Study On The Amplification Effect And Mechanism Of Mitochondrial DNA On Inflammation Of Alveolar Macrophages Induced By Lipopolysaccharide

Objective:To clarify the amplification effect of mitochondrial DNA on the inflammatory response of alveolar macrophages(NR8383)induced by lipopolysaccharide(LPS),transfection of TLR9 siRNA can inhibit the amplification effect of mitochondrial DNA,and to explore the mechanism of mitochondrial DNA(mtDNA)on the inflammatory response of alveolar macrophages induced by LPS.Method:1)The mtDNA was extracted by mitochondrial DNA kit and the purity of mtDNA was detected by agarose gel electrophoresis.2)Alveolar macrophages(NR8383)induced by LPS were intervened by mtDNA with different concentration gradients.The experimental groups were divided into Control group,LPS group,0.25μg/ml mtDNA+LPS group,0.5μg/ml mtDNA+LPS group and 1μg/ml mtDNA+LPS group.The final concentration of LPS was 0.5 μg/ml.The supernatant was collected 24 hours later.Enzyme linked immunosorbent assay(ELISA)was used to detect the content of inflammatory factor TNF-α.To clarify that mtDNA can amplify inflammation of alveolar macrophages induced by LPS and the optimum concentration of mtDNA.3)50nM TLR9 siRNA was transfected into alveolar macrophages in vitro for 48 h.The expression of TLR9 mRNA in alveolar macrophages was detected by RT-qPCR to determine the silencing effect of TLR9.4)The experiment was divided into seven groups: Normal group,Normal+ siRNA-NC group,LPS group,mtDNA group,LPS+mtDNA group,siRNA-NC+ LPS+mtDNA group,TLR9siRNA+ LPS+ mtDNA group.After 48 hours of transfection,NR8383 was stimulated by mtDNA and LPS.Cells and supernatants were collected 24 hours later.The levels of inflammatory factors TNF-α,IL-1β and IL-6 were detected by ELISA.The NF-ΚB nuclear translocation was detected by immunofluorescence.The expression of MyD88 and NF-ΚBp65 were detected by Western Blot.Result:1)Agarose gel electrophoresis showed a single band of mtDNA with a molecular weight of approximately 16.5 Kb.2)MtDNA can amplify the inflammatory effect of LPS,and the amplification effect of mtDNA on LPS is concentration-dependent,with the best inflammatory effect at 0.5μg/ml.3)The down-regulation of TLR9 expression indicated that the transfection was successful.4)Compared with LPS group,the contents of TNF-α,IL-1β and IL-6 in LPS+mtDNA group increased significantly,the nuclear translocation of NF-kB occurred,the expression of MyD88 and NF-kB p65 in protein level increased,which indicated that mtDNA could amplify the inflammatory effect induced by LPS.Compared with LPS + mtDNA group,TLR9 siRNA + LPS + mtDNA group could alleviate the above-mentioned inflammatory reaction and the expression of MyD88,and NF-kBp65 decreased,suggesting that down-regulation of TLR9 could inhibit the inflammatory effect of mtDNA on LPS.Conclusion:1)mtDNA can amplify LPS-induced alveolar macrophages inflammation;2)Down-regulation of TLR9 expression can inhibit the inflammatory response of mtDNA to LPS-induced alveolar macrophages;3)mtDNA may amplify the inflammatory response of LPS to alveolar macrophages through the TLR9/MyD88/ NF-kB signaling pathway.