Hydrogen Peroxide Promotes The Expression Of Angiopoietin Like Protein 4 In Macrophage Through MAPKs Pathways

Objective:This study aimed to explore the effect of hydrogen peroxide?H₂O₂?on Angiopoietin-like protein 4?Angptl4?in primary macrophage cells of mouse and explain the potential mechanism of MAPKs pathways in this process.Methods:The macrophage cell line RAW264.7 was purchased and then cultured under appropriate culture condition.Subsequently,the expression level of Angptl4 was measured under different stimulus concentration?0.25mmol/L,o.5mmol/L?for 24h and using western blot,enzyme-linked immune sorbent assay and immunofluorescence.Then macrophage cells were divided into twelve groups?normal,0.25mM H202,0.5mM H₂O₂,0.25mM H₂O₂+U0126,0.5mM H₂O₂+U0126,U0126,0.25mM H₂O₂+SB203580,0.5mM H₂O₂+SB203580,SB203580,0.25mM H₂O₂+SP600125,0.5mM H₂O₂+SP600125,SP600125?to investigate the possible mechanism of MAPKs pathways in promoting expression of Angpt14 by H₂O₂.U0126,SB203580 and SP600125 are specific inhibitor of ERK1/2,p38 MAPK and JNK respectively.Then the expression level of Angptl4 and p-ERK1/2,ERK1/2,p-p38 MAPK,p38 MAPK,p-JNK and JNK was measured by western blot.Statistical analyses were performed using the Student's t test or analysis of variance when necessary.Results:1.H₂O₂ induces Angptl4 release in RAW264.7 macrophage cells:To study the effect of H₂O₂ on the protein expression of Angptl4,different concentrations of H₂O₂ was added to the macrophage cells.To identify optimum concentration of H₂O₂,0.125mM,0.25mM,0.375mM,and 0.5mM were tried,and the lower concentration?0.125mM?increased Angptl4 protein levels slightly and had no statistical significance.Cells incubated with H₂O₂?0.25mM,0.5mM?for 24 hours had significant up-regulation of Angptl4 protein compared with the control.The immunofluorescence confirmed this result further.Likewise,the detection of Angptl4 secreted into the medium by ELISA was in accord with the above results.The effects of H₂O₂?0.25mM,0.5mM?on cell viability were determined by CCK8 assay.However,a higher concentration H₂O₂ exhibited cytotoxicity to the macrophage cells cultures and in a dose dependent manner.2.Regulation of Angptl4 by H₂O₂ is mediated by MAPKs pathways:To gain insight into the underlying mechanism of H₂O₂-induced ANGPTL4 release,we investigated whether the MAPKs signaling pathways play roles in the process.We found that the phosphorylation protein level of ERK1/2,p38 MAPK and JNK extracted from the cells stimulated by H₂O₂?0.25mM,0.5mM?increased obviously compared with the control.We concluded that a certain concentration of H₂O₂ effectively induced phosphorylation of ERK1/2,p38 MAPK and JNK proteins in the macrophage cells.But this conclusion is insufficient to determine that H₂O₂-induced Angptl4 expression is dependent on MAPKs pathways activation.3.U0126 and SB203580 inhibit H202-induced Angptl4 release in RAW264.7 macrophage cells:To explore this issue further,we next sought to determine whether specific inhibitors for p3 8 MAPK?SB203580?,JNK?SP600125?,and ERK1/2?U0126?have effects on H₂O₂-induced Angptl4 release.As illustrated,preincubation of the cells with U0126?20mM?for 90 minutes to block ERK1/2 activation dramatically inhibited overexpression of ANGPTL4 protein induced by H₂O₂.And likewise,preincubation of cells with SB203580?40mM?for 90 minutes to block p38 MAPK pathway activation,and the effect of H₂O₂ on Angptl4 expression was markedly diminished.We also preincubated cells with SP600125?10mM?for 30 minutes to block JNK pathway activation,and specially,the Angptl4 protein level was not affected.Because of the toxicity to the macrophage cells,we were not able to increase the concentration of SP600125,and at the lower concentration?10mM?had no significant effect on H₂O₂-induced Angptl4 expression.Together these results indicate that pretreatment ofmacrophage cells with inhibitors for p38 MAPK and ERK1/2,but not JNK attenuated H₂O₂-induced Angptl4 release.Conclusion:1.H₂O₂ can decrease Angptl4 expression level in in macrophage cells RAW264.7.2.Regulation of Angptl4 by H₂O₂ is mediated by MAPKs pathways and U0126 and SB203580 inhibit H₂O₂-induced Angptl4 release in RAW264.7.