Effect Of Selenium On Regulation Of DHA On The Expression Of Cytokines And Lipid Metabolism In Mouse Macrophages Induced By LPS

Excessive or persistent inflammation can lead to many diseases,how to effectively control and treat inflammatory diseases is still the focus of attention.While both the studies on the anti-inflammatory effect and mechanism of DHA and selenium?Se?have made remarkable progress,the interaction between n-3PUFAs and selenium has yet to be comprehensively studied.In addition,since the essential fatty acid-derived lipid mediators such as prostaglandins,leukotrienes and lipoxin are involved in the regulation of inflammatory reaction,it is helpful to understand the mechanism of inflammation and find the potential targets for the treatment of inflammatory diseases by studying the metabolic mechanism of PUFAs and its metabolites.Studies have shown that selenium may be involved in the regulation of fatty acid-derived lipid mediators,but its mechanism remians unclear.Therefore,the following experiments were carried out based on mouse macrophage RAW264.7 in this study,the test is divided into 6 groups,cells were treated with 10 ?g/mL DHA?group D?,10 ?g/mL DHA+0.05 ?mol/L sodium selenite?group DS?,10 ?g/mL DHA+1 ?g/mL LPS?group LD?,10 ?g/mL DHA+1 ?g/mL LPS+0.05 ?mnol/L sodium selenite?group LDS?,1 ?g/mL LPS?group L?and 0 ?g/mL DHAA+0 ?g/mL LPS+0?mol/L sodium selenite?group normal?for 24 h respectively,the cells and culture supernatant were collected according to the requirements for each test:1.In order to investigate whether and how selenium effects the regulation of DHA on mRNA and protein expression levels of TNF-a,IL-1?,IL-6 and IL-10 genes in RAW264.7 cells induced by LPS.The mRNA expression levels of TNF-a,IL-1?,IL-6 and IL-10 genes were measured by semi-quantitative reverse transcription PCR,and ELISA was used to test levels of TNF-a,IL-1?,IL-6 and IL-10 in cell culture medium.The results showed that adding sodium selenite?0.05 ?mol/L?not only significantly enhanced the inhibitory effects of DHA?10 ?g/ml?on expression of proinflammatory cytokines TNF-a mRNA?P<0.05?and production of IL-1??P<0.01?,but also extremely significantly enhanced the promoting effects of DHA?10 ?g/ml?on mRNA and protein expression of anti-inflammatory cytokine IL-10 genes in RAW264.7 cells induced by LPS?1 ?g/ml?for 24 h?P<0.01?.The results indicate that Se can enhance the anti-inflammatory effects of DHA on inflammatory response in macrophages induced by LPS.2.In order to investigate the effects of selenium and DHA on the mRNA expression of 5-LOX and COX-2,two of key enzymes converting PUFAs into lipid mediators,in macrophages induced by LPS.The mRNA expression levels of 5-LOX and COX-2 was detected by semi quantitative RT-PCR.Although the results showed that DHA?10 ?g/mL?had no significant effect on 5-LOX mRNA expression in macrophage induced by LPS?1 pg/mL??P>0.05?,it showed a significant downward trend.Adding DHA?10 ?g/mL?and sodium selenite?0.05 p.mol/L?to cells significantly reduced the mRNA expression levels of 5-LOX?P<0.05?;Furthermore,the addition of DHA?10 ?g/mL?alone or together with sodium selenite?0.05 ?mol/L?significantly suppressed expression of COX-2 mRNA in macrophage induced by LPS?1 ?g/mL??P<0.05?,and it also showed an obvious downward trend between LD group and LDS group?P>0.05?.Therefore,it can be conclude that the combination of DHA and selenium can down regulate the mRNA expression of 5-LOX and COX-2 in macrophages induced by LPS,and it can be inferred that selenium may enhance the inhibitory effect of DHA on 5-LOX and COX-2 mRNA expression in macrophages induced by LPS.3.In order to investigate the regulation of selenium and DHA on lipid metabolism and lipid peroxidation in macrophage induced by LPS.The level of malondialdehyde?MDA?in cell culture supernatant was determined to reflect the level of lipid peroxidation.Moreover,LC-MS analysis was used to detect the formation of lipid mediators in group L,group LD,group LDS and group normal cells.Furthermore,LC-MS/MS was used for the analysis of isomers of lipid mediators.According to our research,DHA?10 ?g/mL?not only inhibited the formation of MDA?P<0.01?in macrophage induced by LPS?1 ?g/mL?and the conversion of LTC₄ to LTD₄?P<0.05?,but also increased the level of 15d-PGJ₂?P<0.05?,which has anti inflammatory function,however,it had no significant effect on 9,10-EpOME?P>0.05?,one of the LA-peroxidation products.The combined effect of sodium selenite?0.05 ?mol/L?and DHA?10 ?g/mL?decreased the production of MDA?P<0.01?and 9,10-EpOME?P<0.01?generated by LPS-induced macrophages,enhanced the inhibitory effect on the conversion of LTC₄ to LTD₄,and strengthen the up-regulation of 15d-PGJ₂ level?P<0.01?as well.In addition,the level of LTD₅?less activity than LTD₄?level?P<0.01?has been significantly up-regulated in cells co-cultured with selenium?0.05?mol/L?and DHA?10 ?g/mL?,while the level of 19,20-DiHDPA was significantly down-regulated?P<0.01?.What's more,there were several lipid mediators isomers distinguished by LC-MS/MS analysis method,including AA-derived?namely 5-Oxo-ETE,5S-HETE,11,12-EET,12S-HETE,LTB4,LXA4 and PGE₂?,DHA-derived?namely Maresinsl,Maresins2,14S-HDHA,17S-HDHA,PD1,RvDl,RvD3,RvD4 and RvD6?and EPA-derived?namely RvE2 and RvE3?lipid mediators.Thus,It can be concluded that DHA can inhibit the lipid peroxidation level of RAW264.7 macrophages induced by LPS,and selenium could significantly enhance this inhibitory effect of DHA on lipid peroxidation.DHA can promote the production of 15d-PGJ₂ in macrophages and thus protect cells,Se can enhance the protective effect of DHA.Moreover,DHA can inhibit the conversion of LTC₄ to LTD₄ in LPS-induced-macrophage,combination of Se and DHA not only enhance this inhibitory effect of metabolism,but also conducive to synthesize more LTD₅ and inhibit the conversion of 19,20-EDP into 19,20-DiHDPA in macrophages induced by LPS,It is suggested that the combination of Se and DHA is more beneficial to the anti-inflammatory effect.In addition,the results of LC-MS/MS identification can lay a foundation for further study on the regulation of selenium on lipid mediators by targeted lipidomics.