Effect And Mechanism Of HMGB1 And AMPK Signaling Pathway On LPS-induced Autophagy In A549 Cells

Objective : Objective to investigate the effect of lipopolysaccharide(LPS)on autophagy of human alveolar epithelial cell line A549,and to explore the regulatory effect and mechanism of high mobility group box-1 protein(HMGB1)and amp activated protein kinase(AMPK)signaling pathway on autophagy in A549 cells,to reveal the role of autophagy of alveolar epithelial cells in the development of lung injury induced LPS in acute respiratory distress syndrome(ARDS)at the cellular level,which provides some theoretical and experimental basis for the course and clinical treatment of ARDS.Methods:(1)The expression of LC3B-Ⅱ/ LC3B-in and Beclin-1 in A549 cells was detected by Western blot after LPS stimulation;(2)the effects of different doses of LPS on the survival rate of A549 cells were detected by CCK-8 experiment.The lowest toxic dose of LPS was used to induce A549 cells to construct ARDS in vitro;(3)apoptosis and necrosis were detected by flow cytometry.The ultrastructure of cells was observed by transmission electron microscopy to determine whether there was any difference between them Autophagy and cell death forms;(4)the AMPK inhibitor(compound C)was used to intervene,and the cells were randomly divided into blank group,LPS group,AMPK inhibitor group and AMPK inhibitor plus LPS group with the intervention of the AMPK inhibitor(compound C).The expression level of autophagy was detected by Western blot and transmission electron microscopy to explore the relationship between AMPK signal pathway and LPS induced autophagy and cell death;(5)the HMGB1 was inhibited by si RNA interference,and the cells were randomly divided into blank group,LPS group,Si-HMGB1 group and Si-HMGB1 +LPS group.The expression of autophagy was detected by Western blot and transmission electron microscopy to study the relationship between HMGB1 and LPS induced autophagy and cell death.Results:(1)LPS can induce autophagy of A549 cells,which has a concentration and time dependence.The minimum toxic dose of LPS treatment for 24 hours is 100 μg/ml,and the death form of cells is autophagic cell death;(2)In the AMPK inhibitor intervention experiment,the expression level of p-ampk and autophagy related protein LC3B-Ⅱ / LC3B-Ⅰin LPS group was significantly higher than that in the blank group(P < 0.05);compared with LPS group,the p-ampk and autophagy related protein LC3B-Ⅱ / LC3B-Ⅰ in the AMPK inhibitor plus LPS group decreased significantly,and the expression was between the blank group and LPS group,and the difference was statistically significant(P < 0.05),The autophagy was decreased compared with LPS group;(3)In the experiment of interference technology to inhibit HMGB1 by si RNA,,compared with the blank group,the expression of HMGB1 and LC3B-Ⅱ / LC3B-Ⅰ in LPS group was significantly increased,and the difference was statistically significant(P < 0.05).Compared with LPS group,the expression of HMGB1 and LC3B-Ⅱ / LC3B-Ⅰ in Si-HMGB1 + LPS group decreased significantly,and the expression was between the blank group and LPS group,and the difference was statistically significant(P < 0.05),The autophagy was decreased compared with LPS group.Conclusion:(1)LPS can induce autophagy of A549 cells;(2)LPS can induce autophagic cell death of A549 cells;(3)LPS can induce autophagy and autophagic cell death of A549 cells through AMPK signaling pathway,and inhibition of AMPK signaling pathway can inhibit autophagy formation of A549 cells;(4)HMGB1 can mediate LPS induced autophagy and autophagic cell death of A549 cells.