TFAM调节线粒体DNA逃逸在脓毒症急性肾损伤中的作用及机制研究

Mitochondria injury is the main pathogenesis in sepsis-associated acute kidney injury (SAKI). Studies have demonstrated that mitochondria DNA (mtDNA) can escape from mitochondria to cytoplasm as damage associated molecular patterns (DAMPs), which resulting organ damage. Transcription factor A mitochondrial (TFAM) plays a role in regulating the function of mtDNA, however, the certain mechanism and the role in the development of SAKI are unclear. In our previously studies, we found that: 1. Mitochondrial permeability transition pore was opened in RTECs following sepsis; 2. TFAM/mtDNA were redistributed inside and outside between mitochondria of renal tubular epithelial cell (RTECs); 3. RhTFAM could up-regulate the expression of TFAM in cytoplasm and improve mtDNA injury and mitochondria injury. Thus, we hypothesis that TFAM regulate mtDNA escape to mediating mitochondria injury relying on mitochondrial permeability increase is the pathogenesis of SAKI. According to these results, with molecular biological techniques, our study focus on the mechanism of mtDNA escape regulated by TFAM and the role of rhTFAM confronting the development of SAKI by using knock-out mice, siRNA and other methods. This study has very important significance with regard to further elucidation of mechanisms of SAKI and highlighting the need to develop new treatment targets.

线粒体损伤是脓毒症急性肾损伤（SAKI）重要的发病机制。研究证实，脓毒症时线粒体DNA（mtDNA）发生逃逸并通过损伤相关分子模式（DAMPs）参与器官功能损害，而mtDNA功能受线粒体转录因子A(TFAM)调控，但TFAM 调控mtDNA逃逸的机制及该机制在SAKI发生发展中的作用尚待明确。本项目组前期发现：1. SAKI线粒体通透性转化孔（mPTP）开放；2. SAKI发生TFAM/mtDNA重分布；3. 重组TFAM（rhTFAM）上调胞内TFAM表达并修复mtDNA和线粒体损伤。我们假设TFAM调节mtDNA逃逸介导线粒体损伤依赖线粒体通透性增加是SAKI的发病机制。本项目拟根据前期研究为切入点，运用基因敲除、siRNA等方法，从体内外探讨TFAM调节mtDNA逃逸在SAKI中的机制及rhTFAM对抗SAKI的作用。这对阐明SAKI发病机制及推动新的治疗靶点具有重要意义。

线粒体损伤是脓毒症急性肾损伤（SAKI）重要的发病机制。研究证实，脓毒症时线粒体DNA（mtDNA）发生逃逸并通过损伤相关分子模式（DAMPs）参与器官功能损害，而mtDNA功能受线粒体转录因子A(TFAM)调控，但TFAM 调控mtDNA逃逸的机制及该机制在SAKI发生发展中的作用尚待明确。本项目组前期发现：1. SAKI线粒体通透性转化孔（mPTP）开放；2. SAKI发生TFAM/mtDNA重分布；3. 重组TFAM（rhTFAM）上调胞内TFAM表达并修复mtDNA和线粒体损伤。我们假设TFAM调节mtDNA逃逸介导线粒体损伤依赖线粒体通透性增加是SAKI的发病机制。本项目拟根据前期研究为切入点，运用基因敲除、siRNA等方法，从体内外探讨TFAM调节mtDNA逃逸在SAKI中的机制及rhTFAM对抗SAKI的作用。结果发现：1）LPS刺激HK-2细胞后，细胞内ROS呈明显上升，16h最为明显；mtDNA的mRNA的表达和拷贝数明显下降；TFAM表达增加，TFAM从细胞核向胞浆转移；2）上调TFAM表达，保护了TFAM/mtDNA功能，下游TLR-4/P38MAPK通路受到抑制，减少了SAKI的进展，反正沉默TFAM表达，TFAM/mtDNA功能受到抑制，下游TLR-4/P38MAPK通路上调，启动了炎症反应，发挥了DAMPS作用，加重了SAKI；3）体内制作CLP模型24h处死小鼠，HE、masson染色检测肾脏病理变化，可发现炎症细胞浸润，IHC染色证实CLP组TNF-a表达增强；TFAM表达上升，ROS升高后下降，TLR-4 、MAPK 和P38MAPK的mRNA含量，同样呈现先上升后下降趋势； 4）LPS诱导HK-2细胞发生细胞焦亡；5）TFAM诱导HK-2细胞焦亡，并促进受损细胞分泌 IL-1β和 IL-18; 3) LPS通过上调C/EBPβ表达，诱导TFAM表达；6）C/EBPβ通过上调TFAM表达，促进细胞焦亡；7）：TFAM体内诱导RTECTs发生细胞焦亡。这些结果与目前许多文献结果一致，这些表明TFAM在SAKI中扮演“双刃剑”作用，具有保护和损伤的双重机制，其具体机制有待进一步研究。研究成果对阐明SAKI发病机制及推动新的治疗靶点具有重要意义。