目的 基于蛋白组学和实验验证探讨芍药苷通过紧密连接通路改善胆汁淤积的作用。方法 SD大鼠随机分为对照组、模型组、熊去氧胆酸（60 mg/kg）组和芍药苷高、中、低剂量（200、100、50 mg/kg）组，每组8只。连续7 d ig相应药物，于第4天给药2 h后ig α-萘异硫氰酸酯（60 mg/kg）诱导肝内胆汁淤积大鼠模型。末次给药后，采用试剂盒测定大鼠血清中肝功能指标；苏木素-伊红（HE）染色观察大鼠肝脏组织病理变化；蛋白组学及生物信息学技术筛选芍药苷改善胆汁淤积的关键通路；分子对接确定芍药苷与相关靶点的结合能；qRT-PCR和Western blotting验证大鼠肝组织中关键靶点闭锁小带蛋白-1（zonula occludens-1，ZO-1）、闭合蛋白（Occludin）、密封蛋白1/2/3（Claudin 1/2/3）和连接黏附分子-A（junctional adhesion molecule-A，JAM-A）的表达。结果 药效学结果显示，与对照组比较，模型组大鼠血清中天冬氨酸氨基转移酶（aspartate aminotransferase，AST）、丙氨酸氨基转移酶（alanine aminotransferase，ALT）、碱性磷酸酶（alkaline phosphatase，ALP）、γ-谷氨酰转肽酶（γ-glutamyl transpeptidase，γ-GT）活性及总胆汁酸（total bile acid，TBA）、总胆红素（total bilirubin，TBIL）、直接胆红素（direct bilirubin，DBIL）水平均显著升高（P<0.01），肝组织可见大量炎性细胞浸润，伴随着肝细胞变性坏死，而经芍药苷治疗后上述指标得到显著改善（P<0.05、0.01）。蛋白组学结果表明芍药苷主要通过凋亡途径、免疫炎症过程、代谢通路、细胞周期通路、胆汁酸分泌通路与紧密连接通路等发挥改善胆汁淤积的作用。选择紧密连接通路进行验证，验证结果表明，与对照组比较，模型组大鼠肝组织ZO-1、Occludin、Claudin 1、Claudin 2、Claudin 3和JAM-A mRNA表达水平均显著降低（P<0.01），ZO-1、Occludin和Claudin 3蛋白表达水平均显著降低（P<0.05、0.01），经芍药苷干预后以上紧密连接通路相关蛋白及基因表达显著升高（P<0.05、0.01）。结论 芍药苷可以多途径、多靶点缓解大鼠胆汁淤积，是治疗胆汁淤积的潜在药物，紧密连接通路可能是芍药苷改善胆汁淤积的关键通路。

Objective To investigate the effect of paeoniflorin on cholestasis through tight junction pathway based on proteomics and experimental verification. Methods SD rats were randomly divided into control group, model group, ursodesoxycholic acid (60 mg/kg) group and paeoniflorin high-, medium-and low-dose groups (200, 100, 50 mg/kg), with eight rats in each group. The rat model of intrahepatic cholestasis was induced by ig α-isothiocyanate (60 mg/kg) after 7 d of ig corresponding drugs for 2 h on 4th day. After the last administration, the indexes of liver function in serum of rats were determined by kit. HE staining was used to observe the pathological changes of rat liver. Proteomics and bioinformatics techniques were used to screen the key pathways of paeoniflorin on ameliorating the cholestasis. Molecular docking was used to determine the binding energy of paeoniflorin to the relevant targets. qRT-PCR and Western blotting were used to verify the expressions of key targets such as zonula occludin-1 (ZO-1), Occludin, Claudin 1/2/3 and junction adhesion molecule-A (JAM-A) in liver tissue. Results The pharmacodynamic results showed that compared with control group, activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GT) and levels of total bile acid (TBA), total bilirubin (TBIL), direct bilirubin (DBIL) in serum of rats in model group were significantly increased (P < 0.01). A large number of inflammatory cells infiltrated in liver tissue, accompanied by degeneration and necrosis of hepatocytes, and the above indexes were significantly improved after paeoniflorin treatment (P < 0.05, 0.01). Proteomics results indicated that paeoniflorin mainly exerted its effects in improving cholestasis through apoptosis pathway, immune-inflammatory processes, metabolic pathways, cell cycle pathway, bile acid secretion pathway, and tight junction pathway. The tight junction pathway was selected for validation, and the results showed that compared with control group, ZO-1, Occludin, Claudin 1, Claudin 2, Claudin 3 and JAM-A mRNA expressions in liver tissue of rats in model group were significantly decreased (P < 0.01), ZO-1, Occludin and Claudin 3 protein expressions were significantly decreased (P < 0.05, 0.01), after the intervention of paeoniflorin, the expressions of proteins and genes related to the tight junction pathway were significantly increased (P < 0.05, 0.01). Conclusion Paeoniflorin can relieve cholestasis in rats in multiple ways and multiple targets, and it is a potential drug for treating cholestasis. Tight junction pathway may be the key pathway for paeoniflorin to improve cholestasis.

R285.5

国家自然科学基金资助项目（82274187）；四川省自然科学基金项目（2023NSFSC0687）