四氯化碳诱导的肝纤维化小鼠肝组织和HSC-T6细胞NOX4基因及其蛋白表达的变化

doi:10.3969/j.issn.1672-5069.2021.03.004

摘要/Abstract

摘要： 目的探讨四氯化碳(CCl₄)诱导的肝纤维化小鼠和肝星状细胞(HSC-T6)还原型辅酶烟酰胺腺嘌呤二核苷酸磷酸氧化酶家族氧化酶4(NOX4)基因及其蛋白表达的变化。方法采用腹腔注射四氯化碳(CCl₄)构建10只小鼠肝纤维化模型,采用qRT-PCR法和Western Blot法检测小鼠肝组织NOX4基因和蛋白表达水平。将肝星状细胞(HSC-T6)分为对照组、无意干预组和NOX4-siRNA干预组,后两组分别采用脂质体2000将无意义序列或NOX4-siRNA转染HSC-T6细胞,采用qRT-PCR法和Western Blot法检测HSC-T6细胞NOX4、α平滑肌肌动蛋白(α-SMA)、I 型胶原(Col1a I)、组织金属蛋白酶抑制剂1(TIMP-1)、金属蛋白酶2(MMP-2)、转化生长因子 β1(TGF-β1)、Smad2和Smad3水平,采用DCFH-DA荧光探针法检测细胞活性氧(ROS)含量,采用MTT法检测细胞增殖,使用流式细胞术检测细胞周期和凋亡情况。结果模型小鼠肝组织出现明显的病理学损伤,有大量的胶原纤维沉积;模型组小鼠肝组织NOX4 mRNA水平显著高于对照组(P＜0.05);与对照组细胞比,NOX4-siRNA干预组细胞NOX4 mRNA及其蛋白、ROS、增殖活性、S期细胞比例、α-SMA、Col1a I、TIMP-1、MMP-2、TGF-β1、p-Smad2/Smad2、p-Smad3/Smad3表达水平显著降低,而G0/G1期细胞比例和细胞凋亡率显著升高(P＜0.05)。结论肝纤维化组织NOX4呈高水平,下调NOX4可抑制肝星状细胞的增殖和活化,并促进其凋亡,可能是通过下调TGF-β/Smad信号通路实现的。

关键词: 肝纤维化, HSC-T6细胞, 还原型辅酶烟酰胺腺嘌呤二核苷酸磷酸氧化酶家族氧化酶4, GF-β/Smad信号通路, 小鼠

Abstract: Objective The aim of this experiment was to explore the changes of NADPH oxidase 4 (NOX4) gene and its protein in liver tissues of mice with carbon tetrachloride (CCl₄)-induced liver fibrosis and hepatic stellate HSC-T6 cells.Methods The liver fibrosis model was established by intraperitoneal injection of CCl₄ in ten mice, and the NOX4 mRNA and its protein in liver tissues were detected by qRT-PCR and Western bloting. The HSC-T6 cells were normally cultured and divided into blank, nonsense and NOX4-siRNA-intervened groups, which were transfected by liposome 2000-coated meaningless sequence or NOX4-siRNA in the two latter groups. The expression ofNOX4, α-smooth muscle actin (α-SMA), type I collagen (Col1a I), tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase-2 (MMP-2), transforming growth factor-β1 (TGF-β1), Smad2 and Smad3 in HSC-T6 cells was detected by qRT-PCR and Western bloting. The intracellular reactive oxygen species (ROS) content was detected by DCFH-DA fluorescent probe, the cell proliferation was detected by MTT assay, and the cell cycles and apoptosis were detected by flow cytometry.Results There was a significant pathological damage, with a large amount of collagen fiber deposition in liver tissues of mice in model; the NOX4 mRNA level in liver tissues of mice in model was significantly higher than that in control group (P<0.05); the NOX4 mRNA and its protein, ROS, proliferation activity, percentage of cells in S phase, the α-SMA, Col1a I, TIMP-1, MMP-2, TGF-β1, p-Smad2/Smad2 and p-Smad3/Smad3 expression were significantly decreased, while the percentage of cells in G0/G1 phase, and apoptosis rate were significantly increased (P<0.05) in NOX4-siRNA-intervened group. Conclusion The NOX4 is highly expressed in liver fibrotic tissues, and the down-regulation of NOX4 could inhibit proliferation and activation of HSCs, and promote their apoptosis, which mmight be related to the inhibition of TGF-β/Smad signaling pathway.

Key words: Liver fibrosis, HSC-T6 cells, NADPH oxidase 4, TGF-β/Smad signaling pathway, Mice

彭婕, 李弼民, 雷宇鹏. 四氯化碳诱导的肝纤维化小鼠肝组织和HSC-T6细胞NOX4基因及其蛋白表达的变化[J]. 实用肝脏病杂志, 2021, 24(3): 319-322.

Peng Jie, Li Bimin, Lei Yupeng. Changes of NOX4 gene and its protein in liver tissues of mice with CCl₄-induced fibrosis and in HSC-T6 cells[J]. Journal of Practical Hepatology, 2021, 24(3): 319-322.

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