活反应，产生活性TGF-β，这可能诱导纤维化并抑制肝细胞的增殖，从而解决了诱饵肽在肝炎治疗中的潜在用途。这些结果也表明LAP降解物作为肝纤维化的一种新的生物标志物的潜在用途。

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.