| Objective:In this study,we observed the anti-inflammatory effect of Scutellaria-coptis herb couple on LPS-induced RAW264.7 cells at the cellular level.Proteomics and transcriptome were used to investigate the possible chronic inflammation mechanism of SC against type 2 diabetes mellitus in RAW264.7 cells and KKAy mice liver tissue.Method:1.Macrophages with 1ug/mL LPS to induce an inflammation model.(1)MTT assay was used to detect the effect of SC on the viability of RAW264.7 cells.(2)LDH kit was used to detect the effect of SC on LDH content in RAW264.7 cells.(3)The concentration of interleukin-1 beta(IL-1?)in macrophage supernatant by ELISA.(4)q RT-PCR was used to detect the expression of interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-?)in macrophages.(5)Western blot was used to detect the expression of TLR4 and My D88 in macrophages.(6)The translocation of NF-kappa B in macrophages was determined by immunofluorescence assay.2.Proteomic analysis: nano-LC-LTQ/Orbitrap-MS technology was used to analyze the effects of SC on RAW264.7 cells induced by LPS and liver tissue of KKAy diabetic mice.String and DAVID databases were used for bioinformatics analysis of differential proteins: GO(Gene Ontology)analysis,KEGG Pathway enrichment analysis,protein interaction analysis and so on.3.Transcriptionomic analysis: RNA-seq method was used to analyze the effect of SC on LPS-induced RAW264.7 cells.GO annotation function classification,KEGG pathway enrichment,Cytoscape network analysis were used to identify the expression level of differentially expressed genes in RAW264.7 cells.Fluorescence quantitative PCR was used to verify the expression level of differentially expressed genes in RAW264.7 cells.Result:1.(1)MTT assay results of SC on RAW264.7 cell viability: Compared with blank group,different concentrations of SC had inhibitory effect on RAW264.7 cells,but there was no significant difference(P > 0.05).(2)LDH kit was used to detect the effect of SC on LDH content of RAW264.7 cells: Compared with blank group,there was no significant difference in LDH content of RAW264.7 cells in different doses SC on drug-containing serum(P > 0.05).(3)The expression of IL-1? in RAW264.7 inflammation model was measured by ELISA.Compared with the blank group,the expression of IL-1? in the model group was significantly increased(P < 0.01);compared with the model group,the expression of IL-1? in both SC and pioglitazone groups was significantly decreased(P < 0.05).(4)The expression of IL-6 and TNF-? in macrophages was detected by q RT-PCR.Compared with the blank group,the expression of TNF-? and IL-6 in the model group increased significantly(P < 0.01).Compared with model group,SC group can reduce the expression of TNF-? and IL-6.In three dosage groups of SC,the expression of TNF-? decreased significantly(P < 0.01);in SC-M and SC-L group,the expression of IL-6 decreased significantly(P < 0.05),but there was no significant change in SC-H group.(5)Western blot was used to detect the expression of TLR4 and My D88 protein in RAW264.7.Compared with the blank group,the expression of TLR4 and My D88 protein in the model group increased significantly(P < 0.01).Compared with the model group,SC could reduce the expression of TLR4 and My D88 protein;the expression of My D88 protein in SC-H,SC-M and SC-L groups decreased significantly(P < 0.01);the expression of TLR4 protein in SC-L group decreased significantly(P < 0.01);the expression of TLR4 protein in SC-H group was significantly different(P < 0.01).(6)Cellular immunofluorescence assay was used to detect the translocation of NF-kappa B in macrophages.Compared with the blank group,the model group translocated NF-?B into the nucleus.Compared with model group,SC high,middle and low dose groups and pioglitazone group can inhibit the translocation of NF-?B.2.The results of cell proteomics showed that there were 950 differentially expressed proteins in SC and model groups,which were consistent with the trend of blank group.GO analysis showed that the differentially expressed proteins were mainly involved in biological processes,including cell processes,metabolic processes,organic metabolic processes,biological regulation,drug metabolic processes,catabolism and so on.Molecular functions of differentially expressed proteins include protein binding,catalytic activity,nucleotide binding,drug binding and anionic coordination.The main differentially expressed proteins include cytoplasm,extracellular,intracellular and organelle.DAVID online software was used to analyze the pathway enrichment of differentially expressed proteins.The signaling pathways involved in differentially expressed proteins include glycolysis and glycogenesis,metabolic pathway,MAPK signaling pathway,glycerol metabolism,c AMP signaling pathway,HIF-1 signaling pathway,Rap1 signaling pathway,VEGF signaling pathway,influenza A,renal cell carcinoma and so on.Using string online analysis,950 differentially expressed proteins were input and 341 effective proteins were obtained.The interaction graph showed that the interaction between differentially expressed proteins was complex.Many nodal proteins interacted with other proteins,and some differentially expressed proteins were scattered.Through analysis,differential proteins related to type 2 diabetes mellitus such as CD14,Ptgs2(COX-2),Caspase 8 and MAPK14 were screened.3.The results of proteomics of liver tissue showed that 2412 proteins were screened out,of which 637 were up-regulated and 1775 were down-regulated.GO analysis of all differentially expressed proteins was performed using String software.Biological Process,in which differentially expressed proteins were up-regulated,mainly included cell composition,organelles,DNA conformation changes and cell processes.The main Molecular functions of up-regulated differentially expressed proteins include ATPase activity,nucleotide binding and small molecule binding.Cellular Components of up-regulated differentially expressed proteins include DNA packaging complexes,protein-DNA complexes,organelle parts,protein-containing complexes,chromosome parts,etc.The results showed that down-regulated differential proteins were mainly involved in biological processes,including small molecule metabolism,organic acid metabolism,drug metabolism,cell processes,etc.The main Molecular functions of down-regulated differential proteins include catalytic activity,binding,anionic coordination,drug binding,ATP binding,etc.Cellular Components of down-regulated differential proteins include cell parts,organelle parts,cytoskeleton,myelin sheath and so on.DAVID online software was used to analyze the pathway enrichment of differentially expressed proteins.The results showed that the main pathways involved in differentially expressed proteins were metabolic pathways,inflammation-related signaling pathways and some disease pathways.Up-regulation of differential proteins involved in signaling pathways includes metabolic pathways such as cysteine and methionine metabolism,phenylalanine metabolism,central carbon metabolism in cancer,and disease pathways such as systemic lupus erythematosus,alcoholism,viral carcinogenesis,etc.Down-regulation of differential proteins involved in signaling pathways mainly includes metabolic pathways such as metabolic pathways,ascorbic acid and aldehyde acid metabolism,carbon metabolism,retinol metabolism,inflammation-related signaling pathways such as MAPK signaling pathway,m TOR signaling pathway,AMPK signaling pathway and some disease pathways such as chemical carcinogenesis,drug metabolism-cytochrome P450,Alzheimer's disease,etc.Using string online analysis,637 differentially expressed proteins were inputted into the experimental group and the model group,and 321 effective proteins were obtained.Some nodal proteins interacted with other proteins,and many differentially expressed proteins were scattered.By inputting 1775 down-regulated differential proteins,864 effective proteins were obtained.The interaction map showed that there were many scattered differential protein spots.Some nodal proteins interacted with other proteins,and the interaction of differential proteins was more complex.Through analysis,differential proteins related to type 2 diabetes mellitus such as Apo E,PIK3,AKT2,m TOR,TRAF6 and NF-kappa B were screened.4.Transcriptomics results: There were 2816 differential genes in the model group and the blank group,of which 1864 genes were up-regulated and 952 genes were down-regulated.There were 292 differential genes in the model group and SC group,which up-regulated 273 genes and down-regulated 19 genes.These differentially expressed genes were classified by GO enrichment.Upregulation differentially expressed genes were classified by GO into cell processes,metabolic processes and biological regulation in biological processes,cell organelles and membranes in cellular components,and binding,catalytic and molecular sensor activities in molecular functions.The down-regulated differential genes are classified into biological processes by GO,which are cell processes,stimulus responses and biological regulation.The down-regulated differential genes are classified into cellular components and molecular functions,which are identical to the up-regulated differential genes.Differential gene pathways in blank group and model group include TNF signaling pathway,NF-kappa B signaling pathway,MAPK signaling pathway,hepatitis B and so on.Differential gene pathways in SC group and model group include metabolic pathway,arachidonic acid metabolism,drug metabolism-cytochrome P450,PPAR signaling pathway and so on.STRING database was used to obtain the close connection network between different genes by homology with known proteins.Only a few scattered points were distributed nearby.Through bioinformatics analysis,33 common differential genes between SC and model group were identified.22 genes with consistent up-regulation and 5 down-regulation were screened.The results of q RT-PCR validation of three genes,S100A8,Apoc2 and Lpin1,were as follows: Compared with the blank group,the expression of S100A8 in model group was significantly up-regulated(P < 0.01),the expression of Apoc2 and Lpin1 was significantly up-regulated(P<0.01).Compared with model group,SC group could enhance the expression of S100A8 and decrease the expression of Apoc2 and Lpin1.In SC group,the expression of S100A8 was significantly different(P < 0.01);the expression of Apoc2 and Lpin1 was significantly different(P < 0.01);in B group,the expression of Apoc2 was significantly different(P < 0.01),and the expression of Lpin1 was not changed significantly.The trend was consistent with the results of transcriptome.Conclusion:1.SC has a role in reducing the expression of inflammatory factors such as TNF-?,IL-6,IL-1? and TLR4,MYD88 protein,affecting the translocation of NF-?B,suggesting that SC has a certain anti-inflammatory effect.2.Proteomics studies have shown that SC can participate in metabolic pathways and inflammation by regulating CD14,Ptgs2(COX-2),Caspase8,MAPK14,Apo E,PIK3,AKT2,TRAF6,NF-?B and so on.SC may fight type 2 diabetes by participating in metabolic pathways,inflammation-related signaling pathways and some disease pathways3.Transcriptionomic studies showed that SC may regulate type 2 diabetes mellitus at the genetic level by regulating S100A8,Apoc2,Lpin1,SGLT1 and CBS genes,participating in metabolic pathways,arachidonic acid metabolism,drug metabolism-cytochrome P450,PPAR signaling pathways.4.Through comprehensive analysis,it is concluded that Scutellaria-coptis herb couple can improve inflammation pathway through TLR4/My D88/PI3K/Akt/NF-kappa B signaling pathway through multi-target and multi-channel,which has reached the goal of improving type 2 diabetes mellitus. |
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