Protective effect of lipoxin A4 on hyperoxia injury of mouse lung epithelial cells / 中华实用儿科临床杂志

ABSTRACT

Objective To investigate the protective effects of lipoxin A4 (LXA4) on the lung epithelial cells (MLE-12) in mice with hyperoxia injury.Methods MLE-12 cells were cultured in vitro and divided into air group,air + LXA4 group,hyperoxia group and hyperoxia + LXA4 group.The receptor of LXA4 (ALX) was verified by using reverse transcription-polymerase chain reaction (RT-PCR).MLE-12 cells were exposed to hyperoxia (＞ 850 mL/L oxygen concentration) for 12 h followed by pretreatment of 1 nmol/L,10 nmol/L and 50 nmol/L LXA4 for 1 h,6 h,12 h and 24 h.Quantitative real-time PCR (qRT-PCR) was applied to analyze the heme oxygenase 1 (HO-1) expression to determine the optimal concentration and the optimal pretreatment time of LXA4.The cell morphology was observed by using inverted microscope.The survival rates and cell viability were determined by using Trypan Blue stain and cell counting kit-8 (CCK-8).The superoxide dismutase (SOD) level was determined by using hydroxylamine method.The expressions of mRNA and protein of HO-1 were measured by using qRT-PCR,western blot and immunofluorescence assay,respectively.The interleukin-6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) were determined by using enzyme-linked immunosorbent assay.Results ALX was expressed in MLE-12 cells.The optimal intervention concentration and time of LXA4 was 10 nmol/L for 12 h.Compared with air group [(84 ± 5) ％,1.22 ± 0.27,(5.33 ± 1.16) kU/L],the cell survival rate,viability and SOD level of hyperoxia group [(66 ± 8) ％,0.67 ± 0.21,(2.38 ± 0.65) kU/L] decreased,and the differences were significant (t =3.98,2.55,4.86;P =0.01,0.03,0.00);compared with the hyperoxia group,the cell survival rate,viability and SOD level of hyperoxia + LXA4 group [(88 ± 5) ％,1.43 ± 0.05,(6.50 ± 0.19) kU/L] significantly increased,and the differences were significant (t =4.83,3.52,6.78;P =0.01,0.02,0.00).The HO-1 mRNA and protein expression of hyperoxia group (0.57 ± 0.03,1.31 ± 0.11) increased as compared to air group (0.13 ± 0.03,0.24 ± 0.10),and the differences were significant (t =8.00,10.10;all P =0.00);the HO-1 expression of hyperoxia + LXA4 group (0.78 ± 0.08,1.82 ± 0.09) significantly increased as compared to hyperoxia group,and the differences were significant (t =3.94,8.82,all P=0.00).The levels of MCP-1 and IL-6 of hypemxia group [(1 025.18 ±35.51) rig/L,(1 136.65 ±160.01) ng/L] significantly increased as compared to air group [(467.63 ± 13.69) ng/L,(470.03 ± 118.22) ng/L],and the differences were significant (t =16.51,7.48;all P =0.00);the MCP-1 and IL-6 of hyperoxia + LXA4 group [(640.25 ± 61.03) ng/L,(655.48 ± 88.57) ng/L] significantly decreased as compared to hyperoxia group,and thedifferences were significant (t =11.40,5.40,all P =0.00).Conclusions LXA4 can attenuate hyperoxia-induced injury in MLE-12 cells.The protective role of LXA4 in the hyperoxia-induced cell injury is related to the up-regulation of HO-1 expression and down-regulation of IL-6 and MCP-1 levels.