Kallistatin调节肝星状细胞EMT抑制肝纤维化发展的机制研究

Hepatic fibrosis is one of common diseases which is seriously harmful to human health and may develop liver cirrhosis and even liver cancer. Recently, it was reported that Kallistatin inhibited hepatic fibrosis in mouse model. We found that plasma Kallistatin was decreased in hepatic fibrosis patients. Moreover, Kallistatin reversed Hepatic stellate cells (HSCs) mesechymal phenotype, up-regulated E-cadherin and down-regualted N-cadherin, α-SMA as well as collagen I. Furthermore, the epithelial-mesenchymal transition (EMT) of HSCs is closely related to hepatic fibrosis. Therefore, our preliminary data suggest that Kallistatin may be an approach for the inhibition of hepatic fibrosis via inversing EMT of HSCs. This study was aimed to further illustrate the role and mechanism of Kallistatin in hepatic fibrosis: (1) To define whether Wnt/β-catenin signaling pathway is involved in the regulation of HSCs EMT; (2) To validate whether inversion of HCSs EMT is mediated by membrane receptor LRP6. Overall, this study will elucidate the molecular mechanism by which Kallistatin regulates EMT and hepatic fibrosis, thus providing a new candidate drug for the treatment of hepatic fibrosis.

肝纤维化是严重危害人类健康的常见病之一,将最终发展为肝硬化，乃至肝癌。最新研究报道Kallistatin能抑制小鼠肝脏的纤维化，我们最近研究显示肝纤维化病人血浆Kallistatin水平降低；进一步研究发现Kallistatin逆转肝星状细胞间皮表型并上调E-cadherin、下调N-cadherin和α-SMA，抑制I型胶原的表达；而肝星状细胞EMT与肝纤维化密切相关，提示Kallistatin可能通过逆转肝星状细胞EMT抑制肝纤维化发展。本项目拟进一步探讨其分子机制：（1）明确Kallistatin是否通过Wnt/β-catenin信号通路调节肝星状细胞的EMT；（2）明确LRP6是否是介导Kallistatin调节肝星状细胞EMT的膜受体。旨在阐明Kallistatin 调控EMT和肝纤维化的分子机制，从而为kallistatin治疗肝纤维化提供新的候选药物。

激肽释放酶结合蛋白（Kallistatin）与体内许多生理、病理状态有关，如癌症、炎症、组织纤维化、糖尿病等。我们首次发现肝纤维化病人血浆中Kallistatin水平降低，提示其与肝纤维化密切相关。文献报道CCl4诱导的小鼠肝纤维化模型中，Kallistatin重组蛋白可通过抑制小鼠肝脏细胞因子TGFβ的表达来保护肝脏；细胞上发现Kallstatin可以清除肝星状细胞内的H2O2从而发挥抗氧化损伤功能。但是，关于Kallistatin调节EMT来逆转肝纤维化方面尚未见相关报道。为此本项目设计相关内容并取得以下重要结果：（1）免疫组织化学染色发现，相比于人正常肝脏组织标本，随着肝纤维化分期增加，人肝脏组织Kallistatin表达逐渐下调，EMT间皮标志物Vimentin表达逐渐上调；并且Kallistatin表达与Vimentin的表达负相关（r=0.213， p<0.05）；提示Kallistatin表达水平与肝纤维化时EMT及肝纤维化分期存在相关性；（2）此外我们构建了过表达Kallistatin的腺病毒，HE和Sirrus Red染色显示大鼠尾静脉注射Kallistatin腺病毒后能够逆转CCl4所诱导的大鼠肝脏纤维化和肝脏损伤，抑制肝纤维化EMT间皮标志物Vimentin和α-SMA的表达，并且调控Wnt/β-Catenin信号通路的关键蛋白β-Catenin的表达。以上结果表明，在肝纤维化中，Kaliistatin可能通过β-Catenin发挥抑制EMT从而逆转肝纤维化的作用；（3）最后，在机制方面，我们发现Wnt/β-Catenin信号通路和TGFβ/Smad信号通路均参与Kallistatin调节肝星状细胞EMT。本项目阐明了Kallistatin调节EMT和肝纤维化发展的分子机制，为治疗肝纤维化提供新的候选药物，同时信号通路和关键调控分子的阐明也为寻找新的肝纤维化治疗有效干预靶点提供重要线索。