MAP1S调控TLRs信号通路介导同型半胱氨酸引起泡沫细胞自噬的分子机制

Homocysteine (Hcy) is an independent risk factor of atherosclerosis (As), its harmfulness is large, but the mechanism is not clear. The cell autophagy is involved in the regulation of cardiovascular disease based on As lesions and previous study also found that toll-like receptors (TLRs) can activate the cell autophagy, MAP1S plays a "switch" role in the regulation of autophagy, so it can be used as potential target for intervention, but molecular mechanisms of MAP1S caused cell autophagy by regulating TLRs pathway has not been reported. This topic proposed on the basis of copying Hcy animal model and cell model, detecting the expression and distribution of MAP1S and determine the subtypes of TLRs by organizing tissue membrane hybridization to clear their roles in Hcy induced As; constructing the activated state TLR4 plasmid and transferred into cells, Immunofluorescence technique was used to analyze the formation and changes of LC3 aggregate to explore the roles of MAP1S and TLR4 in autophagy; specific antibodies were used to locate the position and expression of MyD88 and LC3 in cells, Immune coprecipitation was used to detect the changes of transshipping MyD88 to LC3II promoting autophagy body mature, to explore the mechanism of Hcy affects MyD88 locate in the LC3 combined autophagy through MAP1S, to provide potential drug targets for the prevention and treatment of As.

同型半胱氨酸(Hcy)是动脉粥样硬化(As)的独立危险因子，危害性大，但机制未清。文献报道细胞自噬参与了以As病变为基础的心血管疾病的调控，且前期研究提示Toll样受体(TLRs)可激活细胞自噬，而MAP1S在调控细胞自噬中起到“开关”作用，可作为潜在干预靶位，但MAP1S调控TLRs通路作用于接头蛋白介导Hcy引起泡沫细胞自噬的机制未见报道。本课题拟采用组织膜杂交等检测组织中MAP1S的表达与分布情况并确定TLRs的亚型，明确其在Hcy引起As中的作用；构建激活状态的TLR4质粒并转染细胞，免疫荧光技术分析LC3聚集体的形成及变化，探讨MAP1S及TLR4在细胞自噬中的作用；利用特异性抗体对细胞内MyD88和LC3进行定位并检测其含量，免疫共沉淀观察将MyD88转运至LC3II促自噬体成熟的变化，探讨Hcy经MAP1S调控MyD88结合于自噬体引起As的机制，为Hcy的防治提供潜在药靶。

背景：同型半胱氨酸(Hcy)是动脉粥样硬化(As)的独立危险因子，危害性大，但机制未清。文献报道细胞自噬参与了以As病变为基础的心血管疾病的调控，且前期研究提示Toll样受体(TLRs)可激活细胞自噬，而MAP1S在调控细胞自噬中起到“开关”作用，可作为潜在干预靶位，但MAP1S调控TLRs通路作用于接头蛋白介导Hcy引起泡沫细胞自噬的机制未见报道。. 内容：复制ApoE-/-鼠高Hcy血症As模型，Western blot检测自噬相关蛋白p62、LC3B的表达，观察泡沫细胞自噬在Hcy引起As中的作用；免疫荧光观察小鼠血管主动脉组织与泡沫细胞中MAP1S表达水平的改变；过表达MAP1S后Western blot检测自噬相关基因LC3B与Beclin-1的蛋白表达情况；qRT-PCR及Western blot检测泡沫细胞中TRL4的表达情况；过表达MAP1S后检测TLR4的表达，阐明MAP1S调控TLR4在Hcy诱导泡沫细胞自噬中的作用；进一步采用qRT-PCR和Western blot检测泡沫细胞中TLRs通路接头蛋白MyD88 mRNA与蛋白的表达，双荧光素酶报告系统与蛋白质免疫共沉淀(Co-IP)分析MAP1S与Myd88相互作用，揭示Hcy通过MAP1S调控TLRs通路接头蛋白MyD88引起As的作用机制。. 结果：首先，小鼠血管主动脉与泡沫细胞中自噬相关蛋白LC3B的表达降低，p62的表达升高；其次，qRT-PCR及Western blot结果显示MAP1S表达水平显著降低，且过表达MAP1S后自噬水平明显增加；进一步研究显示TRL4在Hcy诱导的泡沫细胞中表达降低，过表达MAP1S后TRL4表达水平升高；最后，我们发现Hcy处理的泡沫细胞中MyD88明显降低，双荧光素酶报告系统与Co-IP结果显示MAP1S与Myd88相互作用，且敲除MyD88后自噬水平显著升高。. 该项目共发表SCI论文2篇，中文期刊3篇，宁夏科技进步奖1项。. 意义：阐明Hcy引起As的机制，寻找致病环节，为As这一全球重大疾病的防治工作提供更多研究资料。