Analysis Of Material Metabolism In Macrophages Regulated By NMAAP1 And The Regulatory Effect Of Emodin

Macrophages are a heterogeneous population,including myeloid-derived macrophages and tissue-resident macrophages.They can be activated by lipopolysaccharide alone or with Th1 cytokines such as IFN-γ,Th2 cytokines such as IL-4 and IL-13,and polarized in two distinct directions of phenotype and function.In the pathological process,they play a role in antimicrobial defense,anti-tumor immunity.Moreover,they are related with obesity,asthma and allergies,atherosclerosis,fibrosis,wound healing and autoimmunity.BCG can activate macrophages to up-regulate the expression of NMAAP1 protein.The previous results found that the protein can regulate the polarization of macrophages to M1 and play a role in killing tumor cells by secreting killer factors.The cellular material metabolism,such as sugar metabolism,amino acid metabolism,fat metabolism,etc.,can affect the polarization of macrophages in the direction of M1,and members of this research group have also found that NMAAP1 can regulate the sugar metabolism of macrophages,whether it can regulate other substances metabolism is unclear.Emodin is a trihydroxyanthraquinone herb found in several Chinese herbal medicine,including rhubarb and fleece-flower root.Emodin has been shown to reduce the severity of experimental disease models,including arthritis,liver damage,atherosclerosis,myocardial ischemia,and cancer.The previous work of our research group found that emodin can inhibit the inflammatory polarization of macrophages induced by LPS,but whether it can regulate the expression and secretion of inflammatory factors in M1 macrophages caused by NMAAP1 is still unclear.Therefore,this study firstly used bioinformatics to analyze the transcriptome of NMAAP1 high-expressing macrophages,and explored whether NMAAP1 affects the related genes of macrophage material metabolism,including carbohydrate metabolism,amino acid metabolism and lipid metabolism;secondly,it explores the role of lactic acid in NMAAP1 regulating the secretion of inflammatory factors by macrophages;finally,it is explored whether the anti-inflammatory effect of emodin is related with NMAAP1.Research methods1.Bioinformatics analysis of the transcriptome of macrophages with high expression of NMAAP1.2.The role of lactic acid in the regulation of macrophages by NMAAP1We cultured NMAAP1 high-expressing RAW264.7 cells and the control group and seeded 2*10~5 cells in a 12-well plate.The next day,FX11(80μM),a lactic acid inhibitor,was added to each well.After 24 hours,the cell culture supernatant was collected,and the kit was used to detect lactic acid,and the ELISA kit was used to detect the concentration of TNF-α.We collected the cells,extracted m RNA,and used q RT-PCR to detect the expression of TNF-α,NMAAP1,iNOS,Arg1 and HIF-1α.3.Emodin inhibits the inflammatory macrophage effect by down regulated NMAAP1 expressionWe cultured NMAAP1 high-expressing RAW264.7 cells and control group,and emodin(40μM)was added and the supernatant was collected after 24h.The ELISA kit was used to detect the concentration of TNF-α;q RT-PCR was used to detect the expression of NMAAP1 and TNF-α.The supernatant was collected and added to the MCA207 cell culture system.After 24 hours,the cell viability was detected by the CCK-8 kit.Experimental results1.NMAAP1 could regulate genes related to sugar metabolism,amino acid metabolism and lipid metabolism in RAW264.7 macrophage cells with high expression of NMAAP1.The expression of hexokinase(HK)is increased.HK is also involved in the metabolism of fructose and mannose.When the expression of NMAAP1 increased,cystathionineβ-synthase and cystathionineγlyase also increased,indicating that NMAAP1 can regulate the metabolism of serine and methionine.In the metabolism of arginine,after the high expression of NMAAP1,the expression of acetaldehyde dehydrogenase is up-regulated,which promotes the metabolism of arginine.Moreover,NMAAP1 up-regulates acetaldehyde dehydrogenase,3-phosphoglycerol O-acyltransferase,phosphatidic acid phosphatase and diacylglycerol kinase,and participates in regulating the metabolism of glycerolipids.Finally,in the high expression of NMAAP1 cells,we found that the expression of acid ceramidase is elevated,which is related to sphingomyelin metabolism.2.NMAAP affects the expression and secretion of macrophage cytokines through lactic acidAfter adding the lactic acid inhibitor FX11(80μM)to the high expression of NMAAP1 cells,the lactic acid release increased by NMAAP1 were significantly inhibited by FX11.It inhibited the expression and secretion of TNF-αat the gene and protein level.After adding FX11to RAW264.7 cells with high expression of NMAAP1,it can inhibit iNOS gene expression and up-regulate Arg1 gene expression.In addition,it was also found that FX11 down-regulated HIF-1αgene expression.3.The regulation of emodin on the effect of NMAAP1When NMAAP1 high-expressing RAW264.7 cells are added with emodin(40μM),it can inhibit the gene expression of NMAAP1 and TNF-α.Emodin reduced the effect of NMAAP1 in promoting macrophages to kill tumor cells to a certain extent.ConclusionNMAAP1 can affect Hexokinase,cystathionineβ-synthase and cystathionineγlyase,acetaldehyde dehydrogenase,3-phosphoglycerol-O-acyltransferase,phosphatidic acid phosphatase and diacylglycerol kinase and acid Ceramidase and play important roles in the sugar metabolism,amino acid metabolism,and lipid metabolism.NMAAP1 affected the expression and secretion of TNF-αin macrophages through lactic acid.On the one hand,emodin exerts anti-inflammatory effects by directly reducing the expression of TNF-α,on the one hand,it is achieved by reducing the gene expression of NMAAP1.