TLR4调控Rab26介导溶酶体成熟在肺微血管内皮屏障损伤中的机制研究

Impaired the barrier function of pulmonary microvascular endothelial cells (PMECs) is key pathogenesis in acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS) occurrence. Our previous study showed that LPS suppressed the expression of Rab26 protein. Over-expression Rab26 by Rab26 adenovirus suppressed LPS induced activation of TLR4 and it’s downstream signal pathway, partly reversed the damage of PMECs barrier function. Further study demonstrated that LPS induce DNA methylation of Rab26 in promoter region. Furthermore, Rab26 could anchor at lysosomes in PMECs. Here is our hypothesis: Excessive activation of TLR4 induce Rab26 promoter DNA methylation, suppress the expression of Rab26 mRNA and protein and Rab26-mediated lysosomes maturation, resulted in blockage TLR4 degrading in lysosomes. Thus, abnormal accumulation of TLR4 at cytoplasm and cell surface could active the down stream signal pathway, such as MyD88 dependent signal pathway, as a consequence permeability of PMECs have been damaged. By using these techniques including TLR4-/- Rab26-/- gene kick-out mice, co-IP (Co-Immunoprecipitation), to investigate the novel mechanism of Rab26-mediated lysosomes maturation to depredate TLR4 at lysosome and TLR4 induced Rab26 promoter DNA methylation; The mechanism would be clarified about the TLR4 regulated Rab26 mediated lysosome maturation to impair PMECs barrier function. This study will provide the new targets and strategies for treatment of clinical diseases ALI/ARDS.

肺微血管内皮细胞(PMECs)屏障功能损害是ALI/ARDS发生的关键病理改变。我们发现LPS抑制Rab26的表达，过表达Rab26可抑制LPS诱导的TLR4及其介导的信号通路激活，逆转PMECs屏障功能损害。进一步研究发现LPS诱导Rab26启动子甲基化，同时Rab26可锚定在溶酶体膜上。故推测，激活TLR4引起Rab26启动子甲基化，抑制Rab26表达及其介导的溶酶体成熟，阻碍TLR4溶酶体内降解，导致TLR4在胞内和胞膜异常聚集并持续性激活下游信号通路，最终使得PMECs屏障功能的损害。本项目应用TLR4-/- Rab26-/-双敲除鼠、CO-IP等方法研究Rab26调控溶酶体成熟对TLR4的降解以及TLR4调控Rab26启动子甲基化的关键机制。阐明TLR4调控Rab26介导的溶酶体成熟在PMECs屏障损伤中的关键作用和机制，为了防治ALI/ARDS提供新靶点和新策略。

既往的表明Rab26在急性肺损伤中发挥着关键作用，但机制不明确。我们研究表明TLR4可通过上调DNMT3B，进而促进Rab26启动子的甲基化，进而抑制Rab26的表达。相反地，Rab26通过调控自噬体、溶酶体的生成，进而影响内化后TLR4在溶酶体的降解，发挥负性调控的作用。我们还发现Rab26通过控制肺部炎症反应、中性粒细胞的募集和组织浸润，进而在急性肺损伤中起保护作用。Rab26通过调控CD36从细胞将转运至细胞膜和NOX2转移到自噬相关吞噬体上，进而发挥调控巨噬细胞吞噬凋亡细胞的作用。急性肺损伤时损伤组织损伤后释放eCIRP后，通过抑制Rab26介导的EPOR转运至细胞膜，进而促进巨噬细胞的炎症反应。上述研究进一步阐释了Rab26在急性肺损伤的关键作用和机制，为ALI/ARDS 防治提供关键靶位和新策略。