茵栀黄方利湿退黄作用药效物质、作用机制及其配伍规律研究

Neonatal jaundice is one of the main diseases for babies. Hyperbilirubinemia is the clinical manifestations of this disease occurred.Bilirum encephalopathy and legacy sequela will be resulted if hyperbilirubinemia cannot be treated timely, which will bring to heavy burden for social and family. Phototherapy and drug therapy (enzyme inducers, traditional Chinese medicine) are often used. The use of enzyme is restricted due to side effects and tolerability, while traditional Chinese medicine is widely favored by clinicians. The presciption of Yinzhihuang is a classical traditional Chinese medicine, widely used in neonatal jaundice/hyperbilirubinemia, and obtain better clinical curative effect. However, the mechanism of action, effective substituents and its prescription compostion rules are not clear. Based on this, the endogenous cholestasis jaundice rat model will be used to investigate the relationships of drug amount and effect, the prescription composition rules. After the diiferent drugs adminstration to rat model in different periods, the proteomics technology will be used to analyze the different expression of liver cell membrane protein and find the different protein, and the different protein are identified. Through analysis the function of different protein, the mechanism of action of Yinzhihuang and its prescription composition rules will be demonstrated. Moreover, the serum drug chemical fingerprint technology and chemometrics algorithm associated quantitative spectrum activity relationship will be adopted to screen the potential effective substituents in Yinzhihuang prescription, and the potential compounds will be seperated by chromatographic methods and identified by spectrum analysis. The pharmacodynamics of the selected compounds will be further tested with rat model used in this project.

新生儿黄疸是新生儿室内收治的主要疾病之一，临床表现为高胆红素血症，如不及时救治可能导致胆红素脑病，遗留后遗症，给社会和家庭带来沉重负担。目前主要采用光疗和药物治疗（酶诱导剂、中药），酶诱导剂由于副作用大且耐受性差，其使用受到限制，而中药则广泛受到临床医生的青睐。茵栀黄方是经典的利湿退黄中药，广泛用于新生儿黄疸/高胆红素血症的治疗，临床疗效确切。然而，其发挥利湿退黄作用的作用机制、药效物质及其方剂配伍规律尚不清楚。基于此，本项目拟采用内源性胆汁淤积黄疸大鼠模型对茵栀黄方的量效关系、配伍规律进行研究；采用蛋白质组学技术对不同配伍茵栀黄方干预后的肝细胞膜转运蛋白组进行分析，寻找差异蛋白并进行鉴定，通过差异蛋白的功能分析，进一步阐释茵栀黄利湿退黄的作用机制及配伍规律；采用血清药物化学谱与化学计量学算法相关联构建定量谱效关系，筛选出潜在药效物质，并进行药效化合物鉴定及药效学验证。

茵栀黄方在治疗小儿黄疸中有显著的疗效，但其作用机制及配伍规律尚不清楚。本项目采用炔雌醇诱导制备大鼠胆汁淤积型黄疸模型，以模型大鼠血清中胆酸盐、胆红素含量及组织病理学变化为药效学指标，对一定剂量范围的茵栀黄全方不同用药周期后的药效学进行考察，建立茵栀黄全方退黄作用的量-时-效关系，并在此基础上从转运体表达变化角度对茵栀黄方退黄的作用机制进行分析；用茵栀黄的不同组方对胆汁淤积大鼠模型干预后，对各组大鼠生化指标、肝细胞膜转运蛋白及病理组织进行分析，初步对茵栀黄方的配伍规律进行阐释。研究结果显示，模型组与正常组相比，各项血清生化指标呈现上升的趋势，但以直胆红素（DBIL）及总胆汁酸（TBA）的升高最为显著（P＜0.05）。茵栀黄方干预14 d后，血清中DBIL及TBA较生理盐水组显著下降（P＜0.05），且茵栀黄方高剂量（2.68 g/kg/d）干预14 d后，血清中总胆红素（TBIL）及DBIL与低、中、超高各剂量组相比，均显著降低（P < 0.05）。模型组与正常组相比，转运体Na+依赖性的牛磺胆酸共转运多肽（Ntcp）、有机阴离子转运多肽（Oatp2)、胆盐输出泵（Bsep）及多药耐药蛋白2（Mrp2）表达均显著下降（P＜0.05），而多药耐药蛋白3（Mrp3）, 多药耐药蛋白4（Mrp4）表达均显著上升（P＜0.05），茵栀黄方干预14 d后，Mrp2，Mrp3, Oatp2及Ntcp的表达较对照组显著升高（P＜0.05）。用茵栀黄的不同组方对胆汁淤积大鼠模型干预14 d后，君药茵陈对Mrp2，Mrp3, Oatp2及Ntcp有显著的调节作用，促进了胆红素经胆汁的排泄，臣药栀子增强了茵陈对Mrp3和Oatp2上调的作用，佐药金银花和黄芩增强了茵陈对Oatp2上调的作用，但逆转性的调节了茵陈对Mrp3和Ntcp的调节作用。总之，茵栀黄方对介导胆盐或胆红素肝摄取和外排的转运蛋白（Mrp2，Mrp3, Oatp2及Ntcp）的调控是其退黄利胆的重要机制之一，且君药茵陈对介导胆红素的转运体有显著的调节作用，而臣药与佐药可加强和/或逆转君药的调节作用。