抑制MLCK负调控Bcl-2蛋白对急性肺损伤活化中性粒细胞的作用及机制研究

Acute lung injury(ALI) is characterized in appearance of SIRS in lung. Previous study indicated adhesion,migration and activation of polymorphonuclear leukocyte(PMN) is the provocation of imflammation waterfall,our study also demonstrate that Edaravone and Raloxifene can protect and treat ALI by release migration and confinement of PMN. Nature reports adhesion,migration and activation of PMN are associated with non-muscle myosin light-chain kinase(nmMLCK,MLCK isoforms)mediation. Based on previous excellent research outcome,we further use inhibitor of MLCK/nmMLCK to accomplish targets including blocking kinase activition,suppression of adhesion and migration of PMN ,and representing the mechanism of regulation of apoptosis delay by Bcl-2 signal pathway or not. We make use of several techniques, including MicroPET to quantitatively analyze activation of PMN, cell microtubule sucking from engineering mechanics technique to account adhesive power between cells and Bcl-2 transfection to PMN technique to analyze inversion of apoptosis delay as well, in order to fully clarity ultraearly interruption and regulation mechanism of adhesion,migration,activation and apoptosis delay of PMN by inhibiting MLCK/nmMLCK,further manifest irrefutable therapeutical effect. This study may be likely to ultraearly,successfully interrupt cascade reaction of imflammation waterfall and clinical symptoms＇rapid aggravation in ALI .

急性肺损伤(ALI)系SIRS反应在肺部的表现，研究发现中性粒细胞（PMN）粘附和迁徙是ALI炎症瀑布链的超早期激发点。我们此前亦发现Edaravone和Raloxifene可减轻PMN肺内迁徙扣押来实现对ALI的保护治疗；Nature报道：PMN粘附迁徙与非肌性肌球蛋白轻链激酶(nmMLCK，MLCK亚型)介导密切关联。我们在原基础进一步采用MLCK/nmMLCK抑制剂靶向阻断其活性, 抑制PMN粘附迁徙活化，阐明和论证能否同时通过Bcl-2负调控PMN的凋亡延迟来发挥效应的猜想；运用MicroPET技术定量分析PMN过度活化、工程学细胞微管吸吮技术分析靶向细胞间粘附力及Bcl-2转染PMN技术分析凋亡延迟的逆转效应等，充分阐述抑制MLCK/nmMLCK超早期对PMN粘附迁徙活化压制和凋亡延迟的负调控新机制，本研究极可能对ALI瀑布样级联反应激化和临床症状的迅速恶化超早期干预和成功阻断

急性肺损伤（ALI）是一种由中性粒细胞为主要类型炎症细胞介导的炎症性疾病，临床表现为急性、进行性低氧型呼吸衰竭综合征。目前用于治疗炎症性疾病的临床药物主要是通过稳定炎症细胞细胞膜，阻止炎症细胞脱颗粒来实现。本研究首次通过抑制MLCK诱导中性粒细胞凋亡，清除炎症，从而缓解小鼠急性肺损伤模型的炎症。我们首先通过分离大鼠原代中性粒细胞，使用ML-7体外诱导原代中性粒细胞，流式细胞术检测细胞凋亡结果表明，ML-7可以时间依赖性诱导细胞凋亡；Wright-Giemsa染色以及免疫荧光染色也进一步表明，ML-7诱导下，中性粒细胞细胞核皱缩成团，发生凋亡；检测相应时间点磷酸化MLC、Bcl-2、Bax以及磷酸化p38蛋白表达水平，结果表明ML-7可以在下调MLC磷酸化水平的同时，降低Bcl-2/Bax比例，以及磷酸化p38表达上升。在体动物实验表明，对LPS诱导的小鼠急性肺损伤模型给予ML-7干预治疗，可以显著诱导BALF液中中性粒细胞发生凋亡，同时能够降低BALF液中炎症细胞浸润比例以及相应炎症指标。相应的研究结果已撰写论文，准备投稿中。我们首次同时通过体内及体外实验阐明了，抑制MLCK可以诱导中性粒细胞发生凋亡进而缓解炎症。对急性肺损伤的临床治疗以及早期干预，起到一定的提示与指导作用。