基于影响TLR4-NLRP3炎症小体信号通路对总丹参酮治疗肺炎作用机制研究

Pneumonia is a common disease. Under the circumstance of the abuse of antibiotics to lead to antimicrobial resistance, to sniff out effective components from traditional Chinese medicines that could alleviate the inflammatory syndromes and decrease the damage to organisms is an effective and practical strategy. In terms of our recent investigation, we found that the therapeutic effect of Salvia Miltiorrhiza Bunge (Danshen) on pneumonia in the clinic was significant. However, due to the unknown pharmacodynamic material basis and mechanism of action of Danshen, its wide application is limited in the clinic. In this study, our previous data indicated that total tanshinones (TTN) exhibited a significant anti-inflammatory effect in vitro and in vivo. (1) TTN significantly decreased LPS-induced TNF-α, IL-6, and IL-1β release. (2) In stark contrast to antibiotics and hormone, the initial mechanism exploration demonstrated that ROS and NLRP3/Caspase-1 inflammasome were involved in the process of TTN’s anti-inflammation. (3) The result of molecular docking of tanshinone IIA, the major ingredients of TTN, and Toll-like receptor 4 (TLR4) showed that tanshinone IIA suppressed TLR4 dimerization, which suggested that the anti-inflammatory effect of TTN may be through suppression of TLR4 dimerization. Collectively, using the appropriate evaluation model of pharmacodynamics in vitro and in vivo, this project aims to corroborate the pharmacodynamic material basis for Danshen’s treatment with pneumonia, which will further unravel the hallmarks and the mechanism of Danshen’s effect. Furthermore, this study will provide with a scientific theoretical foundation and experimental basis for Danshen’s application in the clinic of treatment with pneumonia.

肺炎是一种常见的疾病，在滥用抗生素导致细菌耐药的严峻形势下，从中药中寻找不仅能改善炎性症状，又能减少机体损伤的有效组分，成为了新的研究热点。而近期调研发现，丹参在肺炎的临床治疗上效果显著，但由于其物质基础及其分子机制不清楚，限制了其在肺炎治疗中的应用。本课题组前期研究发现，总丹参酮具有明显的抗炎作用：（1）总丹参酮体内外能明显减少TNF-α、IL-6、IL-1β炎症因子释放；（2）在机制初步研究中发现，与抗生素及激素不同，总丹参酮抗炎作用涉及到ROS以及NLRP3/Caspase-1炎症小体；（3）分子对接结果显示总丹参酮主要成分丹参酮IIA与TLR4结合，抑制TLR4形成二聚体，从而达到抗炎作用。因此，本课题拟采用体内外药效学评价模型，明确丹参治疗肺炎的药效物质基础，阐明其治疗肺炎的作用特点和分子机制，为丹参在肺炎治疗中的临床应用提供科学理论依据和实验基础。

丹参是唇形科植物丹参(Salviae Miltiorrhizae Bge.)的干燥根和根茎。丹参酮(Tanshinone)是丹参的主要脂溶性有效成分，包括丹参酮I (Tanshinone I) 、丹参酮IIA (Tanshinone IIA)、隐丹参酮 (Cryptotanshinone，CPT)、5，16-二氢丹参酮(15，16-Dihydrotanshinone，DHT)等60多个化学成分。急性肺损伤（ALI）引起的肺炎是一种严重威胁人类健康的疾病，当发展到重度(氧合指数<200)时，称为急性呼吸窘迫综合征(ARDS)。全球每年有300多万ARDS患者，占重症监护病房(ICU)入院人数的10%。ALI/ARDS患者的平均死亡率为35%-46%。尽管进行了数十年的研究，ALI 的发病机制至今尚未明确。丹参酮类成分对细胞中亚硝酸盐含量有明显抑制作用，并显著减少促炎细胞因子TNF-α、IL-6、IL-1β、ROS和NO的释放量。此外，LPS显著促进NF-κB通路活化和核转位，并使MAPK、PI3K通路蛋白磷酸化水平明显上调，这一作用被丹参酮类成分抑制。PLCγ2通路蛋白PLCγ2、PIP2、IP3和DAG蛋白表达受丹参酮类成分调节。进一步研究表明，丹参酮类成分能显著上调Nrf2通路活化从而发挥抗氧化作用。Western blotting实验发现丹参酮类成分能显著下调细胞中Nek7、NLRP3、cleaved-Caspase-1以及成熟的IL-1β蛋白表达。免疫共沉淀和免疫荧光实验结果表明丹参酮类成分抑制Nek7/NLRP3复合物形成。Cellular thermoshift assay结果表明丹参酮和Nek7有直接结合作用。进一步的研究发现，Nek7沉默后抑制了NLRP3炎症小体的激活。体内实验：小鼠肺部Nek7基因的敲除明显改善了ALI的病理变化，并能抑制 NLRP3炎症小体通路蛋白表达以及IL-1β的释放。血常规数据表明，丹参酮类成分能减少血液中Lymphocyte、Neutrophil和WBC数量；MPO试剂盒分析结果说明，肺组织中MPO活性受丹参酮类成分抑制。本课题拟通过对丹参中丹参酮类成分进行研究，深入探讨其对肺炎的治疗作用及其分子机制。为肺炎相关疾病的防治提供新的策略，为明确丹参中治疗肺炎的物质基础提供科学依据。