Effect Of HIF-1?/BNIP3 Pathway On Autophagy And Apoptosis Of Neutrophils-like Cells

Objective:To investigate the HIF-1?/BNIP3 pathway on the autophagy and apoptosis of neutrophils-like cells from the cellular level,in order to provide a theoretical basis for the study of the pathogenesis of neutrophilic asthma.Method:1.Neutrophil-like cells induced differentiation: HL-60 cells were cultured and the logarithmic cells were added into 1.3%DMSO for 4days.The morphology of the cells was detected by Diff staining,and the expression level of CD11 b was detected by flow cytometry.The detection results determine whether the induction is successful or not;2.The model of neutrophils-like cells in IL-8 culture was constructed:HL-60 cells were induced to differentiate into neutrophils-like cells and then cultured with 0 ng / ml,100 ng / ml,200 ng / ml,400 ng /ml,800ng/ml IL-8 for 24 h.Flow cytometry was used to detect the apoptosis rate of neutrophils-like cells.The IL-8 concentration of the lowest apoptosis group was selected and the neutrophils-like cells were cultured with the selected concentration for 0 h,6 h,12 h,24 h,48 h to detect the apoptosis rate of neutrophils-like cells in each group.The group with the lowest apoptosis rate was selected for the next experiment.3.To verify the effect of HIF-1 ? regulating BNIP3 on neutrophils-like cells autophagy and apoptosis: divided into 6 groups: 1.control group2.IL-8 group,3.IL-8+HIF-1? group,4.IL-8+HIF-1?-vector group,5.IL-8+HIF-1?si RNA group,6.IL-8+HIF-1?si RNA-vector group.Western-blot and RT-q PCR method were respectively used to detect the expression of HIF-1 ?,BNIP3 and LC3? in each group,flow cytometry was used to detect the apoptosis rate of neutrophils-like cells,and immunofluorescence was used to detect the neutrophils-like cells expression of intracellular LC3 ?.Results:1.The HL-60 cells were induced by 1.3% DMSO for 4 days,and the nuclei were identified as renal and lobulated by Diff staining.The expression of CD11 b was detected by flow cytometry.The fluorescence intensity in the induction group was significantly higher than that in the control group(p < 0 01),indicating that neutrophils-like cells induction was successful in the induction group.2.The apoptosis rate of neutrophils-like cells in each group was detected by flow cytometry.It was found that the apoptosis rate of cultured with 400ng/ml for 24 h group was the lowest(p < 0.05).3.By Western-blot,RT-q PCR,flow cytometry,immunofluorescence assay revealed that:(1)The expression of HIF-1 ?,BNIP3 and LC3? in IL-8 group was significantly higher than that in control group from the western-blot and RT-q PCR results(P < 0 05),the apoptosis rate of neutrophils-like cells was decreased by flow cytometry(P < 0 05),and the expression of LC3?was increased revealed by immunofluorescence.(2)Compared with the control group,IL-8 group and IL-8 + HIF-1?-vector group,the expression of HIF-1?,BNIP3 and LC3? were increased in IL-8+ HIF-1? group(P < 0 05),The results of flow cytometry showed that the apoptosis rate of neutrophils-like cells was decreased(P < 0.05),and the expression of LC3? was increased revealed by immunofluorescence.(3)Compared with the control group,IL-8 group and IL-8 + HIF-1?si RNA-vector group,the expression of HIF-1 ?,BNIP3 and LC3?was lowest in IL-8 + HIF-1?si RNA group(P < 0 05),The results of flow cytometry showed that the apoptosis rate of neutrophils-like cells increased(P < 0.05),and the expression of LC3? decreased revealed by immunofluorescence.Conclusion:1.IL-8 increased neutrophils-like cells autophagy and decreased apoptosis.2.The HIF-1?/BNIP3 pathway can induce the increase of autophagy and the inhibition of apoptosis in neutrophils-like cells.3.Il-8 may induce increased autophagy and inhibit apoptosis of neutrophils-like cells by the pathway of HIF-1? / BNIP3.