The Mechanism Of Maresin1 Inhibiting LPS-induced Inflammatory Response In Macrophages And Mast Cells

Objective:The study aim to explore the protective effect of Maresin1(MaR1)on the inflammatory response in macrophages cells RAW264.7 and mast cells P815 induced by lipopolysaccharide(LPS),and to understand its mechanism of action.Method:RAW264.7 and P815 cells were cultured and grouped,and given different concentrations of MaRl(1nm,10nm and 100nm)and 50μg/mL dexamethasone(Dex)pretreated cells for 3 hours,then stimulated with LPS for 21 hours.The release levels of inflammatory cytokines TNF-α、IL-6 and IL-1β in the cell supernatant were determined by enzyme-Linked immunosorbent assay(ELISA).Real-time fluorescent quantitative PCR(QRT-PCR)was used to detect the mRNA expression levels of inflammatory cytokines TNF-α、IL-6 and IL-1β in RAW264.7 cells and P815 cells.The expression of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),NF-κB/P65 and P-NF-κB/P65 proteins in RAW264.7 cells and P815 cells were detected by Western blotting.Result:(1)Compared with the control group,the release levels of inflammatory cytokines TNF-α,IL-6 and IL-1β in supernatant of RAW264.7 cells and P815 cells in LPS group were significantly increased,with statistical significance(P<0.01);Compared with LPS group,the release levels of inflammatory cytokines TNF-α,IL-6 and IL-1β in supernatant of cells in all MaR1 groups decreased gradually with the increase of MaR1 concentration,and the differences were statistically significant(P<0.05).When MaR1 concentration reached 100nm,there was no statistical difference in the release levels of inflammatory cytokines TNF-α,IL-6 and IL-1β in supernatant of cells compared with Dex group(P>0.05).(2)Compared with the control group,the mRNA expression levels of inflammatory cytokines TNF-α,IL-6 and IL-1β in RAW264.7 cells and P815 cells in LPS group were significantly increased,with statistical significance(P<0.01);Compared with LPS group,the mRNA expression levels of inflammatory cytokines TNF-α,IL-6 and IL-1β in different concentrations of MaRl group were decreased in a dose-dependent manner,and the differences were statistically significant(P<0.05).When MaR1 concentration reached 100nm,the mRNA expression levels of inflammatory cytokines TNF-α,IL-6 and IL-1β showed no significant difference compared with dexamethasone group(P>0.05).(3)Compared with the control group,the protein expression of TLR4,MyD88,NF-κB/P65 and P-NF-κB/P65 in RAW264.7 cells and P815 cells in LPS group was significantly increased(P<0.01).Compared with LPS group,the protein expression of NF-κB/P65 in RAW264.7 cells in 1nm MaR1 group was only slightly decreased,and the difference was not statistically significant(P>0.05),while the protein expression of TLR4,MyD88 and P-NF-κB/P65 in RAW264.7 cells in 1nm MaR1 group was significantly decreased.The difference was statistically significant(P<0.05).Compared with LPS group,TLR4 protein expression in P815 cells in lnm MaRl group was only slightly decreased,and the difference was not statistically significant(P>0.05).However,the protein expression of MyD88,NF-κB/P65 and P-NF-κB/P65 in P815 cells in 1nm MaR1 group was significantly decreased,with statistical significance(P<0.01).Compared with LPS group,the expression of TLR4,MyD88,NF-κB/P65 and P-NF-κB/P65 in RAW264.7 and P815 cells were significantly decreased in other MaRl groups(ALL P<0.01).Conclusion:1.MaR1 can reduce the LPS-induced release of inflammatory cytokines TNF-α,IL-6 and IL-1β in murine macrophagesand mast cells,suggesting that MaRl has a protective effect on LPS-induced inflammation in macrophages and mast cells.2.MaR1 can down-regulate the mRNA expressions of inflammatory cytokines TNF-α,IL-6 and IL-1β in LPS-inducedmurine macrophagesand mast cells in different degrees,indicating that MaRl can inhibit macrophages and mast cells from the transcriptional level Inflammatory cytokine production.3.MaRl can down-regulate the expression of TLR4,MyD88,P-NF-κB/P65 and NF-κB/P65 proteins in LPS-induced murine macrophagesand mast cells,suggesting that MaRl can inhibit the activation of TLR4/MyD88/NF-κB signaling pathway in macrophages and mast cells.4.By inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway in macrophages and mast cells,MaR1 further regulates the gene and protein expressions of inflammatory cytokines TNF-α,IL-6 and IL-1β in macrophages and mast cells,thus playing a protective role in lPS-induced inflammation in macrophages and mast cells.