Xinguang Yin, Huixing Yi, Xiaowei Wen, Xiaojun Wu, Linghua Yu
Centre for Gastroenterology and Hepatology, The First Affiliated Hospital of Jiaxing College, Jiaxing, Zhejiang Province, PR China.
Intensive Care Unit, The Second Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang Province, PR China.
J Surg Res. 2015 Feb;193(2):598-605. doi: 10.1016/j.jss.2014.08.009. Epub 2014 Aug 10.
The development of liver fibrosis is the key stage toward a number of mortal complications of liver diseases, including cirrhosis and hepatocellular carcinoma. Canonical Wnt pathway is crucial in diverse biological processes and mediates the progression and regression of liver fibrosis. As a potent Wnt pathway agonist, roof plate-specific spondin-1 (R-spondin1) protein's role in the hepatic fibrosis has not been well elucidated. The purpose of this study was to investigate whether R-spondin1 contributed to hepatic stellate cells (HSC) activation, the key event in liver fibrogenesis.
Tissue microarrays of human fibrotic liver samples, hepatocellular carcinoma samples, and normal hepatic tissue samples were constructed and immunostained for R-spondin1. Protein expression and transcriptional level of freshly isolated mice HSC were analyzed by Western blot assay and real-time polymerase chain reaction, respectively. Exogenous stimulation with recombinant R-spondin1 and Dickkopf-1 was performed to investigate the functionality. Nuclear β-catenin level and T-cell specific transcription factor activity were analyzed, and HSC proliferation was tested by Methyl-Thiazol-Tetrazolium bromide assay.
Overexpression of R-spondin1 was observed in both fibrotic liver tissues and culture-activated HSC. Coculture with recombinant R-spondin1 induced a dose-dependent increase in both the transcription factor activity and the protein level of α-smooth muscle actin, collagen I, and nuclear β-catenin. Additionally, Dickkopf-1 repressed R-spondin1's effect on HSC.
These findings suggested that R-spondin1 might argument liver fibrogenesis by enhancing the canonical Wnt pathway.
肝纤维化的发展是导致多种肝脏疾病致命并发症（包括肝硬化和肝细胞癌）的关键阶段。经典Wnt信号通路在多种生物学过程中起关键作用，并介导肝纤维化的进展和消退。作为一种有效的Wnt信号通路激动剂，顶板特异性类朊蛋白1（R-spondin1）蛋白在肝纤维化中的作用尚未得到充分阐明。本研究的目的是探讨R-spondin1是否促成肝星状细胞（HSC）激活，这是肝纤维化形成中的关键事件。
构建人纤维化肝脏样本、肝细胞癌样本和正常肝组织样本的组织芯片，并对R-spondin1进行免疫染色。分别通过蛋白质印迹分析和实时聚合酶链反应分析新鲜分离的小鼠HSC的蛋白表达和转录水平。进行重组R-spondin1和Dickkopf-1的外源性刺激以研究其功能。分析核β-连环蛋白水平和T细胞特异性转录因子活性，并通过甲基噻唑四氮唑溴盐法检测HSC增殖。
在纤维化肝组织和培养激活的HSC中均观察到R-spondin1的过表达。与重组R-spondin1共培养诱导转录因子活性以及α-平滑肌肌动蛋白、I型胶原和核β-连环蛋白的蛋白水平呈剂量依赖性增加。此外，Dickkopf-1抑制R-spondin1对HSC的作用。
这些发现表明，R-spondin1可能通过增强经典Wnt信号通路促进肝纤维化。
