Toll样受体4介导的自噬通过调节气道平滑肌细胞生理行为参与哮喘发病的机制研究

Autophagy involves many cell physiology and pathophysiology process, plays an important role in keeping cellular homeostasis and involves in the initiation and development of many diseases. We previously found that in airway smooth muscle tissue of ashmatic mice, autophagic protein LC3II expression enhanced and autophasomes increased, on the other hand, autophagy could regulate vascular smooth muscle cell (VSMCs) phenotype and behaviors, implying that autophagy could regulate ASMCs cell behaviors in asthma. It has been reported that Toll-like receptor 4 (TLR4) enhanced ASMCs proliferation, migration and secretion while inhibited its apoptosis, on the other hand, TLR4 could induce many cell autophagy, implying that TLR4 may regulate ASMCs behavior by inducing its autophagy. NF-кB serves as a TLR4 downstream signal molecule, also participate in regulation of smooth muscles autophagy and cell behavior. So it is hypothesized that through activation NF-кB, TLR4 induces ASMCs autophagy to regulate the cell behavior in airway remodeling in asthma. In this project, isolated mouse primary ASMCs cells will be used for in vitro study, we first evaluate the effect of autophagy on ASMCs behavior, then through activating or inactivating TLR4, study the role of TLR4 in regulating ASMCs autophagy and cell behavior, further detect the function of NF-кB in ASMCs autophagy and cell behaviors, overall we will explore whether TLR4 regulates ASMCs behaviors through NF-кB activated autophagy at molecular and cellular level. For in vivo study, we will establish asthmatic model by ovalbumin in normal and TLR4 knockout mice, detecting the impact of TLR4 defect on ASMCs autophagy, cell behavior and asthma attack. Exploring the effect and mechanism of TLR4 in regulating autophagy and cell behavior of ASMCs at animal level. This project will be focused on studying the effect and mechanism of autophagy in regulating ASMCs behavior, to explore new therapeutic target for asthma.

自噬参与细胞生理病理学进程，与疾病的发生、发展关系密切。我们前期研究发现哮喘小鼠气道平滑肌组织中自噬蛋白LC3II表达升高，自噬体形成增加，提示自噬可能调节哮喘气道平滑肌细胞（ASMCs）生理，但调控机制尚不清楚。Toll样受体4（TLR4）和其下游信号核因子кB（NF-кB）能够诱导多种细胞自噬，而韦江红等人发现它们能够调节ASMCs增殖、凋亡、迁移和分泌等生理行为。据此我们提出假设：TLR4通过激活NF-кB诱导自噬调节ASMCs生理行为，参与哮喘发病。本项目拟利用离体细胞，在分子、细胞水平明确自噬对哮喘ASMCs生理行为的影响，探索TLR4/NF-кB信号对ASMCs自噬的作用和对ASMCs生理行为的调控机制。利用TLR4基因缺陷小鼠，建立哮喘模型，检测TLR4缺失对ASMCs自噬、生理行为和哮喘发病的影响。探索TLR4调节ASMCs自噬参与哮喘发病的机制，为哮喘治疗提供新的靶点。

背景：自噬参与细胞生理病理学进程，与疾病的发生、发展关系密切。TLR4能够调节多种细胞自噬，那么TLR4能否通过调节自噬，参与哮喘发病，尚未有人研究。本课题首次利用TLR4基因敲除小鼠，探索TLR4基因缺失对哮喘气道炎症和气道重塑的作用，并通过体外研究探索TLR4对自噬及ASMCs生理行为的调节作用。内容：本课题利用大鼠原代ASMCs，首先通过抑制或诱导自噬，检测其对ASMCs生理行为的影响；然后通过激活或沉默TLR4，检测ASMCs自噬和生理行为的变化；通过激活NF-κB检测其对ASMCs自噬和生理行为的影响，抑制NF-κB探索TLR4对ASMCs自噬和生理行为的调节是否依赖于NF-κB。最后，利用TLR4基因敲除小鼠建立哮喘模型，观察气道高反应性、气道炎症和气道重塑情况，评价TLR4基因缺失对哮喘发病的影响，为哮喘的临床治疗提供新的靶点。重要结果：（1）抑制自噬能够抑制ASMCs增殖，促进ASMCs凋亡；诱导自噬能够抑制ASMCs凋亡。（2）激活TLR4能够诱导ASMCs自噬，沉默TLR4能够抑制ASMCs自噬。（3）激活TLR4同时抑制自噬，能够显著逆转TLR4激活对ASMCs增殖和迁移的促进作用，并促进凋亡。（4）TLR4基因缺失抑制气道炎症浸润。（5）TLR4基因缺失抑制转录因子GATA3，促进T-bet基因和蛋白水平的表达。（6）TLR4基因缺失抑制哮喘小鼠气道粘液、胶原沉积和气道平滑肌特异性标记蛋白α-SMA的表达。关键数据：沉默TLR4能够通过抑制自噬，进而抑制ASMCs增殖和迁移，促进ASMCs凋亡，从而达到抑制哮喘气道炎症和气道重塑的效果。科学意义：本研究发现，TLR4能够通过自噬调节ASMCs生理行为，从而参与哮喘发病，为哮喘的临床治疗提供新的靶点。

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