| 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 |
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| 引用本文： | 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. DOI: 10.3969/j.issn.1672-5069.2021.03.004 |
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| 作者姓名： | 彭婕 李弼民 雷宇鹏 |
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| 作者单位： | 330000 南昌市南昌大学第一附属医院消化科 |
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| 基金项目： | 江西省教育厅科学技术研究基金资助项目(编号:180009)。 |
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| 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。
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| 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 |
| 收稿时间： | 2021-02-01 |
Changes of NOX4 gene and its protein in liver tissues of mice with CCl4-induced fibrosis and in HSC-T6 cells |
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| 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 Clinical Hepatology, 2021, 24(3): 319-322. DOI: 10.3969/j.issn.1672-5069.2021.03.004 |
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| Authors: | Peng Jie Li Bimin Lei Yupeng |
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| Affiliation: | Department of Gastroenterology, First Affiliated Hospital, Nanchang University School of Medicine, Nanchang 330000, Jiangxi Province, China |
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| 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,theα-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. |
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| Keywords: | Liver fibrosis HSC-T6 cells NADPH oxidase 4 TGF-β/Smad signaling pathway Mice |
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