弗林蛋白酶靶向同源性磷酸-张力蛋白调控线粒体自噬及肝纤维化进展

doi:10.6040/j.issn.1671-7554.0.2024.1106

摘要/Abstract

摘要： 目的探讨弗林蛋白酶在肝纤维化发生发展中的调控作用及相关分子机制。方法将40只C57小鼠随机分为弗林蛋白酶过表达组和对照组,每组20只,腹腔注射四氯化碳诱导小鼠肝纤维化动物模型,诱导8周。弗林蛋白酶过表达组小鼠在四氯化碳诱导4周后,尾静脉注射弗林蛋白酶过表达转染质粒至实验结束,对照组尾静脉注射无意义对照质粒,观察弗林蛋白酶调控小鼠肝纤维化进展的作用效应;采用Nycodenz密度梯度分离法分离C57小鼠肝脏原代星状细胞并进行体外培养;采用免疫组织化学/免疫荧光染色法检测弗林蛋白酶在肝纤维化/肝硬化组织的表达及定位;采用Western blotting法检测组织及细胞蛋白表达水平;采用CCK8法检测弗林蛋白酶处理后肝星状细胞的增殖活力;采用Transwell法检测不同处理组细胞迁移能力变化;采用流式细胞法及免疫荧光染色法检测不同处理组细胞线粒体膜电位变化。结果弗林蛋白酶在肝硬化组织及活化的原代肝星状细胞中呈显著阳性。弗林蛋白酶处理组肝星状细胞活化、增殖及迁移能力显著受到抑制(P<0.05);对照组肝星状细胞活化、增殖以及迁移能力未受影响。弗林蛋白酶处理组肝星状细胞线粒体膜电位显著下降,对照组无明显下降(P<0.05);敲低同源性磷酸酶-张力蛋白(phosphatase and tensin homolog-long, PTEN-L)表达后肝星状细胞线粒体膜电位也显著下降(P<0.05),敲低对照组线粒体膜电位无明显改变。四氯化碳化学诱导实验中,弗林蛋白酶过表达组小鼠肝纤维化病变程度较轻,对照组小鼠出现较严重的肝纤维化组织学改变。结论弗林蛋白酶通过靶向作用PTEN-L,增强肝星状细胞线粒体自噬,抑制其活化及小鼠肝纤维化进展。

关键词: 肝星状细胞, 肝纤维化, 弗林蛋白酶, 线粒体自噬, 同源性磷酸酶-张力蛋白

Abstract: Objective To investigate the regulatory role of Furin in the progression of liver fibrosis and the molecular mechanisms involved. Methods Forty C57 mice were randomly divided into the Furin overexpression group and the control group, with 20 mice in each group. The mice were intraperitoneally injected with CCL4 to induce animal models of liver fibrosis for 8 weeks. Four weeks after CCL4 induction, mice in the Furin overexpression group received tail vein injections of Furin expression transfection plasmids until the end of the experiment, while the control group was injected with empty control plasmids. The effect of Furin on the progression of liver fibrosis was investigated. Primary hepatic stellate cells from C57 mice were isolated by the Nycodenz density gradient separation method and cultured in vitro. The expression and localisation of Furin in liver fibrosis/cirrhosis tissues were detected by immunohistochemistry / immunofluorescence staining assay. Protein expression levels in tissues and cells were detected by Western blotting. The proliferation activity of Furin-treated hepatic stellate cells was detected by CCK-8 assay. Changes in cell migration ability of each group by different treatments were detected by Transwell assay. Changes in mitochondrial membrane potential of cells in different treatment groups were detected by flow cytometry and immunofluorescence staining. Results Furin was significantly upregulated in cirrhosis tissues and activated primary hepatic stellate cells. The activation, proliferation and migration abilities of hepatic stellate cells in the Furin treatment group were significantly inhibited(P<0.05), while those in the control group were not affected. The mitochondrial membrane potential of hepatic stellate cells in the Furin treatment group was significantly decreased, while that in the control group was not significantly changed(P<0.05). The mitochondrial membrane potential of stellate cells also decreased significantly in PTEN-L knockdown experiments(P<0.05), while there were no significant changes in the control group. In CCl₄-induced liver fibrosis mice, the severity of liver fibrosis was milder in Furin overexpression group mice, while the control group mice showed more severe histological changes of liver fibrosis. Conclusion Furin targets PTEN-L to enhance hepatic stellate cell mitophagy and inhibit hepatic stellate cell activation and progression of liver fibrosis in mice.

Key words: Hepatic stellate cells, Liver fibrosis, Furin, Mitophagy, Phosphatase and tensin homolog-long

中图分类号:

R575

宋彦威,付振美,徐静怡,马铭泽,孙琳琳. 弗林蛋白酶靶向同源性磷酸-张力蛋白调控线粒体自噬及肝纤维化进展[J]. 山东大学学报 (医学版), 2025, 63(4): 59-68.

SONG Yanwei, FU Zhenmei, XU Jingyi, MA Mingze, SUN Linlin. Furin regulates mitophagy and liver fibrosis progression through phosphatase and tensin homolog-long in mouse[J]. Journal of Shandong University (Health Sciences), 2025, 63(4): 59-68.

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