基于HMGB1信号通路研究黄葵"解毒散瘀"对造影剂肾损伤的快速干预作用及入肾成分对其疗效的贡献度

The pathogenic mechanism of iodinated contrast-induced acute kidney injury (CI-AKI) is mainly associated with oxidative stress and inflammatory response induced by renal ischemia and direct nephrotoxicity. It holds that“intermingled toxin and blood stasis”is the main pathological link of CI-AKI in TCM，and emphasizes method of resolving toxin and dissolving stasis in treatment. Abelmoschus manihot is a representative herb which has strong effect on resolving toxin and dissolving stasis. Moreover, our pre-experiment found that HMGB1 play a pathogenic role in apoptosis, ROS generation and increased inflammatory cytokines induced by iodine contrast media iohexol, which can be alleviated by acute oral high dose of Abelmoschus manihot extract! Therefore, further study and more evidence are needed to clarify the role of HMGB1 and mechanism of acute protective efficacy induced by Abelmoschus manihot in CI-AKI..In this project, we use molecular biology, UPLC-MS-MS and the previously-established CI-AKI animal model and cell model et al, from three levels: the animal, cell and molecular level to focus on determining 1) HMGB1-TLR4-NF-κB/ inflammatory cytokines pathway in iodine contrast media induce renal tubular epithelial cells damage; 2) protective efficacy of Abelmoschus manihot and its regulation on HMGB1 pathway in CI-AKI ; 3) absorbed bioactive compounds(ABCs) of Abelmoschus manihot in CI-AKI rats’ blood and kidney tissues, evaluating their contribution to the protective efficacy and regulation of HMGB1 pathway according to the equation “ABC efficacy ÷ Abelmoschus manihot efficacy ×100%” , screening the most representative ABCs to further validate protective mechanism of Abelmoschus manihot in the vitro experiment, the conclusion from above experiment will highlight therapeutic material basis and molecular mechanism of acute renal protective induced by Abelmoschus manihot.

造影剂急性肾损伤(CI-AKI)发病机制与肾缺血及直接肾毒性诱发的氧化应激及炎症反应有关，HMGB1介导造影剂导致肾小管上皮细胞损伤。中医学认为“毒瘀互结”是本病的主要病理环节，预实验运用解毒散瘀的黄葵高剂量一次灌胃即能有效下调HMGB1并干预CI-AKI发生，我们推测黄葵的急性药效是通过抑制HMGB1介导的TLR4-NF-κB信号途径，但缺乏证据且分子机理远不清楚。本课题拟应用分子生物学、液质联用等技术，基于整体、细胞和分子水平三个层次，采用既往建立的CI-AKI动物和细胞模型，从造影剂-HMGB1-TLR4-NF-κB通路-炎性因子途径研究黄葵及吸收成分(ABCs)抗CI-AKI肾小管上皮细胞凋亡的分子机制，通过"ABC疗效÷母方疗效×100%"的简单公式定量药效物质对母药的疗效贡献度，采用高贡献度的ABCs加入离体实验进行验证，开辟黄葵急性作用的药效物质基础和分子机制研究的新途径。

造影剂急性肾损伤(CI-AKI)发病机制与肾缺血及直接肾毒性诱发的氧化应激及炎症反应有关，HMGB1介导造影剂导致肾小管上皮细胞损伤。. 本项目课题组基于动物、细胞、分子三个层次，首先验证了CI-AKI动物模型肌酐、尿蛋白、尿素氮、HMGB1、HMGB1 mRNA及TLR4、NF-κB、氧化应激、炎性因子、凋亡蛋白均显著升高。HMGB1抑制剂glycyrrhizin与黄葵总黄酮提取物800mg/kg对上述指标具有显著的干预效果，证实了CI-AKI发生的关键靶点为HMGB1。.我们测试了黄葵入肾活性吸收成分（ABCs）主要为芦丁、金丝桃苷、异槲皮苷、棉皮素-8-O-葡萄糖苷、杨梅素、槲皮素-3-O-葡萄糖苷、槲皮素。综合分析肾组织损伤指标、肾功能指标、HMGB1表达等数据，筛选出了槲皮素、异槲皮苷、金丝桃苷为能代表黄葵干预CI-AKI药效的有较高相似度，分析出槲皮素与黄葵药效具有最高相似度，定量了槲皮素对黄葵调控HMGB1靶点药效的贡献度为72%，可以代表黄葵作为细胞实验验证用的ABC。. 我们基因测序对CI-AKI大鼠的lncRNA相关靶点和作用网络进行富集分析，初步预测差异化表达的lncRNA NEAT1与本课题组关注的HMGB1有调控关系，故我们采用慢病毒介导的shRNA基因沉默技术，进一步深度研究HMGB1介导CI-AKI上游信号通路。我们使用黄葵药效高贡献度的入肾ABC槲皮素来进行干预，测试出槲皮素的IC50值为163.25μΜ。在CI-AKI模型中，HIF-1α、lncRNA NEAT1和HMGB1上调。槲皮素可以显著通过抑制HIF-1α来改善细胞损伤和凋亡。靶向lncRNA NEAT1的HIF-1α沉默改善了细胞损伤和凋亡。沉默lnc NEAT1可通过抑制HMGB1的表达改善CI-AKI细胞损伤和凋亡并下调下游炎性信号分子。我们的研究证实了lncRNA NEAT1/HMGB1信号通路是介导CI-AKI的核心通路，黄葵入肾ABC槲皮素通过抑制HIF-1α对lncRNA NEAT1/HMGB1信号通路的影响，发挥了类似于母方黄葵的药效，改善CI-AKI模型中的细胞损伤和凋亡。. 我们的研究揭示了CI-AKI发病的分子机制以及黄葵对CI-AKI干预作用的药效物质基础，对未来肾脏保护类中药防治CI-AKI的药效机制及活性成分研究有一定的参考价值