目的 研究大黃（Rhei Radix et Rhizoma，R）-枳實炭納米類成分（Aurantii Fructus Immaturus Carbonisatum nano-components，AFIC-NCs）對肝內(nèi)膽汁淤積癥（intrahepatic cholestasis，IC）的治療作用及作用機(jī)制。方法 從枳實炭中提取分離出AFIC-NCs并利用納米材料表征方法分析其形態(tài)結(jié)構(gòu)、光學(xué)性質(zhì)和官能團(tuán)性質(zhì)等。將48只雄性C57BL/6J小鼠隨機(jī)分為對照組、模型組、熊去氧膽酸（ursodeoxycholic acid，UDCA，100 mg/kg）組及R-AFIC-NCs高、中、低劑量（22.66、11.33、5.67 mg/kg）組，每組各8只。連續(xù)給藥7 d，并于第5天igα-萘異硫氰酸酯（α-naphthalene isothiocyanate，ANIT）誘導(dǎo)IC小鼠模型。給藥結(jié)束后檢測各組小鼠血清中丙氨酸氨基轉(zhuǎn)移酶（alanine aminotransferase，ALT）、天冬氨酸氨基轉(zhuǎn)移酶（aspartate aminotransferase，AST）、堿性磷酸酶（alkaline phosphatase，ALP）、直接膽紅素（direct bilirubin，DBIL）、間接膽紅素（indirect bilirubin，IBIL）、總膽紅素（total bilirubin，TBIL）和總膽汁酸（total bile acid，TBA）的水平；觀察小鼠肝臟和膽囊外觀并計算肝臟指數(shù)；采用蘇木素-伊紅（hematoxylin eosin，HE）染色觀察肝組織病理變化；TUNEL法檢測肝細(xì)胞凋亡；檢測各組小鼠肝臟中腫瘤壞死因子-α（tumor necrosis factor-α，TNF-α）、白細(xì)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）及還原型谷胱甘肽（glutathione，GSH）含量；采用Western blotting法檢測肝組織中內(nèi)質(zhì)網(wǎng)應(yīng)激（endoplasmic reticulum stress，ERS）相關(guān)蛋白葡萄糖調(diào)節(jié)蛋白78（glucose-regulated protein 78，GRP78）和C/EBP同源蛋白（protein kinase RNA-like endoplasmic reticulum kinase，CHOP）的表達(dá)。結(jié)果 表征結(jié)果顯示，AFIC-NCs呈類球形顆粒，晶格間距為0.18 nm，表面含有羥基、羰基等官能團(tuán)。R-AFIC-NCs各劑量組均能顯著下調(diào)肝內(nèi)膽汁淤積癥模型小鼠肝功能指標(biāo)ALP、ALT、AST及膽汁淤積指標(biāo)DBIL、IBIL、TBIL、TBA（P＜0.05、0.01）；改善肝組織損傷，減少肝細(xì)胞凋亡（P＜0.05）；降低炎癥因子TNF-α、IL-1β和IL-6的水平（P＜0.05、0.01），提高肝臟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各劑量組還能降低GRP78和CHOP蛋白的表達(dá)（P＜0.05、0.01）。結(jié)論R-AFIC-NCs可能通過抑制內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志蛋白GRP78和CHOP的表達(dá)，減輕肝內(nèi)膽汁淤積癥引起的細(xì)胞凋亡和氧化應(yīng)激反應(yīng)，從而改善其肝臟功能。

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

國家中醫(yī)藥管理局岐黃學(xué)者支持項目（90020172120064）；中央高?；究蒲袠I(yè)務(wù)費專項資金資助項目（2024-JYB-JBZD023，2024-JYB-JBZD040，2024-JYB-JBZD-045）