目的 系统探讨茵栀黄颗粒对胆汁淤积性肝病（CLD）中铁死亡的调控效应及其潜在分子机制。方法 分别采用胆总管结扎（BDL）术与Mdr2^-/-小鼠构建CLD模型，将小鼠随机分为对照组、模型组及茵栀黄颗粒低、高剂量[3.51（临床等效剂量）、7.02 g·kg^-1]组。通过计算肝、脾指数，结合肝组织苏木素-伊红（HE）染色、Masson染色及普鲁士蓝染色，检测血清总胆汁酸（TBA）、丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）水平，系统评估茵栀黄颗粒对CLD的改善作用；采用实时荧光定量PCR（qRT-PCR）技术检测肝组织中肝纤维化相关基因（Fn1、Col1a1、Acta2、Krt19）、炎症因子基因白细胞介素1β（Il-1β）、胆汁酸代谢相关基因[法尼醇X受体（Nr1h4）、小异源二聚体伴侣蛋白（Nr0b2）]、胆酸合成关键基因胆固醇7α-羟化酶（Cyp7a1）、胆汁酸分泌转运蛋白编码基因胆盐输出泵（Abcb11）及胆汁酸摄取排泄相关基因[钠-牛磺胆酸协同转运蛋白（Slc10a1）、多药耐药相关蛋白3（Abcc3）]的mRNA表达水平。通过网络药理学方法，构建茵栀黄颗粒活性成分-作用靶点与CLD及铁死亡共同靶点的相互作用网络，预测关键调控通路；借助肝组织转录组学测序分析，明确铁死亡通路在CLD模型中的富集特征；采用生化试剂盒检测肝组织氧化应激指标[丙二醛（MDA）、脂质过氧化物（LPO）、还原型谷胱甘肽（GSH）]及铁沉积水平，通过qRT-PCR验证促铁死亡相关基因[长链脂酰辅酶A合成酶4（Acsl4）、铁蛋白重链1（Fth1）、铁蛋白受体1（Tfrc）、细胞色素b-245 β链（Cybb）]、铁死亡保护相关基因[谷胱甘肽过氧化物酶4（Gpx4）、半胱氨酸/谷氨酸逆向转运体（Slc7a11）、甘氨酸转运蛋白1（Slc6a9）、GSH合成限速酶（Gclc）]及花生四烯酸代谢通路脂氧合酶基因[15脂氧合酶（Alox15）、5脂氧合酶激活蛋白（Alox5ap）、12脂氧合酶（Alox12）]的mRNA表达变化。结果 茵栀黄颗粒虽未显著逆转模型组小鼠肝、脾指数的升高，但高剂量组可显著降低血清TBA、ALT、AST水平（P<0.01、0.001），减轻肝组织铁颗粒沉积（P<0.01），同时降低肝组织MDA、LPO水平（P<0.05、0.01），提升GSH水平（P<0.01）。qRTPCR结果显示，与模型组相比，高剂量茵栀黄颗粒干预后，肝组织中Fn1、Col1a1、Acta2、Krt19、Il-1β、Cyp7a1的mRNA表达显著下调（P<0.01、0.001），而Nr1h4、Nr0b2、Abcb11、Abcc3的mRNA表达显著上调（P<0.01、0.001）；网络药理学分析筛选获得55个茵栀黄颗粒-CLD-铁死亡共有靶点，提示其20种入血活性成分可能通过多成分协同作用靶向铁死亡相关通路；基因集富集分析（GSEA）结果表明，2种胆汁淤积模型组的铁死亡相关基因集均较对照组显著富集；qRT-PCR验证显示，高剂量茵栀黄颗粒可显著下调促铁死亡基因Acsl4、Tfrc的mRNA表达（P<0.01、0.001），上调铁死亡保护基因Gpx4、Slc7a11、Slc6a9、Gclc的mRNA表达（P<0.05、0.01、0.001）。结论 茵栀黄颗粒可有效缓解BDL及Mdr2^-/-小鼠模型的肝脏纤维化、胆汁淤积及铁死亡病理进程，其作用机制可能与调控铁死亡相关基因表达及改善氧化应激水平密切相关。

Objective To systematically investigate the regulatory effect of Yinzhihuang Granule on ferroptosis in cholestatic liver disease（CLD） and its potential molecular mechanism. Methods CLD models were established by bile duct ligation（BDL） and Mdr2^-/- gene-deficient mice. Mice were randomly divided into the control group, model group, and low-high-dose(3.51, 7.02 g·kg^-1) Yinzhihuang Granule intervention groups. The improvement effect of Yinzhihuang Granule on CLD was evaluated by calculating the liver and spleen coefficients, combined with hematoxylin-eosin（HE） staining, Masson staining, and Prussian blue staining of liver tissue, and by detecting the levels of serum total bile acid（TBA）, alanine aminotransferase（ALT）, and aspartate aminotransferase（AST）. The mRNA expression levels of liver fibrosis-related genes（Fn1, Col1a1, Acta2, Krt19）, inflammatory factor gene interleukin-1β（Il-1β）, bile acid metabolism-related genes [farnesoid X receptor（Nr1h4）, small heterodimer partner protein（Nr0b2）], key genes in bile acid synthesis [cholesterol 7α-hydroxylase（Cyp7a1）], bile acid secretion transporter-encoding gene bile salt export pump（Abcb11）, and bile acid uptake and excretion-related genes [sodium-taurine bile acid cotransporter（Slc10a1）, multidrug resistanceassociated protein 3（Abcc3）] in liver tissue were detected by real-time fluorescence quantitative PCR（qRT-PCR） technology. The interaction network of active components of Yinzhihuang Granule-action targets and common targets of CLD and ferroptosis was constructed by network pharmacology methods to predict key regulatory pathways. The enrichment characteristics of the ferroptosis pathway in the CLD model were clarified by liver tissue transcriptome sequencing analysis. The levels of oxidative stress indicators [malondialdehyde（MDA）, lipid peroxides（LPO）, reduced glutathione（GSH）] and iron deposition in liver tissue were detected by biochemical kits, and the mRNA expression changes of ferroptosis-promoting genes [long-chain acyl-CoA synthetase 4（Acsl4）, ferritin heavy chain 1（Fth1）, transferrin receptor 1（Tfrc）, cytochrome b-245 β chain（Cybb）], ferroptosis-protective genes [glutathione peroxidase 4（Gpx4）, cystine/glutamate antiporter（Slc7a11）, glycine transporter 1（Slc6a9）, glutamate-cysteine ligase（Gclc）], and arachidonic acid metabolism pathway lipoxygenase genes [15-lipoxygenase（Alox15）, 5-lipoxygenase-activating protein（Alox5 ap）, 12-lipoxygenase（Alox12）] were verified by q RT-PCR. Results Although Yinzhihuang Granule did not significantly reverse the increase in liver and spleen coefficients in the model group, the high-dose group significantly reduced the levels of serum TBA, ALT, and AST（P ＜0.01, 0.001）, reduced iron particle deposition in liver tissue（P ＜0.01, 0.001）, and simultaneously decreased the levels of MDA and LPO in liver tissue（P ＜0.05, 0.01）, and increased the level of GSH（P ＜0.01）. qRT-PCR results showed that compared with the model group, after high-dose Yinzhihuang Granule intervention, the mRNA expression of Fn1, Col1a1, Acta2, Krt19, Il-1β, and Cyp7a1 in liver tissue was significantly downregulated（P ＜0.01, 0.001）, while the mRNA expression of Nr1h4, Nr0b2, Abcb11, and Abcc3 was significantly upregulated（P ＜0.01, 0.001）. Network pharmacology analysis screened out 55 common targets of Yinzhihuang Granule-CLD-ferroptosis, suggesting that its 20 blood-active components may target ferroptosis-related pathways through multi-component synergy. The results of gene set enrichment analysis（GSEA） indicated that the ferroptosis-related gene sets in the two cholestasis model groups were significantly enriched compared with the control group. qRT-PCR verification showed that highdose Yinzhihuang Granule could significantly down-regulate the mRNA expression of ferroptosis-promoting genes Acsl4, Tfrc,（P ＜0.01, 0.001）, and up-regulate the mRNA expression of ferroptosis-protective genes Gpx4, Slc7a11, Slc6a9, and Gclc（P ＜0.05, 0.01, 0.001）. Conclusion Yinzhihuang Granule can effectively alleviate liver fibrosis, cholestasis and ferroptosis in BDL and Mdr2^-/- mouse models, and its mechanism of action may be closely related to the regulation of ferroptosis-related gene expression and the improvement of oxidative stress levels.

R285.5;R575.5

国家自然科学基金优秀青年科学基金资助项目(82322075)