胆管结扎与再通对大鼠肝内细胞表型和NOX4蛋白表达的影响

Effect of bile duct ligation and recanalization on rat hepatocyte epithelial-mesenchymal phenotype and NOX_4 protein expression

目的设计并建立胆管结扎及结扎后再通的大鼠肝纤维化模型,观察胆管结扎及再通对大鼠肝组织内细胞上皮-间质表型和氧化应激相关蛋白表达的影响。方法 12只雄性Wista大鼠随机分为假手术组、胆管结扎2周组、胆管结扎4周组和胆管结扎2周后再通2周组,通过组织HE染色、Masson染色等方法评估模型大鼠肝纤维化病变程度;通过免疫组化和Western blot等方法并检测上皮、间质标记蛋白及氧化应激相关蛋白的表达情况。结果相较于假手术组,随着胆管结扎时间的延长,模型组大鼠肝纤维化明显加重,α-SMA、collagen I、NOX4和Vimetin等蛋白表达显著升高,而E-cadherin表达则明显降低;结扎后再通组大鼠肝纤维化较单纯胆管结扎4周组大鼠明显减轻,NOX4及间质细胞标记蛋白表明显低于单纯结扎4周组,内皮细胞标记蛋白表达较单纯结扎4周组则显著升高。结论胆管结扎可上调大鼠肝内间质表型相关蛋白及NOX4蛋白的表达,同时抑制上皮表型相关蛋白的表达;当实施再通手术后,原胆管结扎大鼠肝内上皮表型相关蛋白表达增加,而间质表型相关蛋白及NOX4蛋白的表达明显减少。

Objective To observe epithelial-mesenchymal phenotypes and oxidative stress related protein expressions of the liver cells in a rat model of liver fibrosis induced by bile duct ligation and recanalization. Methods Twenty-four male Wistar rats were randomized into 4 groups, including a sham-operated group, two bile duct ligation groups with ligation for 2 and 4weeks, and a bile duct ligation group with a 2-week ligation followed by a 2-week recanalization. HE staining and Masson staining were used to assess liver fibrosis in the rats, and immunohistochemistry and Western blotting were employed to detect expressions of the epithelial and mesenchymal marker proteins and oxidative stress-related proteins. Results Compared with the sham-operated group, the rats with bile duct ligation showed obvious liver fibrosis, which worsened as the ligation time extended, accompanied by significantly increased expression of α-SMA, collagen I, NOX4 and vimetin and reduced Ecadherin expression. Compared with the rats with bile duct ligation for 4 weeks, the rats in bile duct ligation-recanalization group showed obviously lessened liver fibrosis, significantly lowered expressions of NOX4 and mesenchymal cell maker proteins, and enhanced expressions of epithelial cell marker proteins. Conclusion Bile duct ligation up-regulates mesenchymal phenotype-related proteins and NOX4 protein expression and down-regulates the expression of epithelial phenotype-related proteins, and these changes can be reversed by subsequent bile duct recanalization.