Mechanisms Of Lipoxin A₄ Protects Lung Epithelial Cell Against Hyperoxia Injury

Background:Studies have shown that lipoxin A4(LXA4)and activation of LXA4 receptor protected several organs against inflammation and hyperoxia-induced injury.In the previous studies,we have demonstrated that LXA4 induced heme oxygenase-1(HO-1)overexpression in cardiomyocytes and renal tubular epithelial cells exposed to oxidative stress.Our previous study also confirmed that LXA4 inhibits the expression of inflammatory cytokines IL-6 and IL-1? in glomerular mesangial cells of rat.Therefore,we hypothesize that LXA4 may prevent cellular injury in a murine cell model of bronchopulmonary dysplasia(BPD)via HO-1 overexpression and downregulation of proinflammatory cytokines.Objective:To determine whether LXA4 increases expression of HO-1 and inhibits production of interleukin 6(IL-6)and monocyte chemotactic protein l(MCP-1)in LXA4-imparted protection on hyperoxia-induced injury in murine lung epithelial cells(MLE-12),and what are the mechanisms involved in LXA4-inhibited IL-6 and MCP-1 expression.Methods:MLE-12 cells were exposed to air or hyperoxia(85%O2)with or without pretreatment with 0,1,10,50nmol/L LXA4 for 0,1,6,12,24h,Zinc protoporphyrin IX(ZnPP-IX),IL-6,anti-IL-6,MCP-1,anti-MCP-1,inhibitors of p38 mitogen-activated protein kinase(p38 MAPK),Protein Kinase B(AKT)and extracellular signal-regulated kinase 1/2(ERK1/2)signaling pathways.The expression of lipoxygen receptor(ALX)was detected by RT-PCR in MLE-12 cells.The cell survival rates were determined by Trypan blue dye,cell viability was determined by Cell Counting Kit8,apoptosis rates were detected by Flow cytometry,The superoxide dismutase(SOD)level was measured by hydroxylamine method.Expressions of HO-1 mRNA were analyzed by using real time RT-PCR.The levels of IL-6 and MCP-1 in supernatants were measured by ELISA.Expressions of proteins of HO-1,P-p38MAPK,total p38 MAPK,P-AKT,total AKT,P-ERK1/2 and total ERK1/2 were determined by Western blot.The expression of HO-1 and its location were detected by immunofluorescence.Results:MLE-12 cells express the lipoxygen receptor(ALX).The best experimental conditions for LXA4 were lonmol/L pretreatment for 12 h.LXA4 significantly increased the cell survival rates,cell viability,SOD levels and HO-1 expressions,reduced the apoptosis rates,and inhibited the MCP-1 and IL-6 levels induced by hyperoxia in the cells.ZnPP-IX,inhibitor of HO-1,blocked LXA4-imprated protection on cell viability in cells exposed to hyperoxia.Exogenous addition of IL-6 and MCP-1 can significantly reduce cell viability and cause cell damage.Hyperoxia-induced elevated levels of MCP-1 and IL-6 can be inhibited by LXA4.Anti-IL-6 and anti-MCP-1 improved the cell viability in cells exposed to hyperoxia.Inhibition of p38 MAPK and ERK1/2 blocked the expressions of MCP-1 and IL-6 induced by hyperoxia.LXA4 inhibited the activation of the p38 MAPK and ERK1/2 induced by hyperoxia,and increased the activation of AKT signaling pathway which was inhibited by hyperoxia..Conclusion:LXA4 attenuates hyperoxia-induced injury in MLE-12 cells via upregulation of expressions of HO-1.The protection of LXA4 on the hyperoxia-induced cell injury is related to downregulation IL-6 and MCP-1 levels via inhibition of p38 MAPK and ERK1/2 signaling pathways.