HO-1对内毒素急性肺损伤时线粒体运动平衡适应的调控作用及机制研究

Ectogenic treatment such as mechanical ventilation and anti-infection for indotoxin-induced acute lung injury (ALI)isn't satisfactory. Proposers have endeavoured to investigate endogenous defense mechanism to prevent endotoxin-induced ALI and achieved some degree of success which demonstrated that HO-1 is an important medium of endogenous defense system and possesses the antioxygenation and defense function. Mobilizing the endogenous defense system to regulate the change of cell ultrastructure and resist the death of cells is an intervention based on the physiopathologic mechanism of endotoxin-induced ALI. Mitochondrial dysfunction is an important element during endotoxemia, and its dynamic equilibrium of fusion and fission defines the function of mitochondrion, but the mechanism of which is still undefined. The effect of change of mitochondrial dynamics during endotoxin-induced ALI has not been reported. Combined with endogenous defense mechanism of organ, proposers put forward a hypothesis which is "Adaption of Mitochondrial Dynamic Equilibrium" in mitochondrial dynamic. From the aspects of function and form,we will adopt different methods to investigate if HO-1 regulates the mitochondrial dynamic equilibrium by modulating the mitochondrial dynamics related protein to resist endotoxin-induced ALI, using the rat model of LPS-induced endotoxin ALI and cell attack model with LPS. The results will provide theoretical basis for preventation of endotoxin-induced ALI.

内毒素急性肺损伤(ALI)的机械通气、抗炎等外源性治疗效果多不理想。我们致力于探讨自身内源性保护机制以防治内毒素ALI并取得一定成果：证实HO-1在内毒素ALI中发挥重要内源性抗氧化防御功能。调动机体内源性保护系统以调节细胞超微环境变化、抵抗细胞死亡发生，是基于内毒素血症发病机制的干预措施。线粒体功能障碍是内毒素血症发病机制的重要环节，其融合/分裂运动动态平衡决定线粒体有效功能，但调控机制尚未阐明；线粒体动力学改变在内毒素ALI中的作用尚未见报道。我们从线粒体动力学角度入手，结合机体内源性保护机制，提出"线粒体运动平衡适应"假说，拟通过LPS诱导内毒素ALI大鼠模型和LPS细胞攻击模型，从功能、形态等方面，采用分子生物学方法，探索HO-1是否通过调节线粒体运动相关蛋白来调控线粒体融合/分裂运动，维持其动力学平衡以抵抗内毒素ALI，力求为防治内毒素ALI提供理论依据。

内毒素急性肺损伤(ALI)是临床常见急危重症，其防治一直是临床难题。本项目通过成功建立内毒素ALI大鼠模型及LPS攻击肺泡上皮细胞、巨噬细胞、肺动脉内皮细胞模型，观察了内毒素血症时细胞内线粒体运动状态与细胞凋亡的关系及线粒体血红素氧合酶1（HO-1）表达与线粒体运动相关蛋白变化的相关性及可能机制。研究发现：在内毒素致大鼠急性肺损伤时，HO-1受到诱导表达上调后，线粒体融合相关蛋白Mfn1、Mfn2和OPA1的mRNAs及蛋白的表达减少，而线粒体分裂相关蛋白Drp1及Fis1的mRNAs及蛋白的表达上调；HO-1/CO可上调LPS诱导肺泡上皮细胞线粒体融合相关蛋白Mfn1、Mfn2和OPA1表达，促进线粒体融合，减轻肺泡上皮细胞损伤；在内毒素攻击肺泡巨噬细胞、肺泡上皮细胞及肺动脉内皮细胞中，CORM-2上调HO-1表达的同时，能够改善线粒体的动力学相关蛋白的表达进而发挥内源性的保护作用；进一步对机制的研究发现HO-1/CO通过激活多条信号通路（p38MAPK、PI3K/Akt、PKC-α）减轻内毒素诱发细胞氧化应激的损伤，同时增加线粒体融合蛋白Mfn1、Mfn2、OPA1的表达，降低线粒体分裂蛋白Drp1、Fis1的表达水平；此外ROS清除剂预处理能够降低内毒素攻击细胞模型中ROS的生成，使线粒体融合蛋白Mfn1、Mfn2及OPA1表达水平上调，而分裂蛋白Drp1及Fis1表达水平下调。据此可以得出结论，HO-1/CO可能通过p38MAPK、PI3K/Akt、PKC-α多条通路影响线粒体形态功能调控机制，进而影响线粒体形态学改变，从而在超微结构水平影响细胞的代谢状态，最终发挥其内源性保护作用，减轻内毒素急性肺损伤，为临床内毒素ALI的防治提供了重要的理论依据。