龙胆苦苷调控PPAR-α改善胆汁淤积性肝损伤的药效机制研究  

Pharmacodynamic mechanism study of gentiopicroside modulating PPAR-αto ameliorate cholestatic liver injury

作　　者：冯帅霞 徐莹[2] 韩涵 张彤[1] FENG Shuaixia;XU Ying;HAN Han;ZHANG Tong(School of Pharmacy,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;School of Traditional Chinese Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学中药学院,上海201203 [2]上海中医药大学中医学院,上海201203

出　　处：《上海中医药杂志》2024年第2期84-91,共8页Shanghai Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(82173946,82274066,82004162);上海市自然科学基金项目(21ZR1460500)。

摘　　要：目的 探讨龙胆苦苷通过过氧化物酶体增殖物激活受体-α(PPAR-α)通路对胆汁淤积性肝损伤的影响及作用机制。方法 (1)48只C57BL/6J雄性小鼠随机分为正常组、模型组[α-萘基异硫氰酸酯(ANIT),灌胃]、熊去氧胆酸组(200 mg/kg,灌胃)以及龙胆苦苷低、中、高剂量组(50、100、200 mg/kg,灌胃),每组8只。各组小鼠连续给予相应干预措施7 d。第5天给药6 h后,正常组灌胃给予空白橄榄油溶液,其余各组小鼠灌胃给予ANIT诱导胆汁淤积性肝损伤。给药结束后,观察小鼠体质量变化;记录肝脏质量,计算肝脏指数;检测小鼠血清中天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)、总胆汁酸(TBA)、总胆红素(TBil)水平;苏木精-伊红(HE)染色观察肝组织病理变化;实时荧光定量逆转录聚合酶链式反应(RT-qPCR)法检测肝组织PPAR-α、核转录因子-κB(NF-κB)、肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)mRNA表达;Western blot法检测小鼠肝脏中PPAR-α、细胞色素P4503A4(CYP3A4)、肉碱棕榈酰转移酶2(CPT2)以及组成型雄甾烷受体(CAR)蛋白表达。(2)采用L02人肝细胞进行体外实验,建立牛磺鹅去氧胆酸钠(TCDC)胆汁淤积性肝细胞损伤模型,采用10、50、100μmol/L龙胆苦苷进行干预。测定细胞上清液中AST、ALT、TBA的含量;RT-qPCR法验证肝细胞中PPAR-α、NF-κB、TNF-α、IL-1β mRNA表达;Western blot法检测肝细胞中PPAR-α、CYP3A4、CPT2以及CAR蛋白表达。(3)另在TCDC胆汁淤积性肝细胞损伤模型基础上,采用PPAR-α激动剂(非诺贝特)和抑制剂(GW6471)进行PPAR-α拮抗实验。Western blot法检测肝细胞中PPAR-α、CYP3A4、CPT2、CAR蛋白表达。结果 (1)龙胆苦苷可下调胆汁淤积性肝损伤小鼠血清中AST、ALT、TBA、TBil含量(P<0.05),并改善小鼠肝脏病变情况。(2)龙胆苦苷可上调胆汁淤积性肝损伤模型小鼠肝组织和模型细胞中PPAR-α mRNA水平,降低促炎细胞因子NF-κB、TNF-α、IL-1β的mRNA水平�Objective To investigate the effect and mechanism of gentiopicroside on cholestatic liver injury through peroxisome proliferator-activated receptor‑α(PPAR‑α)pathway.Methods①A total of 48 C57BL/6J male mice were randomly divided into normal group,model group[α‑naphthylisothiocyanate(ANIT),gavage],ursodeoxycholic acid group(200 mg/kg,gavage),gentiopicroside low,medium and high dose group(50,100,200 mg/kg,gavage),with 8 mice in each group.The mice were given the corresponding drug by gavage for 7 consecutive days.On the fifth day,6 h after administration,the normal group was given blank olive oil solution by gavage,and the mice in other groups were given ANIT by gavage to induce cholestatic liver injury.The changes of mice body mass were observed;the liver mass was recorded and liver index was calculated;serum levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),total bile acid(TBA)and total bilirubin(TBil)were detected;hematoxylin-eosin(HE)staining was used to observe the pathological changes of liver tissue;the mRNA expressions of PPAR-α,nuclear transcription factor-κB(NF-κB),tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in liver tissue were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-qPCR);the protein expressions of PPAR-α,cytochrome P-4503A4(CYP3A4),carnitine palmitoyl transferase 2(CPT2)and constitutive androstane receptor(CAR)in mice liver were detected by Western blot.②In vitro experiments were conducted using L02 human hepatocytes to establish taurocholate deoxycholate(TCDC)-induced cholestatic hepatocyte injury,and gentiopicroside(10,50,100μmol/L)was used for intervention.The contents of AST,ALT and TBA in cell supernatant were determined;The mRNA expressions of PPAR-α,NF-κB,TNF-αand IL-1βin hepatocytes were verified by RT-qPCR;The protein expressions of PPAR-α,CYP3A4,CPT2 and CAR in hepatocytes were detected by Western blot.③PPAR-αantagonism experiment was performed using a PPAR-αagonist(fenofib

关 键 词：胆汁淤积 肝损伤 肝硬化 龙胆苦苷 胆汁酸代谢 炎症反应 中药研究 

分 类 号：R285.5[医药卫生—中药学]