Roles Of TLR4/ NF-?B Signaling Pathway Against Inflammatory Responses Induced Mastitis By Lipopolysaccharide

Mastitis, generally defined as the inflammation of the mammary gland, is a costly and complex disease associated with variable origin,severity, and outcome depending on the environment,pathogen,and host. Mastitis is caused when pathogenic bacteria enter the sterile environment of the mammary gland,often as a result of disruption of physical barriers such as the teat, requiring prompt and appropriate host defenses to prevent colonization and subsequent disease pathology. On the other hand, those categorized as contagious pathogens can readily be spread from the infected quarters to other quarters of the same cow, or other cows and include Staphylococcus aureus (S.aureus) and Streptococcus agalactiae. Cow factors including age, stage of lactation, and somatic cell score (SCC) historyare known to influence the occurrence of mastitis infection. The diverse pathogens that can cause mastitis induce different immune responses in the mammary gland,and therefore, the host requires highly specific pathogen-dependent responses for protection.In this experiment, we selected Chinese Holstein cattle and bovine mammary epithelial cells for the study,focused on the mechanism of TLR4/NF-?B signaling pathway with mastitis; We also studied the difference of structure in normal and mastitis mammary gland by histological examination; Besides, gene expression analysis of TLR4/NF-?B signaling pathway using gene chip technology were performed; We used the method of in vitro cultured bovine mammary epithelial cells, discussed the effects of biochemical inhibitor and NF-?B-siRNA on the expression of TLR4/NF-?B signaling pathway genes.The use of in vitro methods to study the biochemical inhibitor and NF-?B-siRNA on LPS epithelial cell signaling pathways expression of key genes; Moreover, we used comparative proteomic approach to uncover the activation and inhibition proteins of LPS stimulation of the mammary epithelial cells.1. Altered molecular expression of the TLR4/ NF-?B signaling pathway in mammary tissue of Chinese Holstein cattle with mastitisToll-like receptor 4 (TLR4) mediated activation of the nuclear transcription factor ?B(NF-?B) signaling pathway by mastitis initiates expression of genes associated with inflammation and the innate immune response. In this study, the profile of mastitis-induced differentialgene expression in the mammary tissue of Chinese Holstein cattle was investigated by Gene-Chip microarray and bioinformatics. The microarray results revealed that 48 genes associated with the TLR4/NF-?B signaling pathway were differentially expressed. Of these genes, 19 were up-regulated and 29 were down-regulated in mastitis tissue compared to normal, healthy tissue. Statistical analysis of transcript and protein level expression changes indicated that 10 genes, namely TLR4?MyD88?IL-6 and IL-10 were up-regulated,while,CD14?TNF-??MD-2?IL-1??NF-?B and IL-12 were significantly down-regulated in mastitis tissue in comparison with normal tissue. Analyses using bioinformatics database resources, such as the Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis and the Gene Ontology Consortium (GO) for term enrichment analysis, suggested that these differently expressed genes implicate different regulatory pathways for immune function in the mammary gland.2. Effects of biochemical inhibition of NF-?B on the expression of bovine mammary epithelial cells TLR4 /NF-?B signaling pathway related genesLipopolysaccharides (LPS),the main component of the outer membranes of Gram-negative bacteria, play a key role in producing an inflammatory response. The LPS signals mainly via the Toll-like Receptor 4 (TLR4) lead to the activation of nuclear transcription factor ?B (NF-?B) to induce the production of pro-inflammatory mediators.The research used the method of in vitro cultured bovine mammary epithelial cells,discussing the effects of inhibition of NF-?B on the expression of bMECs TLR4 /NF-?B signaling pathway genes. The results showed that according to the tissue pieces culture, we isolated high purity bMECs.We also cultured the cells with LPS and Bayll-7082. The results show that the TLR4/NF-?B signaling pathway is activated by the LPS. The BMECs stimulate an inflammatory response that is characterized by an increased mRNA expression of TLR4?NF-?B?MyD88?IL-1??IL-6?TNF-? and increased protein levels of TLR4?NF-?B?IL-1??TNF-? in epithelial cells. The knockdown of NF-?B using the biochemical inhibition of NF-?B, reduced LPS-induced TLR4?NF-?B?MyD88?IL-1? IL-6 and TNF-?expression.3. Effects of NF-?B knockdown on the expression of bovine mammary epithelial cells TLR4 /NF-?B signaling pathway related genesIn order to make results more reliable, we also inhibited the NF-?B by RNA interference. The research discussed the effects of NF-?B-siRNA on the expression of bMECs inflammation signaling pathway genes. We isolated high purity bMECs with NF-?B-siRNA. However, the treatment with siRNA in LPS-induced bovine mastitis resulted in the translocation of NF-?B surrounding the nucleus. The levels of TNF-a and IL-1? were efficiently inhibited by siRNA in the control group compared with the LPS group. Transfection with the NF-?B siRNA reduced the fold induction of TLR4?NF-?B?MyD88 and IL-6, while the expression levels of CD14?IL12 and MD-2 of the TLR4/NF-?B signaling pathway were increased in the control group compared with the LPS-treated group. The results were consistent with the chemical inhibition experiment.4. Comparative proteomic analysis of activation and inhibition p65 in bovine mammary epithelial cellsIn this study, our objective was to identify differentially regulated proteins on bovine mammary epithelial cells (bMECs) at three group (bovine mammary epithelial cells, bovine mammary epithelial cells with LPS, bovine mammary epithelial cells with LPS and the NF-?B inhibitor Bay11-7082) using isobaric tag for relative and absolute quantification(iTRAQ) technique and liquid chromatography-tandem mass spectrometry (LC-MS/MS).We compared the protein expression profiles of LPS with the corresponding adjacent normal cells, LPS with the NF-?B inhibitor with the corresponding adjacent LPS from BMECs. It was determined that 8 proteins were significantly down-regulated at LPS compared with control, and were up-regulated at bay11-7082 compared with LPS, 9 proteins up-regulated at LPS compared with control, and down-regulated at bay11-7082 compared with LPS. Protein ontology analysis showed that those significantly differentially expressed proteins were mainly associated with cellular process,metabolic process and biological regulation. The protein protein interaction between p65 with difference expressed proteins in LPS vs control group showed a total of 225 points and 717 interaction relations.The protein protein interaction between p65 with difference expressed proteins in bayl 1-7082 vs LPS group showed a total of 56 points and 77 interaction relations.As stated above, the conclusions are as follows:1. The microarray results revealed that 48 genes associated with the TLR4/NF-?B signaling pathway were differentially expressed. These genes were involved in toll-like receptors signaling pathways, MAPK signaling pathways and cell apoptosis process. The results show that the mammary tissues with mastitis will release the cytokines and chemokines through the TLR4 / NF-?B signaling pathway.2. The TLR4/NF-?B signaling pathway was activated by LPS in bovine mammary epithelial cell and raised the expression of inflammatory cytokines leading to cell damage.The knockdown of NF-?B using the biochemical inhibition and siRNA of NF-?B, reduced LPS-induced inflammatory cytokines as IL-1??IL-6 and TNF-? expression and the expression of related genes of the signaling pathway.3. The proteomic revealed 17 differentially expressed proteins in the activation and inhibition wite bMECs. We selected 13 genes in the protein protein interaction between p65 with difference expressed proteins in LPS vs control group with high degree and stress, and 9 genes in the protein protein interaction between p65 with difference expressed proteins in bay11-7082 vs LPS group with high degree and stress.