靶向调控β-arrestin2表达对肝纤维化星状细胞胶原产生的影响及机制

Liver fibrosis is a common end-result of a wide variety of chronic liver diseases following various types of injuries. It is often associated with severe morbidity and significant mortality. The activation of hepatic stellate cells (HSC) plays an essential role in the fibrosis progression. The activated HSC is distinguished by accelerated proliferation and enhanced production of extracellular matrix (ECM) components. Currently, clinical reports suggested that advanced liver fibrosis is potentially reversible. Thus, it is essential to develop therapeutic strategies to counteract liver fibrosis. β-arrestin2 is multifunctional adaptor protein. Mounting evidence suggests that, in addition to regulation of G protein-coupled receptors signals, β-arrestin2 also serve as modulators in a number of intracellular signaling pathways, which play important roles in the regulation of various cellular functions. Though, the role of β-arrestin2 expression in the pathology of hepatic fibrosis remains unclear.Our previous studies have shown that β-arrestin2 depletion in HSC reduces the platelet derived growth factor BB(PDGF-BB) promoted activation of ERK1/2 pathway, thereby inhibited the proliferation of HSC. Although our studies indicate β-arrestin2 depletion can diminish HSC proliferation, little is known about its participation in the modulation of the collagen production. This study was designed to investigate the role of β-arrestin2 in liver fibrosis and the production of collagen by HSC. To that end, we will choose human samples, which are biopsies from chronic hepatitis patients showing different degrees of fibrosis or control liver samples showing normal histology or minimal change, to determine the possible relationship between β-arrestin2 expression and the degree of fibrosis. Meanwhile, porcine serum (PS) induced liver fibrosis model in rats was used in this study. At different time points, the dynamic expression of β-arrestin2, collagen levels and type III transforming growth factor β receptor(TβRIII) signaling were detected to determine possible relationship between β-arrestin2 and collagen production in vivo. In vitro studies, the dynamic expression of β-arrestin2 in transforming growth factor β1(TGF-β1) stimulated HSC, believed to be a critical component of collagen production, was assessed. Furthermore, the small interfering RNA (siRNA) technique and overexpression plasmid were used to explore the effect of β-arrestin2 on the collagen production of HSC and the TβRIII signaling. Thus, to elucidate the underlying mechanisms of β-arrestin2 mediated collagen production of HSC. We next constructed lentiviral vector targeting β-arrestin2 gene by RNA interference and its effects on PS induced liver fibrosis rats will be explored. Altogether, the present studies will be helpful to discover some new mechanisms underlying the liver fibrosis, and develop a novel therapy target for treatment of hepatic fibrosis.

肝星状细胞（HSC）过度增殖并产生以胶原为主的细胞外基质是各种原因引起肝纤维化的中心环节。课题组前期研究发现，敲除β-arrestin2可抑制HSC增殖，但其在HSC胶原产生中的作用及机制尚不清楚。我们推测β-arrestin2可调节HSC胶原产生参与肝纤维化的病理过程。本项目分别采用肝纤维化临床标本和动物模型，分析β-arrestin2表达与纤维化严重程度的关系。在细胞水平通过RNA干扰或过表达质粒调控HSC β-arrestin2表达，观察胶原合成相关指标的变化，并深入探讨其对转化生长因子 III型受体信号通路的调节，揭示β-arrestin2参与胶原合成的崭新机制。在此基础上，构建β-arrestin2基因RNA干扰慢病毒载体，在整体动物水平研究下调β-arrestin2表达对肝纤维化的影响。本项目不仅能为肝纤维化的病理分子机制提供新的依据，而且能为肝纤维化治疗的新靶点提供实验依据。

肝星状细胞（HSC）过度增殖并产生以胶原为主的细胞外基质是各种原因引起肝纤维化的中心环节。课题组前期研究发现，敲除β-arrestin2可抑制HSC增殖，但其在HSC胶原产生中的作用及机制尚不清楚。本课题在以往工作的基础上，试图阐明β-arrestin2在肝纤维化胶原产生中的作用及其可能机制。首先，免疫组化观察了临床肝纤维化患者标本中β-arrestin2的表达与疾病严重程度的关系，发现随着肝纤维化程度的加重，β-arrestin2表达升高。其次，建立猪血清诱导的免疫性肝纤维化大鼠模型，WB检测发现Collagen I、III随着造模时间延长而表达升高，TβRIII表达降低；相关性分析结果表明，Collagen I、III的表达与β-arrestin2表达呈现正相关，TβRIII与β-arrestin2的表达呈负相关。体外研究以TGF-β1刺激的HSC作为细胞胶原合成模型，发现随着TGF-β1刺激时间的延长，TβRIII表达降低，β-arrestin2表达升高，p-Smad2/3、p-Akt表达增加。HSC转染β-arrestin2过表达质粒后，细胞中胶原相关蛋白的表达升高，促进Smad2/3和Akt信号通路的激活。HSC转染针对β-arrestin2的siRNA后，TGF-β1的刺激无法增强β-arrestin2与TβRIII的相互作用，转染后细胞中胶原相关蛋白的表达被抑制，Smad2/3和Akt信号通路的激活都被抑制。以上结果提示，β-arrestin2可能通过增强与TβRIII的作用，减弱了TβRIII对Smad2/3、Akt通路的抑制作用，进而促进胶原产生。为了在整体水平验证上述结果，我们进一步使用β-arrestin2基因敲除小鼠，建立四氯化碳诱导的肝纤维化模型，观察敲除β-arrestin2是否对肝纤维化具有保护作用。结果发现，与野生型小鼠相比，β-arrestin2敲除鼠肝脏胶原表达降低，p-Smad2/3、p-Akt的表达降低，提示敲除β-arrestin2对肝纤维化具有保护作用。本课题研究结果提示靶向干预β-arrestin2可发挥抑制HSC增殖和胶原合成的双重作用，干预β-arrestin2可能是治疗肝纤维化的药物新靶点，为研发治疗肝纤维化新药提供了新的靶点和实验依据，具有重要的理论意义和潜在的应用价值。

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