天然来源卤酚类高活性衍生物LM49对LPS诱导的血管内皮炎症MAPK信号通路的调控作用与机制研究

Inhibiting vascular endothelium inflammation is an effective strategy for the treatment of atherosclerosis (AS) and other associated cardiovascular diseases. Severl natural new halophenol derivatives prepared by our group showed strong inhibitory activities on the inflammation of human umbilical vein endothelial cells(HUVECs) and could regulate the action via mitogen activited protein kinase(MAPK) signal pathway. However,the action mechanism is unclear. In the present study, a strong active derivative LM49 is selected as a small molecule probe to investigate its regulatory mechanism on the LPS-induced MAPK signal pathway in vascular endothelium by chemical and cell biology methods. By the proteomics technique, the changed proteins can be founded after treatment with LM49 in the vascular endothelium inflammation. The differential proteins will be investigated through analyzing the protein profiles and identified by MALDI-TOF-MS/MS to find the related target proteins, which will be further verified by RNA interference/overexpression methods. The biological targets can also be recognized by a computer-assisted homology modeling/molecular docking technique. The regulatory mechanism of such small molecule compound as LM49 on the LPS-induced MAPK signal pathway will be compositely analyzed and investigated from molecule-cell-whole animal levels to provide the scientific basis for the further structural optimization of halophenol compounds.

抑制血管内皮炎症反应是防治动脉粥样硬化（AS）等心血管疾病的重要策略。前期发现天然来源卤酚类活性衍生物能显著抑制人脐静脉内皮细胞（HUVECs）炎症反应，其作用可能是通过丝裂原活化蛋白激酶（MAPK）信号通路来调节的，但具体机制还不清楚。本课题以高活性新化合物LM49为探针，应用化学生物学方法，探讨其对脂多糖（LPS）诱导的血管内皮炎症MAPK信号通路的调控作用；通过蛋白质组学研究LM49抑制血管内皮炎症反应前后蛋白质谱的改变，经图像分析后选取差异蛋白斑点进行MALDI-TOF-MS/MS 鉴定目标蛋白，寻找相关的新信号调节靶蛋白，利用RNA干扰/过表达方法进行反向验证，采用同源模建/分子对接（Docking）等计算机辅助技术识别生物靶点，从分子-细胞-整体动物三个层次综合分析探讨该类小分子对调控LPS诱导的血管内皮炎症MAPK信号通路的作用及机制，为其结构优化提供科学依据。

阐明了LM49抑制LPS诱导的血管内皮细胞炎症反应的机制与调控CAV-1及下游MAPK, NF-κB信号通路的关联，从体内外阐明了CAV-1是LM49保护血管内皮细胞损伤的潜在靶蛋白，发现了LM49抑制炎症反应与其可诱导巨噬细胞由M1向M2型极化有关。LM49通过抑制单核细胞与LPS诱导的EA.hy926细胞的粘附作用，抑制粘附蛋白ICAM-1、VCAM-1、E-selection和炎症介质IL-6、IL-1β、MCP-1及TNF-α的表达，从而抑制ERK1/2和p38MAPK磷酸化，降低炎性损伤，保护血管内皮细胞；采用iTRAQ技术对总蛋白表达谱进行分析,对显著差异蛋白CAV1和RHOA进行了验证；发现LM49可通过调控CAV-1/NF-κB信号抑制血管内皮细胞炎症；可抑制小鼠主动脉局部炎症细胞的浸润，及降低血清中TNF-α和IL-6的表达和主动脉中IL-6、IL-1β、MCP-1和TNF-α mRNA表达，抑制LPS诱导的小鼠血管壁炎症；通过抑制CAV-1磷酸化，增加其下游相关细胞连接蛋白Occludin及VE-Cadherin的表达，降低血管内皮细胞单层的通透性；可通过直接作用于CAV-1，抑制c-Src与CAV-1的相互作用，降低CAV-1磷酸化水平，保护血管内皮细胞；可降低EAE模型小鼠脑组织中CAV-1磷酸化水平，增加Occludin及VE-Cadherin蛋白表达，维持BBB结构与功能的完整性，起到对EAE模型小鼠的治疗作用；LM49能够降低M1型巨噬细胞表面标志物CD16/32、NOS的表达，升高M2型巨噬细胞表面标志物CD206、Arg I的表达；可通过上调JAK1、p-JAK1、JAK3、p- JAK3、STAT6和p-STAT6诱导巨噬细胞由M1型向M2型极化，从而起到抗炎作用；LM49还具有明确的抗大鼠II糖尿病肾病（DN）作用，其主要治疗指标尿酸、尿蛋白、尿微量白蛋白、生长转化因子、血糖、低密度脂蛋白等优于目前临床药物卡托普利。此外，以LM49为先导物设计合成了63个新衍生物，其中有23个呈高活性。发表相关学术论文9篇，其中SCI收录5篇，另有多篇较高水平论文正在整理中。授权了中国发明专利4项，申请PCT专利和中国发明专利各1项。