醛固酮拮抗剂对肝纤维化大鼠NOX4蛋白表达的抑制作用

Aldosterone antagonist inhibits fibrosis-induced NOX4 protein expression in hepatic cells and tissues of

目的探讨醛固酮拮抗剂对肝纤维化大鼠NOX4蛋白表达的抑制作用。方法体内实验：雄性Wistar大鼠24只，随机分为3组，每组8只。模型组：用四氯化碳（CCl4）油2．5ml／kg皮下注射，3次／周;安体舒通组：CCl4油注射的同时予以安体舒通20mg／kg灌胃，1次／d；对照组：用橄榄油皮下注射，于4周后处死实验动物取材。用HE染色观察肝组织形态结构；Masson染色进行METAVIR肝纤维化评分；免疫组织化学法检测NOX4蛋白的表达。体外实验：将大鼠HSC-T6细胞株与不同剂量的醛固酮（10-9、10-7、10-5 mol／L，观察剂量效应）作用不同时间（6、12、24h）以观察时间效应。给予醛固酮（Aid）1μ mol／L、依普利酮1μmol／L预处理60min，再给予Ald刺激大鼠HSC-T6细胞，设立阴性对照组。Western blot方法检测NOX4蛋白的表达。采用One-wayA NOVA方差分析法，首先用Levene方法进行方差齐性检验，确定方差齐且整体比较组间差异有统计学意义后进一步做多重比较，多重比较采用LSD法。结果CCl4,组纤维化程度较对照组明显增高（相对表达量为16．060±0．300比2．471±0．160），两组比较，差异有统计学意义。CCl4,＋Sp组纤维化程度较CCl4组低（相对表达量为5．761±0．152比16．060±0．300），两组比较，差异有统计学意义。免疫组织化学结果显示，与对照组相比，CClt组NOX4蛋白表达明显增高（相对表达量为7．231±0．211比1．350±0．252），两组比较，差异有统计学意义。与CCl4组相比，CCl4＋Sp组NOX4蛋白表达下降（相对表达量为4．270±0．242比7．231±0．211），两组比较，差异有统计学意义。Aid刺激HSC-T6细胞后NOX4蛋白表达增强，并呈时间、浓度依赖性，在10-5mol／L浓度刺激24h达到峰值，与对照组相比，Aid组NOX4表达明显增加（相对表达量为0.710±0。011比0．316±0．015），两组比较，差异有统计学意义。与Ald组相比，Ald＋依普利酮组NOX4的表达减少（相对表达量为0．615±0．014比0．710±0．011），两组比较，差异有统计学意义。结论醛固酮拮抗剂通过抑制Ald诱导的氧化应激效应抑制肝纤维化大鼠NOX4蛋白的表达。

To investigate the inhibitory potential of aldosterone antagonist on NOX4 protein expression in hepatic fibrosis by using a rat model of carbon tetracholoride （CC14）- induced hepatotoxicity. Methods Twenty-four male Wistar rats were randomly divided into three equal groups： fibrosis model group （receiving three subcutaneous injections per week of 2.5 ml/kg 40% CC14）; spironolactone （Sp）-treated fibrosis model group （receiving CC14 regimen plus three injections per day of 20 mg/kg Sp in olive oil）; negative-treatment fibrosis model group （receiving CC14 regimen plus three injections per day of olive oil alone）. Unmanipulated rats （receiving no CC14 and no supplemental treatments） served as normal controls. After 4 weeks, liver histology was carried out to assess cytotoxicity （by hematoxylin-eosin staining）, fibrosis （by Masson staining and METAVIR scoring）, and NOX4 protein expression （by imrnunohistochemistry）. In addition, in vitro analyses of immortalized rat hepatic stellate cells, HSC-T6, were performed to evaluate dose-response （10-9, 10-7 and 105 mol/L） and time-response （6, 12 and 24 h） of aldosterone agonist （Aid） and an aldosterone antagonist, cplerenone （EPLE）. Effects on NOX4 protein expression were evaluated by western blotting. Results The fibrosis model group showed significantly more fibrosis than the normal control group （16.060±0.300 vs. 2.471±0.160, P = 0.000]; however, the Sp-treated fibrosis model group showed significantly less CCl4-induced fibrosis （5.761±0.152 vs. model： 16.060± 0.300, P = 0.000）. The fibrosis model group also showed significantly higher NOX4 protein expression in liver tissues than the normal control group （7.231 ±0.211 vs. 1.350±0.252, P = 0.000）, and the Sp-treated fibrosis model tissues showed significantly less CCl4-induced up-regulated NOX4 protein expression （4.270±0.242 vs. model： 7.231 ：i： 0.211, P = 0.000]. Aid induced up-regulated NOX4 protein expression in HSC-T6 cells in dose- and concentration-dependent manners, with the peak expression being induced by the I05 mol/L concentration and 24 h exposure. The Aid-treated cells expressed significantly more NOX4 protein than the untreated con~＇ol ceils （0.710±0.011 vs. 0.316±0.015, P=0.000]. and the EPLE-treated cells showed significantly less Aid-induced up-regulated NOX4 expression （0.615 4±0.014 vs. 0.710 4±0.011, P = 0.000]. Conclusion Aldosterone antagonists inhibit the firbosis-induccd NOX4 protein expression in rat hepatic ceils.