Mechanisms Of Mitochondrial DNA Enlarging Fluid Imbalance And Inflammatory Response In Acute Lung Injury By TLR-9

Backgrounds: Acute lung injury/acute respiratory distress syndrome(ALI/ ARDS)is a life-threating disease with high mortality according to its complicated pathogenesis.Until now,the mechanisms of ARDS has not been fully recognized,but it has been widely accepted that excess inflammatory response majorly contributes to this.This uncontrolled inflammation could damge the integrity of alveolar epithelial cells and vascular endothelial cells,leading to the breakdown of lung fluid metabolism balance and the accumulation of lung fluid,which finally causes severe hyoxemia and ARDS.As a canonical damage-associated molecular patterns(DAMPs),mitochondrial DNA(mt DNA)was found to participate in the neutrophilic inflammatory response.Previous study showed that circulating mt DNAwas highly increased in sepsis patients.These data suggested that mt DNAmight be involved in the pathogensis of ALI,but the detailed mechanism is still not clear.Besides,as a classic receptor of DAMPs,the receptor for advanced glycation end product(RAGE)was found to be involved in actue inflammatory response,but its role in ALI has not been fully recongnized.Therefore,in the current study,the role of mt DNAand RAGE in the pathogenesis of ALI/ARDS was investigated.Methods: Firstly,the levels of mt DNAwere detected in bronchoalveolar lavage fluid(BALF)and serum of ARDS patients and healthy subjects,and its relationship with inflammatory cytokines,albumin lekage and oxygenation index was analyzed.Then,In vivo,alveolar fluid clearance rate(AFC),BALF/ serum albumin,and lung acute inflammatory parameters were measured in wide-type and TLR-9 gene knockout mice following the challenge of lipopolysaccharide(LPS),mt DNAor the combination of them,western-blot was performed to investigate the downstream pathways including ion channel protein and intercellular junction proteins.The role of mt DNAreceptor TLR-9 in the pathogenesis of ALI was explored by comparison between gene knock-out and wild-type mice.Finally,RAGE knockout mice were used to establish LPS-induced ALI model.The inflammatory mediators,AFC,protein leakage,ion channel proteins,and intercellular tight junction proteins were detected and compared with those of wild-type mice to explore whether blocking of RAGE could be a potential therapeutic target of ALI.Results: In the first part of the clinical trial,we found that the copy number of mitochondrial DNA in BALF and serum of ARDS patients was significantly higher than those of healthy volunteers,and it was positively correlated with the release of inflammatory mediators in BALF and serum,as well as protein leakage in the lungs.At the same time,the BALF copy number of mt DNAin ARDS patients was negatively related to the oxygenation index.Secondly,we observed that mt DNAcould amplify LPS-induced impairment of AFC,and promote LPS-induced fluid exudation in the lungs,resulting in the increased accumulation of lung fluid.Along with this,the expression of ion channel protein and intercellular tight junction protein in the lungs of mice was further decreased.Furthermore,mt DNAcould also amplify the acute lung inflammation induced by LPS.However,mt DNAalone did not show the similar effects.Besides,in the TLR-9 gene knockout mice,the amplification effects of mt DNAon liquid imbalance and inflammatory response were both attenuated,and mt DNA-induced down-regulation of ion channel protein and intercellular tight junction protein expression was also significantly restored.Finally,we found that after knocking out the RAGE gene,LPS-induced liquid imbalance and acute lung inflammation were both significantly improved,and the expression of ion channel proteins and intercellular tight junction proteins also rallied.Conclusions: A large amount of mt DNAwas released extracellularly following the influence of ALI/ARDS pathogenic factors.These soluble mt DNAs participated in the pathogenetic process of ALI/ARDS by amplifying the pulmonary inflammatory response and lung fluid imbalance,which depended on its receptor,TLR-9,and was regulated by the expression of ion channel proteins and intercellular tight junction proteins.At the same time,knocking out RAGE could significantly diminish the inflammatory response and the accumulation of fluid in the lungs of ALI.These results suggested that mt DNA/TLR-9 and RAGE may serve as potential therapeutic targets for ALI/ARDS in the future.