Biotherapy Centre, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; Cell-gene Therapy Translational Medicine Research Centre, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
Centre for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-sen University, Guangzhou, China; Department of Hepatic Surgery and Liver Transplantation Centre, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
J Hepatol. 2021 May;74(5):1176-1187. doi: 10.1016/j.jhep.2020.11.016. Epub 2020 Nov 17.
BACKGROUND & AIMS: Liver fibrosis is a wound healing response that arises from various aetiologies. The intermediate filament protein Nestin has been reported to participate in maintaining tissue homeostasis during wound healing responses. However, little is known about the role Nestin plays in liver fibrosis. This study investigated the function and precise regulatory network of Nestin during liver fibrosis.
Nestin expression was assessed via immunostaining and quantitative real-time PCR (qPCR) in fibrotic/cirrhotic samples. The induction of Nestin expression by transforming growth factor beta (TGFβ)-Smad2/3 signalling was investigated through luciferase reporter assays. The functional role of Nestin in hepatic stellate cells (HSCs) was investigated by examining the pathway activity of profibrogenic TGFβ-Smad2/3 signalling and degradation of TGFβ receptor I (TβRI) after interfering with Nestin. The in vivo effects of knocking down Nestin were examined with an adeno-associated virus vector (serotype 6, AAV6) carrying short-hairpin RNA targeting Nestin in fibrotic mouse models.
Nestin was mainly expressed in activated HSCs and increased with the progression of liver fibrosis. The profibrogenic pathway TGFβ-Smad2/3 induced Nestin expression directly. Knocking down Nestin promoted caveolin 1-mediated TβRI degradation, resulting in TGFβ-Smad2/3 pathway impairment and reduced fibrosis marker expression in HSCs. In AAV6-treated murine fibrotic models, knocking down Nestin resulted in decreased levels of inflammatory infiltration, hepatocellular damage, and a reduced degree of fibrosis.
The expression of Nestin in HSCs was induced by TGFβ and positively correlated with the degree of liver fibrosis. Knockdown of Nestin decreased activation of the TGFβ pathway and alleviated liver fibrosis both in vitro and in vivo. Our data demonstrate a novel role of Nestin in controlling HSC activation in liver fibrosis.
Liver fibrosis has various aetiologies but represents a common process in chronic liver diseases that is associated with high morbidity and mortality. Herein, we demonstrate that the intermediate filament protein Nestin plays an essential profibrogenic role in liver fibrosis by forming a positive feedback loop with the TGFβ-Smad2/3 pathway, providing a potential therapeutic target for the treatment of liver fibrosis.
肝纤维化是一种由多种病因引起的创伤愈合反应。中间丝蛋白巢蛋白已被报道参与维持创伤愈合反应中的组织内稳态。然而，关于巢蛋白在肝纤维化中的作用知之甚少。本研究旨在探讨巢蛋白在肝纤维化中的功能和精确调控网络。
通过免疫染色和实时定量 PCR（qPCR）评估纤维化/肝硬化样本中的巢蛋白表达。通过荧光素酶报告基因检测研究转化生长因子β（TGFβ）-Smad2/3 信号诱导巢蛋白表达的情况。通过检查促纤维化 TGFβ-Smad2/3 信号通路的活性以及干扰巢蛋白后 TGFβ 受体 I（TβRI）的降解，研究巢蛋白在肝星状细胞（HSCs）中的功能作用。利用携带靶向巢蛋白的短发夹 RNA 的腺相关病毒载体（血清型 6，AAV6）在纤维化小鼠模型中敲低巢蛋白，检测其体内效应。
巢蛋白主要在活化的 HSCs 中表达，并随着肝纤维化的进展而增加。促纤维化通路 TGFβ-Smad2/3 直接诱导巢蛋白表达。敲低巢蛋白促进了小窝蛋白 1 介导的 TβRI 降解，导致 TGFβ-Smad2/3 通路受损，HSCs 中纤维化标志物表达减少。在 AAV6 处理的小鼠纤维化模型中，敲低巢蛋白可降低炎症浸润、肝细胞损伤和纤维化程度。
HSCs 中的巢蛋白表达受 TGFβ 诱导，并与肝纤维化程度呈正相关。敲低巢蛋白可减少 TGFβ 通路的激活，无论是在体外还是体内均能减轻肝纤维化。我们的数据表明巢蛋白在控制肝纤维化中 HSC 活化方面具有新的作用。
肝纤维化有多种病因，但代表了慢性肝病中的一种常见过程，与高发病率和死亡率相关。在此，我们证明中间丝蛋白巢蛋白通过与 TGFβ-Smad2/3 通路形成正反馈环，在肝纤维化中发挥重要的促纤维化作用，为肝纤维化的治疗提供了一个潜在的治疗靶点。
