Escape Mitochondrial DNA Aggravates A549 Epithelial Cell Injury Induced By Mechanical Stretch Through TLR9-MyD88-NF?B Signaling Pathway

Objective:To investigate the mechanism of escape mitochondrial DN A in A549 epithelial cells induced by mechanical stretch induced by TLR 9-MyD88-NF?B signaling pathway.Methods:A549 cells that were untreated and transfected with lentivir us or drug treated were placed in a mechanical stretch(CS)with a 5%o r 20%tension.PINK1 expression was regulated by lentiviral transfection with PINK1 siRNA and cDNA.Activation and inhibition of TLR9 in A549 cells was regulated by the selective activator ODN 1668 and the inhib itor ODN 2088,while the selective inhibitor ST 2825 blocked the express ion of MyD88.The cultured A549 cells were randomly divided into 10 g roups(n=8):blank control group,5%CS group,20%CS group,PINK1 s iRNA lentivirus transfection+20%CS group,PINK 1 cDNA lenti virus tra nsfection+20%CS group,PINK1 negative control lenti virus transfection+20%CS group,ODN2088+20%CS group,ODN1668+20%CS gr oup,ST2825+20%CS group.The expression of protein and mRNA wa s detected by Western-blot and RT-PCR.The cell damage of CON group,5%CS and 20%CS group was observed by transmission electron micro scope.TNF was detected by ELISA in CON group,5%CS and 20%CS group.-?,IL-6,IL-1? inflammatory factor levels,Stub-RFP-Sens-GFP-LC 3B lentiviral vector was transfected into A549 cells to detect changes in autophagic flow.RESULTS:In this study,IL-1?,IL-6 and TNF-? levels were significantly increased in the 20%CS group compared to the blank control group and the 5%CS group.In 20%CS,overexpression of PINK1 cRNA lentivirus group,Parkin,LC3B-?,p-NF-?Bp65 and COX-? expression were significantly increased,Mfn1 expression was significantly reduced;while in the PINK1 siRNA lentivirus group,The expression of Mfn1 was significantly increased.Upregulation of TLR9 increased the expression levels of MyD88 and p-NF-?Bp65 in 20%CS,but COX-IV was similarly expressed in the control group,the negative control lentivirus group,the MyD88 inhibition group,the TLR9 inhibition group,and the TLR9 activation group..In addition,inhibition of both TLR9 and MyD88 attenuated mtDNA-induced inflammation in A549 cells of 20%CS.Conclusion:Mechanical stretch of 20%cyclic tension activates PINK1/Parkin-mediated mitochondrial autophagy to release mtDNA,and then aggravates A549 cell injury and inflammation through the TLR9-MyD88-NF?B signaling pathway.