目的 研究大黄（Rhei Radix et Rhizoma，R）-枳实炭纳米类成分（Aurantii Fructus Immaturus Carbonisatum nano-components，AFIC-NCs）对肝内胆汁淤积症（intrahepatic cholestasis，IC）的治疗作用及作用机制。方法 从枳实炭中提取分离出AFIC-NCs并利用纳米材料表征方法分析其形态结构、光学性质和官能团性质等。将48只雄性C57BL/6J小鼠随机分为对照组、模型组、熊去氧胆酸（ursodeoxycholic acid，UDCA，100 mg/kg）组及R-AFIC-NCs高、中、低剂量（22.66、11.33、5.67 mg/kg）组，每组各8只。连续给药7 d，并于第5天igα-萘异硫氰酸酯（α-naphthalene isothiocyanate，ANIT）诱导IC小鼠模型。给药结束后检测各组小鼠血清中丙氨酸氨基转移酶（alanine aminotransferase，ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase，AST）、碱性磷酸酶（alkaline phosphatase，ALP）、直接胆红素（direct bilirubin，DBIL）、间接胆红素（indirect bilirubin，IBIL）、总胆红素（total bilirubin，TBIL）和总胆汁酸（total bile acid，TBA）的水平；观察小鼠肝脏和胆囊外观并计算肝脏指数；采用苏木素-伊红（hematoxylin eosin，HE）染色观察肝组织病理变化；TUNEL法检测肝细胞凋亡；检测各组小鼠肝脏中肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）、IL-6、超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malondialdehyde，MDA）及还原型谷胱甘肽（glutathione，GSH）含量；采用Western blotting法检测肝组织中内质网应激（endoplasmic reticulum stress，ERS）相关蛋白葡萄糖调节蛋白78（glucose-regulated protein 78，GRP78）和C/EBP同源蛋白（protein kinase RNA-like endoplasmic reticulum kinase，CHOP）的表达。结果 表征结果显示，AFIC-NCs呈类球形颗粒，晶格间距为0.18 nm，表面含有羟基、羰基等官能团。R-AFIC-NCs各剂量组均能显著下调肝内胆汁淤积症模型小鼠肝功能指标ALP、ALT、AST及胆汁淤积指标DBIL、IBIL、TBIL、TBA（P＜0.05、0.01）；改善肝组织损伤，减少肝细胞凋亡（P＜0.05）；降低炎症因子TNF-α、IL-1β和IL-6的水平（P＜0.05、0.01），提高肝脏SOD和GSH活性（P＜0.05、0.01），降低了MDA水平（P＜0.05、0.01）；此外，R-AFIC-NCs各剂量组还能降低GRP78和CHOP蛋白的表达（P＜0.05、0.01）。结论R-AFIC-NCs可能通过抑制内质网应激标志蛋白GRP78和CHOP的表达，减轻肝内胆汁淤积症引起的细胞凋亡和氧化应激反应，从而改善其肝脏功能。

Objective To investigate the therapeutic effect and mechanism of Rhei Radix et Rhizoma-Aurantii Fructus Immaturus Carbonisatum nano-components (R-AFIC-NCs) on intrahepatic cholestasis (IC). Methods Aurantii Fructus Immaturus Carbonisatum nano-components (AFIC-NCs) were extracted and isolated from Aurantii Fructus Immaturus Carbonisatum and analyzed for their morphological structure, optical properties, and functional group properties using nanomaterials characterization methods. Forty-eight male C57BL/6J mice were randomly divided into control group, model group, ursodeoxycholic acid (UDCA) group (100 mg/kg), and high-, medium-, and low-dose (22.66, 11.33, 5.67 mg/kg) R-AFIC-NCs group, with eight animals in each group. The drugs were administered for seven consecutive days, and the IC model was induced by α-naphthalene isothiocyanate (ANIT) gavage on the fifth day. After treatment, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct bilirubin (DBIL), indirect bilirubin (IBIL), total bilirubin (TBIL), and total bile acid (TBA) were measured. The appearance of liver and gallbladder of the mice was observed and the liver index was calculated; Hematoxylin-eosin (HE) staining was used to evaluate the pathological changes in the liver tissues. The TUNEL assay was used to assess hepatocyte apoptosis. Additionally, the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, superoxide dismutase (SOD), malondialdehy (MDA), and glutathione (GSH) were measured in the livers of the different groups. The expressions of endoplasmic reticulum stress (ERS)-related proteins, glucose-regulated protein 78 (GRP78) and protein kinase RNA-like endoplasmic reticulum kinase (CHOP), were detected by Western blotting. Results The characterization results indicated that AFIC-NCs exhibited a spherical morphology, with a lattice spacing of 0.18 nm, and the surface was enriched with functional groups such as hydroxyl and carbonyl. All dose groups of R-AFIC-NCs significantly reduced the liver function indexes ALP, ALT, AST and cholestasis indexes DBIL, IBIL, TBIL, TBA in mouse modeled with intrahepatic cholestasis (P < 0.05, 0.01). They also ameliorated liver tissue damage, decreased hepatocyte apoptosis (P < 0.05), lowered the levels of inflammatory factors TNF-α, IL-1β, and IL-6 (P < 0.05, 0.01), enhanced hepatic activities of SOD and GSH (P < 0.05, 0.01), and diminished MDA levels (P < 0.05, 0.01). Furthermore, R-AFIC-NCs suppressed the expression of GRP78 and CHOP proteins in all dosage groups (P < 0.05, 0.01). Conclusion R-AFIC-NCs may improve liver function by suppressing the expression of endoplasmic reticulum stress markers GRP78 and CHOP, which may reduce oxidative stress responses and apoptosis by intrahepatic cholestasis.

R285.5

国家中医药管理局岐黄学者支持项目（90020172120064）；中央高校基本科研业务费专项资金资助项目（2024-JYB-JBZD023，2024-JYB-JBZD040，2024-JYB-JBZD-045）