Effects Of Sphingosine-1-Phosphate (S1P) On The Expression And Production Of Inflammatory Cytokines In Mouse Monocyte Macrophages

Macrophages are a group of highly adaptive immune cells that can phagocytose and clear pathogens that invade the body,promote the body's inflammatory response and repair and rebuild damaged tissues.In a specific microenvironment in which the organism is located,macrophages can polarize different phenotypes,namely classically activated M1 macrophages and selectively activated M2 macrophages.Usually in the early stages of pathogen infection,macrophages are activated to polarize into M1 macrophages,and then produce a large number of pro-inflammatory mediators,such as IL-1,IL-6,tumor necrosis factor-?(TNF-?),nitric oxide synthase(induced NOS,iNOS),etc.;also can produce chemokines,such as monocyte chemotactic protein-1(MCP-1),CCL2-4,CXCL8-11 and so on.If inflammation continues to occur,a large number of inflammatory factors will be produced,and macrophages will be polarized to form M2 macrophages to secrete anti-inflammatory factors,such as Arg-1,IL-10,etc.,thereby alleviating the body's inflammatory response.Sphingosine-1-phosphate(S1P)is a bioactive lipid produced by the metabolism of sphingomyelin.It is widely found in blood and lymph.S1 P acts as an extracellular ligand and is involved in S1 P membranes found in almost all cell types.Body binding,activation of a series of downstream signaling pathways to produce important physiological functions,such as involvement in cell proliferation,cell migration,apoptosis,angiogenesis,and immune responses.In this experiment,mouse mononuclear macrophage leukemia cell RAW264.7 was used as experimental material to investigate the effect of S1 P on gene expression and protein production of cytokines related to RAW264.7 cells.Firstly,the effect of different concentrations of S1 P on the proliferation activity of RAW264.7 cells was detected by MTT assay.The results showed that treatment of RAW264.7 cells with 20~500 nmol/L S1 P for 24 h had no effect on the in vitro proliferation of RAW264.7 cells.The cell activity was the strongest at a concentration of 500 nmol/L.Secondly,the expression of S1 P receptor on RAW264.7 cells was explored.Macrophages were amplified by LPS(1.0 ?g/mL)to form M1 macrophages,and then total RNA was extracted,and then qRT-PCR was performed.RAW264.7 cells express S1PR1,S1PR2,and S1PR4 receptors.To investigate the effect of S1 P on the polarization of LPS(1.0 ?g/mL)-stimulated RAW264.7 cells into M1 macrophages,after treatment of M1 macrophages with 500 nmol/L S1 P,cells were harvested and total RNA and protein were extracted.Real-time quantitative reverse transcription-PCR(qRT-PCR)was used to detect the expression of TNF-?,IL-6,MCP-1,iNOS,Arg-1 and IL-10,using enzyme-linked immunosorbent assay(ELISA).The protein production of TNF-?,IL-6,MCP-1,iNOS,Arg-1 and IL-10 was examined.The results showed that 500 nmol/L S1 P can inhibit the expression of TNF-?,IL-6,MCP-1 and iNOS in RAW264.7 cells,reduce their protein production,and enhance the anti-inflammatory cytokine Arg-1.And IL-10 gene expression and elevated protein synthesis.From the above results,it can be preliminarily concluded that a certain concentration of S1 P can inhibit the secretion of pro-inflammatory factors of M1 macrophages and enhance the secretion of anti-inflammatory factors.Therefore,S1 P has a certain anti-inflammatory effect as a foreign substance of cells,and chronic diseases The threat of human health cannot be underestimated,and many chronic diseases are mostly inseparable from inflammation,which provides new research ideas for relieving body inflammation and some chronic inflammatory diseases.