Role And Mechanism Of Released Mitochondrial DNA Mediated Toll-like Recepeter 9-myeloid Differentiation Factor 88 Signaling Pathway Activation In Rats With Ventilator-Induced Injury

Objective: To investigate the role and mechanism of released mitochondrial DNA(mtDNA)in ventilator-induced lung injury(VILI)in rats via Toll like receptor 9(TLR9)-myeloid differentiation factor 88(MyD88)signaling pathway.Methods: 1.Forty adult male Sprague-Dawley rats were randomly divided into four groups(each n=10): spontaneous breathing group,normal tidal volume(NTV)group(VT=8 mL/kg),high tidal volume(HTV)group(VT=40 mL/kg)and inhibitor of TLR9+ HTV group(intraperitoneally pretreated with ODN2088 500μg of each rat,VT=40 mL/kg).Rats in the NTV group,HTV group and ODN+HTV group were allowed to ventilated mechanically with a positive end-expiratory pressure(PEEP)of 0 and the fraction of inspired oxygen(FiO2)at 0.5.After 4 hours of ventilation,the blood in the rat’s hearts was collected and cytokine levels of interleukin(IL-6,IL-1β)and tumor necrosis factor-alpha(TNF-α)in plasma were measured using a commercially available rat enzyme-linked immune sorbent assay(ELISA)kit.Then the rats were sacrificed.The bronchoalveolar lavage fluid(BALF)was collected for a determination of total protein using the bicinchoninic acid(BCA)assay.Some lung tissues were used to detect the wet/dry(W/D)ratio and some were stained with hematoxylin and eosin for histological analysis under a microscope.The protein expression levels of TLR9,MyD88 and nuclear factor(NF)-κB p65 in lung tissues were examined using immunohistochemistry and western blot.2.Forty adult male Sprague-Dawley rats were randomly divided into four groups(each n=10): spontaneous breathing group,normal tidal volume(NTV)group(VT=8 mL/kg),high tidal volume(HTV)group(VT=40 mL/kg)and inhibitor of mtDNA+ HTV group(intraperitoneally pretreated with CQ 30μg of each rat,VT=40 mL/kg).The methods of building animal VILI model and assessing the degree of acute lung injury in rats were same as the steps mentioned above.The mRNA and protein expression levels of mtDNA-encoded cytochrome c oxidase subunit 4(COX-4),TLR9,MyD88 and NF-κB p65 in lung tissues were examined using RT-PCR and western blot.Results: The histopathology of lung tissues indicated that lungs from animals ventilated with HTV developed marked lung inflammation changes,whereas no major histological differences were observed between animals ventilated with NTV or spontaneously breathing.Compared with spontaneous breathing group and NTV group,the ratio of W/D,the concentrations of total proteins(IL-6、IL-1β和 TNF-α)in BALF and cytokine levels in plasma were all significantly increased in the HTV group.The mRNA and protein levels of COX-4,TLR9,MyD88 and NF-κB p65 in the lung tissues were significantly higher in the HTV group.The use of inhibitor of TLR9 could down-regulated expression levels of TLR9,MyD88 and NF-κB p65.While the use of inhibitor of mtDNA could down-regulated expression levels of COX-4,TLR9,MyD88 and NF-κB p65.What’s more,MV rats treated with CQ or ODN2088 had significantly reduced lung inflammation compared to the HTV group.Conclusions: Released mtDNA contributes significantly to VILI by activating the TLR9/MyD88 signaling pathway,resulting in subsequent secretion of NF-κB p65 and the proinflammatory cytokines.