The Relationship Between Several Genes With LPS Signaling Pathway And The Influence Of JYT On The Gene Expression In MECs

Lipopolysaccharide(LPS)is one of the stimulating sources of inflammation.Which is often used to establish an inflammatory model.Previous studies in the laboratory found that 5 ?g/mL LPS could cause inflammation at 6 h in mouse mammary epithelial cells(MECs).However,it is still not clear how LPS activates the LPS-induced signaling pathway in the early inflammatory response.In order to find out the key genes of MECs immune inflammation stimulated by LPS in the early stage and the effect of JYT on key genes,ELISA was used to detect the change of TNF-?,IL-1? and IL-6 content under the stimulation of 5 ?g/mL LPS at 6h.Transcriptome sequencing technology(RNA-seq)was used to detect the differentially expressed genes(DEGs)caused by LPS,GO and KEGG Pathway analysis was used to screen inflammatory immune-related genes,and using real-time fluorescence quantitative PCR(qPCR)to verify partial differential gene correlation.By transfection partial DEGs to investigate the influence of IL-1?,IL-6 and the key target gene NF-?B p65 in LPS signaling pathway.qPCR was used for the changes of mRNA in the differential gene were combined with the combination of JYT and LPS.Result showing that:(1)The inflammatory cytokines TNF-?,IL-1? and IL-6were significantly increased(P<0.05,P<0.01)under stimulation of 5 ?g/mL LPS at 6h.(2)By RNA-seq sequencing,there were 2 058 DEGs mainly involved in 594 biological processes and 117 cell signaling pathways.Eight key DEGs(Cxcl1?Csf2?Csf3?Ccl2?Met?Birc3?Icam1?Cav1)with MECs inflammatory immunity in mice were selected by GO and KEGG Pathway analysis.The result of qPCR was consistent with the sequencing trend of transcriptome,and the change of mRNA after LPS stimulation showed an upward trend.(3)Cxcl1 and Ccl2 siRNA transfection fragments were selected.After Cxcl1 was silenced,the LPS mediated the secretion ofIL-1? was significantly reduced compared with LPS group(P<0.05),and the key target gene NF-?B p65 in LPS signaling pathway was extremely reduced compared with the LPS group(P<0.01);after Ccl2 was silenced,the LPS mediated the secretion of IL-1? and IL-6 was extremely reduced compared with LPS group(P<0.01),and the key target gene NF-?B p65 in LPS signaling pathway was extremely reduced compared with the LPS group(P<0.01).(4)The mRNA expression of Cxcl1,Csf2,Csf3,Ccl2,Met,Icam1 and Cav1 in JYT were extremely significantly reduced(P<0.05,P<0.01).Result suggesting that:(1)When 5 ?g/mL LPS stimulates MECs for 6 h in MECs,it can induce the immune inflammatory response of the cells,which can be used as a study of LPS-mediated inflammatory immune response.(2)After the LPS stimulated MECs in mice,there was a large number of gene changes.Among them,several up-regulated genes(Cxcl1,Csf2,Csf3,Ccl2,Met,Birc3,Icam1,Cav1)were closely related to the LPS signaling pathway.(3)Cxcl1 and Ccl2 were transfected successfully with siRNA.Transfection of Cxcl1 and Ccl2 can affect the expression of NF-?B p65,the key target gene in LPS signaling pathway,and reduce the amount of IL-1? or IL-6 in the cell secretion.Further confirmed Cxcl1 and Ccl2 may be mice MECs LPS mediated signaling pathway on the key genes that can be used as a target genes for more in-depth research,providing a new method for the treatment of mammary gland inflammation and the experimental data.(4)Traditional Chinese medicine JYT in LPS stimulated early mammary epithelial cells play a better anti-inflammatory immune activity,this can provide basic data for the study of the mammary gland epithelial cells of the immune inflammatory signal,and for the clinical prevention caused by LPS provides the theory basis for breast inflammatory diseases,also the prospects of Chinese medicine preparations in terms of anti-inflammatory provide data support.