TLR4信号负性调节分子IRAK-M与PAI-1分子间"串话"在急性肺损伤中的作用及机制研究

Pro-inflammatory/anti-inflammatory mediators imbalance and coagulation/fibrinolysis imbalance are two important pathogenesis links in development of acute lung injury (ALI). The therapeutic effect of anti-inflammation is not satisfied so far. Meanwhile, to establish a new balance between coagulation and fibrinolysis may alleviate the inflammation. Our previous study showed that inflammatory response in LPS-induced ALI was more severe in PAI-1 konckout mice because IRAK-M, a negative regualtor in TLR4 signal pathway, was lower expression. So "cross talk" between IRAK-M and PAI-1 may play an important role on TLR4 signal pathway over-activation and uncontrolled inflammatory response. Recently, several studies showed that macrophage autophagy which can be affected by TLR4 signal pathway maybe is a new target to regulate inflammatory response. According to the mentioned-above data, we will use gene expression regulation techiques to study the role and its molecular mechanism of "cross talk" between IRAK-M and PAI-1.The effect of macrophage autophage on LPS-induced inflammations also was studied in LPS-indeced alveolar macrophages or ALI mouse model.Using autophagy inhibitor and enhancer, we will observe the affect of macrophage autophagy in ALI and "cross talk" between IRAK-M and PAI-1 in PAI-1 knockout mice. Aim of this study is to explore the molecular mechanism and regulation methods of uncontrolled inflammatory response. Results of the study may provide some experimental data in the exploration of new strategies for ALI treatment.

促炎/抗炎失衡和凝血/纤溶失衡是ALI的重要发病环节，单纯抗炎治疗效果不佳，若同时兼顾凝血/纤溶失衡则可能有较好的效果。我们的前期研究显示:纤溶酶原激活物-1(PAI-1）基因敲除小鼠更易发生ALI，并且与TLR4途径负性调控分子白介素1受体相关激酶-M(IRAK-M)下调有关。因此推测IRAK-M与PAI-1分子间"串话"在TLR4途径的过度激活和炎症反应的失控中起重要作用。近期又有研究表明，受TLR4途径影响的巨噬细胞自噬是调控炎症反应的新靶点。在上述基础上，本课题以LPS诱导鼠肺泡巨噬细胞炎症模型为研究对象，采用基因调控技术，研究IRAK-M与PAI-1分子串话的机制以及细胞自噬在其中的作用；在PAI-1基因敲除鼠中，采用自噬抑制剂和增强剂调控自噬，进一步研究巨噬细胞自噬参与IRAK-M和PAI-1分子"串话"的机制及对ALI的作用。为阐明ALI炎症反应失控机制和调控方法提供依据。

急性肺损伤/急性呼吸窘迫综合征（ALI/ARDS）是由各种直接或间接损伤诱发的严重肺部炎症反应，以严重低氧血症为主要表现，死亡率高达30-50%。其发病原因多种多样，促炎/抗炎失衡和凝血/纤溶失衡是ALI的重要发病环节，单纯抗炎治疗效果不佳，若同时兼顾凝血/纤溶失衡则可能有较好的效果。本课题以LPS诱导鼠肺泡巨噬细胞炎症模型为研究对象，采用基因调控技术，研究（1）、PAI-1 在 LPS 诱导肺泡巨噬细胞炎症反应过程中的作用以及相关信号通路。（2）、PAI-1 在 LPS 诱导肺泡巨噬细胞炎症反应过程中对细胞自噬的影响以及相关信号通路。（3）、通过干扰 Beclin1、mTOR的表达调节自噬，观察抑制自噬以及增强自噬对 LPS诱导肺泡巨噬细胞炎症反应以及 PAI-1 表达的影响；调节自噬对 TLR4 相关信号通路的影响。研究结果显示，（1）LPS 刺激肺泡巨噬细胞产生炎症反应，激活了 TLR4/MyD88/NF-κB 信号转导通路，细胞自噬水平增高，P38磷酸化增加。（2）干扰 PAI-1表达，TLR4/MyD88/NF-κB信号通路被抑制，LPS诱导的肺泡巨噬细胞炎症反应减轻，LPS诱导的自噬被抑制，P38磷酸化水平降低。过表达PAI-1，TLR4/MyD88/NF-κB 信号通路活化，LPS 诱导的肺泡巨噬细胞炎症反应加重，增强LPS诱导的细胞自噬以及P38磷酸化。（3）干扰Beclin1表达可以抑制自噬，TLR4/MyD88/NF-κB信号通路被抑制，P38磷酸化水平降低，LPS 诱导的炎症反应减轻，抑制了 PAI-1 的表达。干扰 mTOR表达，细胞自噬水平增加，TLR4/MyD88/NF-κB 信号通路被抑制，P38 磷酸化水平降低，LPS诱导的炎症反应减轻，PAI-1的表达被抑制。（4）增强自噬、抑制自噬均可抑制 PAI-1 表达，减轻 LPS 诱导的肺泡巨噬细胞炎症反应。我们的研究能为 ALI/ARDS的发病机制提供新的理论依据，但也可以看到自噬对炎症调控的复杂性，利用自噬来治疗炎症性疾病仍需要大量的研究。