目的 探究小分子化合物逆转素(reversine,Rev)对胆管结扎(BDL)诱导的大鼠胆汁淤积性肝损害、纤维化、上皮细胞-间充质转化以及胆管反应的影响。方法 雄性Lewis大鼠随机分成三组,每组各5只。按照如下处理:BDL组大鼠行2周的胆管结扎;BDL+Rev组行胆管结扎同时给予腹腔注射逆转素;对照采用假手术(Sham)。2周后获取血液和肝组织。血指标检测总白蛋白(TP)、总胆红素(TBIL)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、γ谷氨酰转肽酶(GGT)。H&E染色检测肝组织病理。Azan染色检测组织胶原蛋白。免疫组化检测肝组织α平滑肌肌动蛋白(α-SMA)、结蛋白(Desmin)、波形蛋白(Vimentin)、细胞角蛋白(CK7,CK19)、β-连环蛋白(β-Catenin)以及上皮细胞粘附分子(EpCAM)蛋白的表达情况。结果 胆管结扎导致肝脏合成的总白蛋白量下降,总胆红素(TBIL)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、γ谷氨酰转肽酶(GGT)水平明显上升,逆转素处理使下降的总白蛋白上升,使上升的总胆红素(TBIL)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、γ谷氨酰转肽酶(GGT)水平向正常水平回复。逆转素可以缓解胆汁淤积引起的肝纤维化,表现为下调BDL引起的胶原蛋白和α-SMA蛋白沉积。逆转素可以抑制胆汁淤积引起的上皮细胞-间充质转化表现为逆转素明显降低BDL导致的Desmin和Vimentin的表达。逆转素可以抑制胆汁淤积引起的胆管反应表现为明显减少CK7和CK19阳性胆管的表达含量。逆转素抑制胆汁淤积引起的胆管反应与调节β-Catenin和EpCAM的表达有关。结论 逆转素可以缓解胆汁淤积引起的大鼠肝损害,具有一定的保护作用。逆转素可以成为一种潜在治疗药物。

Objective To investigate the effect of reversine (REV) on bile duct ligation (BDL) -induced hepatic damage, fibrosis, epithelial-mesenchymal transformation, and ductular reaction in rats. Methods Male Lewis rats were randomly divided into three groups with 5 rats in each group. Bile duct ligation was performed in the BDL group for two weeks. BDL+ REV group was treated with bile duct ligation and intraperitoneal injection of reversine. The control group was Sham operation (Sham). Blood and liver tissue were obtained after 2 weeks. Blood indexes were determined for total albumin, total bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and γ-glutamyl transpeptidase. Hepatic histopathology was detected by H&E staining. Azan staining was used to detect tissue collagen deposition. Immunohistochemistry was used to detect the expression of α-SMA, desmin, vimentin, cytokeratin, β-catenin and epithelial cell adhesion molecule (EpCAM) protein. Results Bile duct ligation resulted in the decrease of total albumin synthesis in liver, and the increase of total bilirubin, glutamic-oxalacetic transaminase, alkaline phosphatase and γ-glutamyltranspeptidase. The levels of total bilirubin, aspartate transaminase, alkaline phosphatase and γ-glutamyltranspeptidase returned to the normal level with reversine treatment. Reversine could alleviate cholestasis-induced liver fibrosis by downregulating BDL-induced deposition of collagen and α-SMA protein. Reversine inhibited cholestasis-induced epithelial-mesenchymal transformation by significantly reducing BDL-induced desmin and vimentin expression. Reversine could inhibit cholestasis-induced ductular reaction by significantly reducing the expression of CK7 and CK19 positive biliary cells. Inhibition of cholestasis induced ductular reaction by reversine was associated with regulation of β-catenin and EpCAM expression. Conclusion Reversine can alleviate liver damage caused by cholestasis in rats and have a protective effect. Reversine may be a potential treatment that need further investigation.

目的 探讨Reversine对人肝星状细胞系LX-2凋亡的影响。方法 设对照组和Reversine干预组,其中Reversine干预组分为7个浓度,分别为1,5,10,20,40,80,120 μg/mL,CCK-8法检测Reversine对LX-2增殖的影响,选取最佳浓度。将细胞重悬在加入5 μL FITC-Annexin V和5 μL PI,用流式细胞仪进行了凋亡率分析,免疫荧光检测凋亡蛋白bcl-2及caspase 3。结果 Reversine可促进LX-2细胞凋亡,随着Reversine浓度增加,LX-2的凋亡可呈剂量依赖关系,其中10 μg/mL为最佳浓度,LX-2细胞的bcl-2蛋白的表达显著下降而cleaved-caspase 3的表达显著上升。结论 Reversine可通过促进caspase-3蛋白活化、抑制bcl-2蛋白表达的方式诱导LX-2凋亡。