蒙药朱如拉及其活性成份通过调控中性粒细胞解偶联蛋白-2的表达及其活性从而发挥抗炎作用的分子机制研究

ZHURULA, the fruit of Fructus Gardeniae, has been traditionally used for treating a variety of acute or chronic inflammation diseases by traditional Mongolian medication due to its low toxicity and significant efficacy. Since there is a limitation of scientific evidences for Anti-inflammatory working mechanism, the economic usage and the development of Fructus Gardeniae are severely restricted. As an important executive of non-specific immunity defense mechanism, polymorphonuclear neutrophil (PMN) plays major role in inflammatory. .Study of PMN immunological function caused by ZHURULA has therefore to be significantly important to understand the inflammatory function. Recent studies have found out ZHURULA extracts, Genipin, which acts as inhibitor of uncoupling protein 2(UCP2) and plays important function in diabetes II. UCP2 is a mitochondrial inner membrane transporter protein. It has been found to participate many disease physiological processes and has strong potential to drug target. Therefore, using UCP2 as a breakthrough point, this study focus mainly on investigating the inflammatory mechanism caused by HURULA, specifically, Genipin. Firstly, the expression and activity of UCP2 were checked in mouse PMN after drug treatment applying qRT-PCR and Western blotting methods. Secondly, the relevant cellular immunological function and cellular factors caused by drugs were analyzed in vivo and in vitro. Finally UCP2 knockout mice were applied for studying the anti-inflammatory mechanism caused by Genipin. This study provides an innovative methodology for traditional national drug research and development. In addition, it would offer scientific evidence for the traditional Mongolian medicine, which gives a step stone for new developed Mongolian drugs.

朱如拉是栀子果实，是治疗急慢性炎症的传统蒙药，毒副作用小，药效显著。由于缺乏抗炎免疫作用分子机制研究，它的市场化和进一步开发利用受到严重制约。中性粒细胞(PMN)是机体非特异防御反应的重要执行者，在炎症反应中扮演重要角色。研究朱如拉对PMN免疫功能的影响，对于了解其抗炎作用有重大意义。研究发现，栀子提取物Genipin在II型糖尿病治疗中作为线粒体解耦联蛋白2(UCP2）的抑制剂发挥主要作用，并参与许多疾病的病理生理学过程，有望成为潜在的药物靶点。因此，本课题将UCP2作为切入点，探讨朱如拉的抗炎免疫作用机制。首先采用qPCR、WB法，分析药物对小鼠PMN中UCP2表达、活性的影响；其次经过体内外实验探讨该药对免疫功能及相关细胞因子的作用；最后利用敲除小鼠阐明朱如拉的抗炎分子机制。这将是新思路、方法在民族药物研究与开发的有效应用，为传统蒙药研究提出科学依据，对新的蒙药开发奠定理论基础。

朱如拉是蒙医用于治疗各种急慢性炎症的、历史悠久的传统蒙药。由于缺乏抗炎免疫作用机制方面的科学依据，影响了它的市场化和进一步开发利用。线粒体是细胞的能量储存器，它的功能异常与多种代谢性疾病的发生发展密切相关。大量的研究表明，线粒体解偶联蛋白2（UCP2）有效反应线粒体的功能，参与许多疾病的病理生理学过程，有望成为潜在的药物靶点。本课题采用急性腹膜炎小鼠模型，以中性粒细胞（PMN）UCP2蛋白作为切入点，探讨了朱如拉及其有效成分Genipin的抗炎免疫作用机制。本研究，首先对栀子进行了提取分离,并配置了不同浓度的朱如拉粗提物（水和乙醇提取）和Genipin水溶液,然后利用ZymosanA制作了急性腹膜炎小鼠模型。之后，采用qRT-PCR、Western blotting法，检测了灌胃不同浓度朱如拉和有效成分Genipin水溶液的小鼠体内PMN细胞中的UCP2基因和蛋白的表达量，其次，通过体内外实验探讨了蒙药朱如拉及其有效成分Genipin（或栀子苷）对细胞迁移和炎症相关细胞因子的作用。第三，通过UCP2基因敲除小鼠的制作以及观察药物对基因缺失小鼠的影响，探讨了Genipin 对急性炎症小鼠PMN细胞上UCP2的部分作用机制。最终实验结果证明，蒙药朱如拉水提物和Genipin水溶液对PMN的免疫功能，主要是细胞趋化作用具有促进作用。同时还发现朱如拉粗提物和Genipin水溶液对Zymosan A引起的急性腹膜炎小鼠模型体内中性粒细胞UCP2的表达及其活性具有抑制作用，并且该过程与调控钙离子的流入相关。这项研究是新思路、新方法在民族药物研究与开发中的有效应用，它为传统蒙药朱如拉的临床用药提出了科学的实验依据，还将为新的蒙药开发奠定了一定的理论基础。