基于“化学成分敲除/敲入--谱效表征”研究黄连抗幽门螺杆菌活性分子群及其整合机制

Rhizoma coptidis proved to exhibit more potent anti-H. pylori activity compared with its major component berberine, and mechanism of action remains to be elucidated. Studies have shown that urease plays a vital role in the pathogenesis of H. pylori, and thiol is of vital importance to for the activation of urease, blockage of binding between urease subunits thiol and Ni2+ in its active site resulted in massive inhibition of urease. Findings of our recent work indicated that the aqueous extract of Rhizoma coptidis could effectively affect the subunit thiols in the active site of urease, significantly inhibiting the urease activity, which was superior to berberine. The result indicated that the anti-H. pylori activity of Rhizoma coptidis may stem from the synergic effect of its multiple components. In the present work, the 'component knock-out/knock-in and spectrum-efficiency characterization' strategy will be introduced to comparatively analyze the anti-H. pylori and urease-inhibitory effect variation by the knockout of specific composition, and shed light on the relationship between the overall efficacy and components of different levels and recognize the effective components. On this basis, enzymatic methods, gene transcription and protein expression assay were employed to further investigate the active components obligatory for the urease-inhibitory effect of Rhizoma coptidis and the underlying molecular mechanism via regulating the urease subunits. This present scheme might contribute to further illuminate the active molecule constitutes and the integrating mechanism of Rhizoma coptidis in its anti-H. pylori activity, and provide promising information for identification of brand-new anti-H. pylori active components and urease inhibitor.

黄连抑制幽门螺杆菌（H. pylori）活性显著强于其主要成分小檗碱，机制有待阐明。研究表明，脲酶是H. pylori的关键致病因子，而巯基对脲酶活性至关重要，阻断脲酶活性位点上的蛋白亚单位巯基与Ni2+结合能有效抑制脲酶活性。课题组研究发现，黄连水提物能结合H. pylori脲酶主要活性位点巯基，有效抑制脲酶活性，作用明显优于小檗碱，提示黄连抗H. pylori活性可能是多种成分协作的结果。本课题拟通过“化学成分敲除/敲入--谱效表征”的方法，比较特定成分敲除前后抗H. pylori及脲酶活性变化，阐析组分层次效应与药效整体差异性的关系，辨识药效成分群。并以巯基介导脲酶活性为切入点，通过酶学、基因转录及蛋白表达研究，考察其调控脲酶蛋白亚基，抑制其活性的分子机理，揭示黄连抗H. pylori的活性分子群及整合效应机制，为新型抗H. pylori活性组分的发现和脲酶抑制剂的研究提供理论支持。

项目背景.黄连抑制HP活性显著强于主要成分小檗碱，机制有待阐明。脲酶是HP的关键致病因子，而巯基对脲酶活性至关重要，阻断脲酶活性位点上的巯基与Ni2+结合能有效抑制其活性。黄连水提物能结合HP脲酶（HPU）活性位点巯基，抑制其活性，作用明显优于小檗碱，提示黄连抗HP活性可能是多种成分协作的结果。..研究内容.1.基于“成分敲除”的药效物质辨识方法的建立；.2.基于“成分敲入”的量效关系研究；.3.黄连活性组分抗HPU机理的研究；.4.不同来源原小檗碱类生物碱的理化、生物活性、药动学比较及立体异构的探讨。..研究结果.1.抑酶作用强弱为：表小檗碱>巴马亭>药根碱>黄连碱>小檗碱，表小檗碱明显优于阳性对照，各化合物与脲酶活性中心及周围不同残基互作，可能是产生活性差异及协同作用的原因。.2.黄连碱为黄连抗HP作用最强的成分，小檗碱次之，巴马汀，药根碱和表小檗碱相对较弱。.3.表小檗碱对HPU和JBU的抑制类型分别为反竞争性和竞争性抑制；巴马亭对两种脲酶的抑制均为非竞争性抑制；黄连碱的分别为混合型和非竞争性抑制。.4.表小檗和黄连碱结合脲酶为缓慢型结合。.5.表小檗碱，巴马亭和黄连碱抑制脲酶的位点为其巯基残基，三者可能通过作用HPU活性中心区域，以氢键结合活性中心不同关键氨基酸残基，影响活性构象，抑制其活性。.6.黄连碱可抑制脲酶成熟过程中辅助蛋白UreG的功能，阻断UreG二聚体形成，促进Ni2+与UreG二聚体的解离，而对GTP无明显影响。.7.黄连与黄柏来源的小檗碱在结晶形态，溶解度，熔点及旋光度方面存在较大差异。不同来源的同一原小檗碱类生物碱在抑酶活性，药动行为方面具有较大差异。..科学意义.研究有助阐明黄连治疗HP相关性胃病的机制，为新型抗HP活性组分的发现和脲酶抑制剂的研究提供依据，并为探讨中药抗HP的机理研究提供新的分子靶点。原小檗碱类生物碱光学活性的发现，可为其结构和生物活性研究提供新的视角和领域。