阮文丽, 范丽丽, 徐慧芬, 宋倩, 何瑞, 刁珩, 张玉琼, 张爱华, 王大朋. 丝裂原诱导基因6在砷致人肝星状细胞活化及细胞外基质沉积中的作用[J]. 环境与职业医学, 2022, 39(2): 200-205. DOI: 10.11836/JEOM21293
引用本文: 阮文丽, 范丽丽, 徐慧芬, 宋倩, 何瑞, 刁珩, 张玉琼, 张爱华, 王大朋. 丝裂原诱导基因6在砷致人肝星状细胞活化及细胞外基质沉积中的作用[J]. 环境与职业医学, 2022, 39(2): 200-205. DOI: 10.11836/JEOM21293
RUAN Wenli, FAN Lili, XU Huifen, SONG Qian, HE Rui, DIAO Heng, ZHANG Yuqiong, ZHANG Aihua, WANG Dapeng. Role of mitogen-inducible gene 6 in the activation of human hepatic stellate cells and deposition of extracellular matrix induced by sodium arsenite[J]. Journal of Environmental and Occupational Medicine, 2022, 39(2): 200-205. DOI: 10.11836/JEOM21293
Citation: RUAN Wenli, FAN Lili, XU Huifen, SONG Qian, HE Rui, DIAO Heng, ZHANG Yuqiong, ZHANG Aihua, WANG Dapeng. Role of mitogen-inducible gene 6 in the activation of human hepatic stellate cells and deposition of extracellular matrix induced by sodium arsenite[J]. Journal of Environmental and Occupational Medicine, 2022, 39(2): 200-205. DOI: 10.11836/JEOM21293

丝裂原诱导基因6在砷致人肝星状细胞活化及细胞外基质沉积中的作用

Role of mitogen-inducible gene 6 in the activation of human hepatic stellate cells and deposition of extracellular matrix induced by sodium arsenite

  • 摘要: 背景 砷是一种环境毒物,长期或过量砷暴露可致机体肝纤维化损伤,其具体机制尚不清楚;丝裂原诱导基因6(Mig-6)在多种疾病或癌症中具有保护作用,但其在砷致肝纤维化损伤过程的作用尚不清楚。

    目的 探讨Mig-6在亚砷酸钠(NaAsO2)致人肝星状细胞(HSC)活化及细胞外基质(ECM)沉积中的作用及机制。

    方法 采用0、1.875、3.75、7.5、15 μmol·L−1NaAsO2处理体外常规培养的人肝星状细胞株 (Lx-2)24 h,另以7.5 μmol·L−1NaAsO2处理Lx-2细胞0、12、24、48、72 h,到达处理终点收集细胞。进一步采用pcDNA3.1(+)/Mig-6质粒转染Lx-2细胞,并在此基础上以7.5 μmol·L−1 NaAsO2处理细胞24 h,另设空白对照组、pcDNA3.1(+)空载体转染组、pcDNA3.1(+)/Mig-6转染组、单独染砷组(7.5 μmol·L−1);收集各组细胞及培养上清,Western blotting法检测Mig-6及Lx-2细胞活化相关蛋白α-平滑肌肌动蛋白(α-SMA)、转化生长因子β1(TGF-β1)表达水平,ELISA法检测培养细胞上清中ECM主要成分透明质酸(HA)、层黏连蛋白(LN)、IV型胶原(COL-IV)、III型前胶原氨基端肽(PIIINP)的分泌水平。

    结果 NaAsO2处理Lx-2细胞后,与对照组(1.000±0.000)比较,3.75、7.5、15 μmol·L−1染砷组Mig-6蛋白表达水平降低(0.561±0.095、0.695±0.048、0.401±0.030)(P<0.05);染砷24、48、72 h后Mig-6蛋白表达水平(0.856±0.036、0.515±0.077、0.491±0.060)均较0 h时(1.000±0.000)降低(P<0.05)。过表达Mig-6后,Lx-2细胞活化相关蛋白检测结果显示,与对照组比较,单独染砷组α-SMA和TGF-β1蛋白表达水平均升高(P<0.05);而与单独染砷组比较,Mig-6过表达联合染砷组α-SMA、TGF-β1蛋白表达水平则降低(P<0.05);ELISA法检测结果显示,与对照组比较,单独染砷组HA、LN、PIIINP、COL-IV分泌水平升高(P<0.05);而与单独染砷组比较,Mig-6过表达联合染砷组HA、LN、PIIINP、COL-IV分泌水平则降低(P<0.05)。

    结论 砷可下调HSC中Mig-6蛋白表达水平,过表达Mig-6可逆转砷暴露所引起的HSC活化及ECM沉积,提示Mig-6在砷所致HSC激活及ECM沉积中具有保护作用。

     

    Abstract: Background Arsenic is a well-known environmental toxicant. Hepatic fibrosis could occur dueto excessive or long-term exposure to arsenic, while associated molecular mechanisms remain undefined. Mitogen-inducible gene 6 (Mig-6) exhibits a protective effect on numerous diseases or cancers. However, the specific role of Mig-6 in the mechanisms of arsenite-induced hepatic fibrosis remains indistinct.

    Objective To investigate the specific role of Mig-6 in the activation of hepatic stellate cells (HSC) and the deposition of extracellular matrix (ECM) induced by sodium arsenite (NaAsO2).

    Methods Human hepatic stellate cells (Lx-2) were treated with 0, 1.875, 3.75, 7.5, and 15 μmol·L−1 of NaAsO2 for 24 h, or with 7.5 μmol·L−1 NaAsO2 for 0, 12, 24, 48, and 72 h. Additionally, Lx-2 cells were transfected by pcDNA3.1(+)/Mig-6, then treated with 7.5 μmol·L−1 NaAsO2 for 24 h; a blank control group, a pcDNA3.1(+)-control group, a pcDNA3.1(+)/Mig-6 group, and an arsenic (7.5 μmol·L−1 NaAsO2) group were also set up. After transfection, the cells and culture supernatants were collected, and the protein levels of Mig-6, α-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1) in Lx-2 cells were identified by Western blotting analysis; moreover, the secretion levels of main ECM components in supernatants such as hyaluronic acid (HA), laminin (LN), collagens IV (COL-IV), and procollagen-III (PIIINP) were tested by ELISA.

    Results The Mig-6 expression decreased in the 3.75, 7.5, and 15 μmol·L−1 NaAsO2 groups (0.561±0.095, 0.695±0.048, and 0.401±0.030) compared to the control group (1.000±0.000) in Lx-2 cells (P<0.05). After administration with 7.5 μmol·L−1 of NaAsO2 for 24, 48, and 72 h, the Mig-6 expression (0.856±0.036, 0.515±0.077, 0.491±0.060) decreased compared with the 0 h group (1.000±0.000) (P<0.05). After over-expression of Mig-6, the results of Lx-2 activation related protein levels showed that compared to the control group, the α-SMA and TGF-β1 expression were up-regulated in the arsenic group (P<0.05); meanwhile, the α-SMA and TGF-β1 in the Mig-6 over-expression combined arsenic exposure group reduced compared to the arsenic (7.5 μmol·L−1) group (P<0.05). The results of ELISA showed that compared with the control group, the HA, LN, PIIINP, COL-IV in the arsenic group were up-regulated (P<0.05); while compared to the arsenic group, the HA, LN, PIIINP, and COL-IV in the Mig-6 over-expression combined with arsenic exposure group were decreased (P<0.05).

    Conclusion Arsenic down-regulates Mig-6 expression in HSC, and over-expression of Mig-6 can reverse the activation of HSC and ECM deposition induced by arsenic exposure. It suggests that Mig-6 plays a protective role in arsenic-induced HSC activation and ECM deposition.

     

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