Apelin-13 Ameliorates LPS-induced Iron Deposition By Inhibiting IL-6/STAT3 Pathway In N9 Microglia

OBJECTIVE: To observe the effect of apelin-13 on the iron deposition of N9 microglia induced by lipopolysaccharide(LPS)in high-iron environment and its mechanism and provide new ideas and clues for the prevention and treatment of neuroinflammation.METHODS: 1.Effects of lipopolysaccharide(LPS)on iron deposition of N9 microglia in high iron environment.The experiments were randomly divided into four groups: Control group,FAC group,LPS group,FAC+LPS group.FAC group: N9 microglia were treated with Ammonium ferric citrate(FAC,100 μM)for 24 h.LPS group: N9 microglia were incubated with LPS(2 μg/mL)for 6 h.In the FAC+LPS group,cells were treated with FAC(100 μM)for 24 h and then incubated with LPS(2 μg/mL)for 6 h.Cell viability was detected by MTT assay.Intracellular iron deposition was observed by Prussian blue staining.Total iron level was detected by colorimetry.CD86 expression of microglia activation marker was detected by flow cytometry.The expression of iNOS and TNF-α in N9 microglia were detected by Western Blot.2.The effect of Apelin-13 on the iron deposition of N9 microglia induced by LPS in high iron environment.The experiments were randomly divided into four groups: FAC group,FAC+Apelin-13 group,FAC+LPS group,FAC+Apelin-13+LPS group.N9 microglia was pretreated with apelin-13(0.1 μM)for 24 h in apelin-13 treatment group.The cells were then treated with LPS(2 μg/mL)for another 6 h.Cell viability was detected by MTT assay.Intracellular iron deposition was observed by Prussian blue staining.Total iron level was detected by colorimetry.CD86 expression of microglia activation marker was detected by flow cytometry.Expression of iNOS,TNF-α,DMT1,Fpn1,FTH1,STAT3 and p-STAT3 in N9 microglia were detected by Western Blot.3.The role of STAT3 signaling in Apelin-13 inhibits iron deposition of N9 microglia induced by LPS in high iron environment.The experiment was randomly divided into seven groups: FAC group,FAC+Apelin-13 group,FAC+LPS group,FAC+IL-6 group,FAC+Apelin-13+LPS group,FAC+IL-6+LPS group,FAC+IL-6+ Apelin-13+LPS group.The IL-6 treatment group was treated with STAT3 agonist(IL-6,100 ng/mL)for 1 h,N9 microglia was then pretreat with apelin-13(0.1 μM)for 24 h.The cells were treated with LPS(2 μg/mL)for another 6 h.The cell viability was detected by MTT assay.Intracellular iron deposition was observed by Prussian blue staining.Total iron level in the cells was detected by colorimetry.RESULTS:1.Effect of apelin-13,FAC and LPS on cell viability of N9 microglia1.1 Apelin-13(0.01,0.1 μM,24 h)had no significant effect on the viability of N9 microglia.Treatment of cells with 1 μmol/L apelin-13 for 24 h reduced significantly the viability of N9 microglia.1.2 FAC(100 μM,24 h)had no significant effect on the viability of N9 microglia.FAC(200,400 μM,24 h)inhibited significantly the activity of N9 microglia.1.3 LPS(1,2,4 μg/mL,6 h)had no significant effect on the viability of N9 microglia.6 μg/mL LPS treatment of cells for 6 h reduced significantly N9 microglia activity.2.Effect of LPS on iron deposition of N9 microglia in high iron environment2.1 Compared with the Control group,the expression of TNF-α,iNOS(P<0.05)and CD86(P<0.01)in the LPS group were significantly increased.Compared with the FAC group,the expression of iNOS,TNF-α and CD86 in the LPS + FAC group were futher significantly increased(P<0.05).2.2 Compared with the control group,the expression of DMT1 in the FAC group was significantly decreased(P<0.01),and the expression of Fpn1(P<0.05)and FTH1(P<0.001)were significantly increased.Compared with the FAC group,the expression of DMT1 of N9 microglia in the FAC + LPS group was significantly increased(P<0.001),the expression of Fpn1(P<0.001)and FTH1(P<0.01)were significantly decreased.The expression of intracellular iron deposition and total iron content of N9 microglia were significantly increased in in the FAC + LPS group(P<0.01).3.Effect of Apelin-13 on iron deposition of N9 microglia in high iron environment.3.1 Compared with FAC + LPS group,the expression of TNF-α(P<0.05),iNOS(P<0.001)and CD86(P<0.01)of N9 microglia in the FAC + LPS + Apelin-13 group were significantly decreased.3.2 Compared with the FAC + LPS group,the DMT1 expression of N9 microglia was significantly decreased in the FAC + LPS+ Apelin-13 group(P<0.01),the expression of Fpn1(P<0.001)and FTH1(P<0.01)were significantly increased.Iron deposition levels and total iron content of N9 microglia were significantly reduced in the FAC + LPS + Apelin-13 group(P < 0.01).4.STAT3 signaling involved in apelin-13 in improving LPS-induced iron deposition in N9 microglia in a high-iron environment4.1 Compared with FAC group,the ratio of p-STAT3/t-STAT3 and p-STAT3/t-STAT3/β-actinof N9 microglia in the FAC + LPS group were significantly increased(P<0.05).Compared with FAC + LPS group,ratio of p-STAT3/t-STAT3 and p-STAT3/t-STAT3/β-actin in the N9 microglia were significantly decreased in the FAC + LPS + Apelin-13 group(P<0.001).4.2 IL-6(100 ng/mL,1 h)had no significant effect on the viability of N9 microglia.Compared with FAC + LPS + Apelin-13 group,IL-6 increased further total iron content(P<0.01)and aggravated iron deposition of N9 microglia.CONCLUSIONS: Apelin-13 may ameliorate LPS-induced iron deposition by inhibiting STAT3 signaling in N9 microglia in the high iron environment.