大黄灵仙胶囊对胆结石胆汁代谢组学特征及胆小管侧膜运转蛋白表达影响的研究

Cholelithiasis pathogenesis is a research focus of cholangiopathy while postoperative calculus remaining and recurring being a major clinical problem. In recent years, researchers in China and overseas have studied from various directions and aspects to explore the causes and pathogenesis of gall-stone. However, little has been done on bile metabonomics analysis. Our research group found that Dahuanglingxian capsule was effective on cholelithiasis prevention and treatment, and potential targeting sites were suggested in research. Moreover, 1H-Nuclear Magnetic Resonance (1HNMR) was used in our research to analyze the serum metabonomics of cholelithiasisi model animals and found the relations between serum lipid metabolite and cholelithiasis. However, it is really difficult to reveal the pathogenesis of cholelithiasis and the targeting sites of Chinese medicine by researches only from one direction as the forming of gall-stone and acting process of complex prescription of Chinese herbs are both extremely complicated. This research program will apply 1HNMR, immunohistochemistry, Western-Blot, and RT-PCR, to analyze the bile metabonomic characteristics of cholelithiasisi model and the protein expression changes of canalicular membrane transporters and to observe the intervention effects of Dahuanglingxian capsule. Through the comprehensive analysis of metabolizing information and biological events, the forming of gall-stone and the mechanism of Dahuanglingxian capsule for prevention and treatment of cholelithiasis will be further revealed, to provide a theory basis and research method for the application of combining Chinese medicine and western medicine for cholelithiasis prevention and treatment.

胆结石形成机制是胆道疾病的研究热点，而胆石病术后结石的残留与复发是临床医疗的难点。近年来国内外学者已从多角度、多方面对胆石成因及其机制进行探讨，但对胆汁代谢组学分析涉及甚少。课题组前期研究表明，大黄灵仙胶囊能有效防治胆结石，并发现其可能作用靶点；还借助核磁共振氢谱技术对胆石病模型动物进行血清代谢组学分析，发现血清脂质代谢产物与胆结石形成的相关性。然而，胆结石的形成及中药复方的作用过程非常复杂，单一角度研究很难真正把握胆结石的形成机制以及中药防治作用的靶点。本项目采用核磁共振氢谱技术结合免疫组化、Western-Blot等方法，分析胆石病模型豚鼠胆汁代谢组学特征以及肝细胞胆小管侧膜转运蛋白表达的改变，并在此基础上观察大黄灵仙胶囊的干预作用，通过综合分析代谢信息与生物学事件，进一步揭示胆结石形成和大黄灵仙胶囊防治胆结石的机制，为胆石病的中西医结合综合防治提供理论基础和研究思路。

国内外学者已从多角度、多方面对胆石成因及其机制进行探讨，但对胆汁代谢组学分析涉及甚少。然而，胆结石的形成及中药复方的作用过程非常复杂，单一角度研究很难真正把握胆结石的形成机制以及中药防治作用的靶点。本项目经调整采用气象色谱-质谱联用技术结合免疫组化、Western-Blot、RT-PCR 等方法，分析胆石病模型小鼠胆汁代谢组学特征和肝细胞胆小管侧膜转运蛋白表达的改变，以及慢性肝损伤兔胆石症模型肝细胞炎症因子、肝组织羟脯氨酸水平变化，并在此基础上观察大黄灵仙胶囊的干预作用，综合分析代谢信息与生物学事件。结果：小鼠胆结石模型：①模型组小鼠成石率为100%，胆囊内见颗粒样或泥沙样沉淀，干预组成石率分别为44.44%、30%，两组均低于M组（P<0.0125）,②经溴化钾压片法红外光谱分析出小鼠胆结石为胆固醇结石；③GC-MS结果：与其他组比较，模型组小鼠胆汁内源性代谢物丙氨酸、柠檬酸、赖氨酸、蛋氨酸等差异均有统计学意义（P<0.05，P<0.01）；④PCR结果：与模型组相比，N、Y、D组小鼠 Abcb11 和 Abcc2均相对升高（均P<0.001）；⑤免疫组化结果：与模型组相比，N、U、Y、D 四组组小鼠 Abcb11和Abcc2蛋白表达均升高（均P<0.001）；⑥wternblotting结果：与模型组相比，N、U、Y、D四组小鼠Abcb11和 Abcc2蛋白表达均升高（均 P<0.001）；与N组相比，D组Abcc2 蛋白表达下降（P=0.002）；兔胆结石模型：①血常规、生化：与对照组相比，治疗组白细胞、中性粒细胞、淋巴细胞、ALT、AST、TBIL等指标均有统计学意义（均P＜0.05）；②免疫组化：与对照组相比，中药干预组肝细胞胞浆或胞膜上棕褐色颗粒沉积表达明显减少，小胆管胆汁淤积减少；③ PCR检测结果：与对照组相比，用药各组的IL-6、EGR-1mRNA的表达量均有不同程度降低（P＜0.05）；④与对照相比，中药干预组肝细胞胞羟脯氨酸含量均有不同程度降低（均为P＜0.05）。结论：大黄灵仙胶囊能够改善小鼠胆汁代谢组学特征、血清生化指标，同时能改善胆囊结石小鼠Abcb11和Abcc2转运蛋白的基因和蛋白表达，使其接近正常生理水平，减少IL-6、EGR-1的基因与蛋白表达量，不同程度地降低慢性肝损伤性胆石病实验兔肝组织中羟脯氨酸含量，从而达到防石治石的功效。