长白山区域朝药肾炎草与独活防治败血症性急性肾功能衰竭的药效物质基础及其作用机理研究

Sepsis is a serious disease accounting for the major of death in intensive care unit. Until now, treatment of severe sepsis is unsatisfactory. Moreover, sepsis is a major cause of acute renal failure in intensive care units, sepsis-related ARF is associated with a 70-80% mortality. Sepsis induces several proinflammatory cytokines including tumor necrosis factor-α (TNF-α), a major pathogenetic factor in sepsis-related acute renal failure, induced via lipopolysaccharide (LPS) signaling through Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB) and activated protein-1 (AP-1). Thus, it is important to research new anti-sepsis related acute renal failure drug through modulation of TLR4, NFκB and AP-1 signaling..Melandrium pubescens Makino. and Aralia continentalis Kitag., known as Korean Chinese folk medicine"Shinyemcao"and "Dolwhal" around Changbai Mountain area, have been found to protect against sepsis-related acute renal failure induced lethality in mice and have renoprotective properties. The purpose of this proposal is to separate compounds from the active part and identify the chemical structures by using chromatographic and spectroscopic methods. And then, isolated compounds will be evaluated for potential utility in combating experimental sepsis-related acute renal failure, inhibiting TNF-α and nitric oxide (NO) levels using both in vivo and in vitro screening assay systems. .In potential mechanisms study, the effect of selected 3-5 active compounds on recognition receptors of LPS signaling pathways, p38 mitogen-activated protein kinase (p38MAPK), AP-1, nuclear factor-κB-p65 (NF-κB-p65) and downstream inflammatory factors TNF-α, NO, cytokines, glomerular filtration rate (GRF), blood urea nitrogen (Bun), Creatinine (Cr) will be observed in sepsis-related acute renal failure mouse model. To investigate the molecular mechanisms in LPS-induced RAW 264.7 macrophages and renal tubular epithelial cells (HK-2 cells), respectively, the effects of 3-5 active compounds on recognition receptors of LPS signaling pathways, the expression of LBP、CD14、TLR4、IKK、IκB、NF-κB mRNA, and protein levels of TLR4、p38、P65、c-jun、c-fos, and the concentration of TNF-α and cytokines will be measured..This proposal will seek to examine the potential protective and therapeutic effects of new agents from Korean Chinese folk medicine around Changbai Mountain area in sepsis-related acute renal failure.

在国家自然科学基金资助下，本课题组从长白山区域民族药中筛选出降低败血症致死率，又具有肾保护作用的朝药肾炎草和独活。基于针对LPS 信号通路关键环节进行目标明确的阻断和调节，以遏止LPS所致器官衰竭等病理发展进程，达到防治败血症性器官衰竭思路，本课题拟利用天然药物化学和药理学方法，活性导向分离出活性化合物，分析其有效部位化合物组合，并进行整体动物药效学和分子水平的活性筛选，找出能降低致死率，又能抑制TNF-α等炎症因子的先导化合物，观察对肾功能衰竭的小鼠保护作用和对LPS刺激巨噬细胞和肾小管上皮细胞产生炎症反应的影响，从识别受体、胞内信号转导分子及核转录因子、下游炎症产物四个层次探讨对LPS诱导的TLR4信号通路上几个重要分子，揭示防治败血症性急性肾功能衰竭作用机理。本研究成果将为防治败血症性急性肾衰基础研究提供新的先导化合物，为长白山区域朝药防治败血症性急性肾功能衰竭新药研发提供理论依据。

本课题组利用天然药物化学方法，分别从朝药肾炎草和独活的活性部位分离和纯化出59个化合物，利用波谱学方法对所分离的化合物1-59进行了化学结构鉴定，其中化合物1-4为新化合物。对化合物1-59进行整体动物和细胞水平的活性筛选，找出即能够降低败血症致死率，又能抑制TNF-α和NO等炎症因子的化合物30，并通过药理学实验观察化合物30对D-GalN/LPS诱导的败血症性急性肾衰竭小鼠的防治作用，通过观察小鼠48小时存活率，测定小鼠血清中TNF-α、NO、IL-1β和IL-6含量和活性，以及对小鼠血清尿素氮和肌酐含量的影响。化合物30在25 mg/kg和50 mg/kg时均可显著性降低败血症小鼠血清TNF-α、NO、IL-1β和IL-6含量，降低小鼠血清尿素氮和肌酐的含量，通过对败血症小鼠急性肾衰竭的保护作用，提高败血症小鼠的存活率。同时研究了化合物30对败血症性肾功能衰竭小鼠的肾组织LPS 信号转导途径的识别受体、NF-κB 信号等通路的影响，化合物30在25和50 mg/kg抗败血症性肾功能衰竭机制与抑制下游炎症因子TNF-α 的释放有关，其作用可能通过下调小鼠肾组织LPS 识别受体CD14、TLR4 的表达、抑制c-jun、c-fos和TNF-α的mRNA表达量和p-p38蛋白的表达，下调NF-κB 通路中信号转导分子IKK、NF-κB的mRNA表达量和p65蛋白的表达，干预LPS识别受体、p38 MAPK/AP-1和NF-κB信号通路而实现。. 本实验还进一步分别探讨了化合物30在12.5、25、50 μM浓度下均可从不同程度对LPS诱导的小鼠巨噬细胞和肾小管上皮细胞中CD14、TLR4、IKK、NF-κB mRNA以及TLR4、p38、c-jun、c-fos蛋白，p65核蛋白的表达有抑制作用，表明化合物30抑制了小鼠巨噬细胞和肾小管上皮细胞内LPS激活的p38 MAPK/AP-1和NF-κB通路的多个信号分子，预防NF-κB、P65的大量转位入核，抑制了NF-κB的活化，进而减少炎症因子的大量失控表达，即化合物30不仅影响LPS诱导小鼠RAW264.7和HK-2细胞内LPS识别受体，而且通过调控p38 MAPK/AP-1和NF-κB信号通路，抑制下游炎症因子TNF-α的释放，最终抑制MODS、败血症等的发生。