【作者】 张程亮；

【导师】 刘东；

【作者基本信息】 华中科技大学 ， 中西医结合药理， 2018， 博士

【摘要】 肝内胆汁淤积（intrahepatic cholestasis,IHC）存在于多种慢性肝病,是由多种原因所致的肝细胞内胆汁酸蓄积和排泄受阻,从而导致肝脏炎性浸润、损伤坏死,如果未经有效干预,可进一步发展成为肝纤维化、肝硬化,甚至肝癌。机体高水平的雌激素是敏感的妊娠期妇女、口服避孕药或绝经后激素治疗人群中IHC的高危因素。IHC发病机制非常复杂,有效治疗药物至今仍然较为缺乏。中医认为IHC一般属于“黄疸”,而肝胆和脾胃湿热是黄疸的主要证型。因此,保肝利胆、清热燥湿是中医药治疗IHC的主要法则。中药黄芩善清热燥湿,在我国的应用已经有千年之久。其主要药效成分黄芩苷具有多种药理学作用,在保肝护肝方面的效果显著,但是其对IHC的疗效、作用机制以及在IHC机体中的药动学特征尚未见研究报道。本研究评价了黄芩苷对IHC大鼠的保护作用、内在机制和药动学特征,为该药的临床应用奠定了理论依据。第一部分黄芩苷对雌激素诱导胆汁淤积大鼠的保护作用研究目的:初步探讨黄芩苷治疗17α-乙炔基雌二醇（17α-ethynylestradiol,EE）诱导的IHC大鼠模型的效果。方法:采用雄性SD大鼠,以5 mg/kg的EE连续5天皮下注射诱导IHC动物模型,分别给予50 mg/kg、100 mg/kg、200 mg/kg的黄芩苷和阳性对照药物熊去氧胆酸（UDCA 40mg/kg）进行干预。实验同时设置给予正常大鼠200 mg/kg黄芩苷的对照组。通过测定大鼠的肝重比、一般情况、胆汁流量评价黄芩苷的利胆效果。大鼠肝脏和回肠组织进行HE染色,部分肝脏组织制作超薄切片行透射电镜观察细微结构改变。取动物血清,测定总胆汁酸（TBA）、总胆红素（TBil）、直接胆红素（DBil）、谷丙转氨酶（ALT）、谷草转氨酶（AST）、碱性磷酸酶（AKP）等生化指标。检测动物肝脏组织中超氧化物歧化酶（SOD）、丙二醛（MDA）、髓过氧化物酶（MPO）等氧化应激指标。结果:黄芩苷可以显著缓解EE连续注射所致的动物一般情况改变、降低肝重比并增加IHC大鼠的胆汁流速。黄芩苷可以有效缓解IHC大鼠血清中TBil、DBil、TBA、ALT、AST、AKP的异常升高,作用呈现剂量依赖性。从HE染色和透射电镜观察结果可见,黄芩苷缓解了EE所致的肝脏和肠道结构破坏及炎性浸润,并减轻了肝细胞超微结构破坏。在抗氧化损伤方面,黄芩苷能够升高IHC大鼠肝脏组织的SOD活性,并减少MDA和MPO含量。黄芩苷对正常大鼠的上述指标均无明显影响。结论:黄芩苷具有显著改善EE诱导IHC大鼠胆汁淤积的效果,减轻了肝脏和肠道损伤,这一作用可能与其抗氧化活性有关。其中,200 mg/kg的黄芩苷效果最佳,且未见明显的毒副反应。第二部分黄芩苷对胆汁淤积大鼠胆汁酸平衡和炎性损伤的干预作用目的:从胆汁酸平衡、炎性损伤和法尼酯X受体（FXR）的角度探讨黄芩苷缓解EE诱导IHC的作用机制。方法:分别给予正常大鼠和EE诱导的IHC大鼠灌胃200 mg/kg的黄芩苷溶液,评价其对胆汁淤积大鼠的保护效果。采用RT-PCR检测以下基因的表达:Cyp7a1、Cyp8b1、Cyp3a2、Sult2a1、Bal、Baat等肝脏胆汁酸合成或代谢酶;Ntcp、Oatp1a1、Oatp1b2、Bsep、Mrp2、Mrp3、Mrp4、Mdr2等肝脏转运体;Tnf-α、Il-6、Il-1β等前炎性因子;肝脏Fxr和靶基因Shp;肠道Fxr和靶基因Fgf15。Western Blotting法检测肝脏中Cyp7a1、Ntcp、Bsep、Mrp2等胆汁酸主要合成酶、转运体和上述前炎性因子表达,以及肝脏Fxr和肠道Fxr的蛋白含量。免疫组织化学染色方法检测肝脏和肠道Fxr的表达。结果:200 mg/kg黄芩苷表现出显著的保肝利胆效果。该药对肝脏Cyp7a1以及Cyp8b1等胆汁酸合成酶的表达无明显影响,但可以上调Cyp3a2、Sult2a1、Bal、Baat的表达;黄芩苷能够有效降低Ntcp和上调Bsep、Mrp2的基因和蛋白表达,对Oatp1b2基因表达无影响,但可上调Oatp1a1、Mrp3、Mrp4和Mdr2的基因表达;黄芩苷可以显著缓解前炎性因子Tnf-α、Il-6、Il-1β的肝脏大量释放。从RT-PCR、Western Blotting和免疫组化结果可见,黄芩苷可以有效上调肝脏和肠道Fxr的基因和蛋白表达,并促进相应靶基因Shp和Fgf15的转录。结论:黄芩苷可能通过激活Fxr,促进胆汁酸的解毒,增加胆汁酸外排并减少摄取,以及减轻肝脏炎性损伤来达到保护肝细胞的作用。第三部分黄芩苷在雌激素诱导胆汁淤积大鼠体内的药动学研究目的:建立液相色谱串联质谱（LC-MS/MS）法分析大鼠血浆中黄芩苷的测定方法,研究黄芩苷在正常大鼠和IHC大鼠体内的药动学特征。方法:采用甲醇沉淀蛋白法处理大鼠血浆样品。色谱条件:C₁₈色谱柱,流动相A为含0.1%甲酸的水溶液,流动相B为甲醇溶液,采用等度洗脱条件A:B=20:80（v/v）,时间5 min,流速为0.8 mL/min,进样体积10μL,柱温30℃;质谱条件为电喷雾离子源（ESI）,正离子模式（+）,多离子反应监测（MRM）,黄芩苷检测离子对m/z 447.0→271.3。在正常大鼠或EE诱导的IHC大鼠体内单次或连续灌服给予200mg/kg的黄芩苷,应用上述测定方法分析黄芩苷的血药浓度,计算相应的药动学参数。结果:所建立的黄芩苷测试方法比较简便,专属性好。在定量范围1.01–506.00μg/mL内线性良好（R²=0.9980）,定量下限为1.01μg/mL。日内、日间精密度的RSD均不超过10.55%,日内、日间准确度均在94.94-109.13%,回收率和稳定性考察结果均符合《中国药典》2015版生物样品定量分析要求。黄芩苷在大鼠体内的药时曲线表现出双峰现象,多次给药后,黄芩苷在正常大鼠体内的AUC和C_max升高,CL和t_1/2减小。与正常大鼠相比,在EE诱导的IHC大鼠体内单次给予黄芩苷,其C_max、AUC显著升高,t_1/2显著延长,CL明显降低。早期连续给予黄芩苷后,与IHC大鼠模型组内单次给药相比,可使AUC下降、CL上升,t_1/2缩短,其药动学特征更加接近正常大鼠体内的药动学特征。结论:与正常大鼠相比,IHC大鼠模型单次给予黄芩苷,其药动学特征表现为暴露量显著增加,清除率下降,但是连续多次给予黄芩苷干预,可以有效降低模型动物体内药物生物利用度增加的现象。这可能与黄芩苷长期给药逆转转运体和代谢酶活性下降,并改善肠道结构及功能有关。更多还原

【Abstract】 Intrahepatic cholestasis（IHC）,existing in many chronic liver diseases,result from an accumulation of bile acid in hepatocytes and impairment of bile flow excretion induced by multiple complicated factors.Without effective intervention,IHC can lead to inflammatory infiltration,hepatic injury and necrosis,and ultimately develop into hepatic fibrosis,cirrhosis and hepatocarcinogenesis.Estrogens in high level are well-known to cause IHC in susceptible women during pregnancy,administration of oral contraceptives,and postmenopausal replacement therapy.As the pathogenesis of IHC is obscure,effective drugs remain scarcity.According to the theory of traditional Chinese medicine,IHC belongs to“jaundice”in which dampness-heat in hepatobiliary system as well as spleen and stomach is the main syndrome types.Hence,protecting liver and gallbladder together with heat-clearing and damp-drying is the crucial therapeutic principle for IHC.Radix Scutellariae is adept in heat-clearing and damp-drying,and has been used for thousands years in China.Baicalin is a bioactive flavonoid isolated from Radix Scutellariae,and has many biological functions,especially with eminent hepatoprotective effects.However,the effect of baicalin for IHC,its possible mechanism and the pharmacokinetics in IHC animals have not been reported before.In the present study,we have investigated the protection of baicalin on IHC rats and the underlying pharmacological mechanism,and the pharmacokinetic characteristics of baicalin in IHC rats was also clarified,which can supply an experimental basis for its clinical application.Part Ⅰ Studies on the protection of baicalin on estrogen-induced IHC ratsObjective:To preliminarily explore the effect of baicalin on 17α-ethynylestradiol（EE）-induced IHC rats.Methods:IHC rat model was established by subcutaneously injection of EE（5mg/kg body weight）daily for five consecutive days in SD rats.Simultaneously,IHC rats were treated by baicalin at 50mg/kg,100mg/kg,200mg/kg or ursodeoxycholic acid（UDCA）at40mg/kg.We also add a control group in which normal rats were given 200mg/kg baicalin.Cholagogic action of baicalin was evaluated by examining the liver index,animal behavior and bile flow.Haematoxylin and eosin（HE）staining was performed to study the pathological injury in livers and ileums.Ultrastructure in hepatocytes was examined by transmission electron microscope（TEM）.The serum levels of total bile acid（TBA）,total bilirubin（TBil）,direct bilirubin（DBil）,alanine aminotransferase（ALT）,aspartate aminotransferase（AST）and alkaline phosphatase（AKP）were determined.Moreover,the content of superoxide dismutase（SOD）,malondialdehyde（MDA）and myeloperoxidase（MPO）in livers was also detected.Results:Baicalin significantly improved the behavioral change,the increase of liver index and decline of bile flow in IHC rats induced by EE injection.Our results showed that baicalin could dose-dependently reduce the abnormal biochemical indexes in serum including TBA,TBil,DBil,ALT,AST and AKP.HE staining and TEM observation demonstrated that the structure damage and inflammatory infiltration of livers and ileums as well as the histopathological injury on ultrastructure of hepatocytes were markedly alleviated by baicalin.Baicalin could enhance SOD activity and decrease the content of MDA and MPO in liver tissue of IHC rats.In addition,there was not any influence of baicalin at 200mg/kg on normal rats.Conclusion:Baicalin possesses the improvement effect of cholestasis on EE-induced IHC rats and alleviates the injury of livers and ileums,owing to its potential anti-oxidative damage.Among them,baicalin at 200mg/kg achieved the most outcomes and has not obvious side effect.Part Ⅱ Intervention of baicalin on bile acid homeostasis and inflammatory injury incholestatic ratsObjective:To investigate the pharmacological mechanism of baicalin on EE-induced IHC via bile acid homeostasis,inflammatory injury and farnesoid X receptor（FXR）.Methods:Normal rats and EE-induced IHC rats were orally treated with 200mg/kg baicalin respectively,to evaluate the protection on cholestasis.RT-PCR was perform to analysis the following genes:（1）enzymes for bile acid synthesis and metabolism:Cyp7a1、Cyp8b1、Cyp3a2、Sult2a1、Bal、Baat;（2）hepatic transporters for bile acid uptake and biliary secretion:Ntcp、Oatp1a1、Oatp1b2、Bsep、Mrp2、Mrp3、Mrp4、Mdr2;（3）proinflammatory factors;Tnf-α、Il-6、Il-1β;（4）hepatic Fxr and its targeted gene Shp;（5）intestinal Fxr and its targeted gene Fgf15.The protein expressions of Cyp7a1,Ntcp,Bsep,Mrp2,the proinfalmmatory cytokines,and Fxr in livers and ileums were determined by western blotting.Fxr in livers and ileums was also evaluated by immunohistochemistry.Results:Baicalin at 200mg/kg showed prominent hepatoprotective and cholagogic effect.The gene expressions of the hepatic enzymes of bile acid synthesis such as Cyp7a1 and Cyp8b1 were not affected by baicalin treatment.Baicalin could upregulate the expressions of Cyp3a2,Sult2a1,Bal and Baat.We found the gene and protein expressions of Ntcp were inhibited while those of Bsep and Mrp2 were increased.There was not any change in the gene expression of Oatp1b2,but Oatp1a1,Mrp3,Mrp4 and Mdr2 were significantly increased by baicalin.Moreover,attenuation of proinflammatory cytokines Tnf-α,Il-6,and Il-1βin livers was observed in IHC rats treated by baicalin.Results of RT-PCR,western blotting and immunohistochemistry confirmed that baicalin could upregulate the Fxr expressions in livers and ileums and promote the transcription of Fxr targeted gene Shp and Fgf15.Conclusion:Baicalin can enhance the detoxification of bile acid,accelerate the excretion of bile acid and diminish its uptake,consequently alleviate the inflammatory injury in livers and confer protection to hepatocytes.And our results show those effects are probablyrelated to Fxr activation by baicalin.Part Ⅲ Study on the pharmacokinetics of baicalin in EE-induced IHC ratsObjective:To establish an analysis method for determination of baicalin in rat plasma by liquid chromatography tandem mass spectrometry（LC-MS/MS）and study the pharmacokinetic characteristics of baicalin in normal and IHC rats.Methods:Plasma samples of rats were prepared by protein precipitation with methanol.Chromatographic condition:samples were analyzed on a C₁₈ column with mobile phase A（0.1%formic acid solution）and mobile phase B（methanol solution）at a ratio of 20:80（v/v）in 5 min,flow rate at 0.8 mL/min,10μL of injection volume and column temperature at30℃.Mass spectrometry condition:the detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source（ESI）in positive ionization mode,m/z 447.0→271.3.Single dose or five consecutive days’Baicalin at 200 mg/kg was orally given into normal or IHC rats and its blood concentration was determined by LC-MS/MS.The pharmacokinetic characteristics and parameters were calculated and recorded.Results:The method for baicalin detection was specific and showed excellent linearity over the concentration ranges of 1.01–506.00μg/mL（R²=0.9980）,and the lower limit of quantitation was 1.01μg/mL.Intra-day and inter-day precision averages were no more than10.55%.Intra-day and inter-day accuracy were between 94.94%and 109.13%,and the results of recovery test and stability test conformed to the requirement in Chinese Pharmacopoeia 2015 Edition.Pharmacokinetic study of baicalin in rats showed the concentration-time curve existed bimodal phenomenon.After multiple treatments,AUC and C_max of baicalin in rats elevated,while its CL and t_1/2 decreased.C_max and AUC of single baicalin in EE-induced IHC rats were dramatically increased,accompanying with reduced AUC,prolonged t_1/2 and slower CL.However,compared with single dose of baicalin in IHC rats,successive pretreatment of baicalin while EE modeling could lower the abnormal AUC,increase CL and shorten t_1/2,thus restoring the pharmacokinetic characteristics of baicalin in normal rats.Conclusion:Compared with single baicalin in normal rats the pharmacokinetic characteristics of single baicalin in IHC rats show elevated exposure and decreased elimination,which can be remitted by consecutive treatment of baicalin.The in-depth mechanism maybe derived from the reversion of baicalin on EE-induced the reduction of hepatic transporters and enzymes as well as intestinal injury.更多还原