Effects And Mechanism Of NF-κB/Snail Signaling Pathway On LPS-Induced Epithelial-Mesenchymal Transition In Goat Mammary Epithelial Cells

Mastitis is a common multiple clinical disease in dairy goat breeding.It could develop into chronic inflammation and cause breast fibrosis if clinical diagnosis or treatment is not timely,and severe disease could even cause sepsis with a extremely high rate to die.Fibrosis is mainly caused by two factors: tissue damage activates myofibroblasts,leading to the accumulation of extracellular matrix(ECM);epithelial cells undergo epithelial mesenchymal transition(EMT)and synthesize pro-inflammatory and pro-fibrosis factors,which act on effector cells through paracrine way,leading to fibrosis.Therefore,tissue damage in mastitis caused by different pathogenic factors may lead to breast fibrosis to a certain extent.Lipopolysaccharide(LPS)is a gram-negative bacterial endotoxin that is widely used to induce inflammatory responses.LPS activates Toll-like receptors(TLR)-related protein transduction signal pathways by inducing an inflammatory response,and promotes the activation of nuclear factor-κB(nuclear factor kappa-B,NF-κB)transcription factors,while promoting secretory cells synthesize and secrete tumor necrosis factor alpha(TNF-α),interleukins,other inflammatory cytokines and transforming growth factor-beta(TGF-β).It is still unclear whether LPS induced inflammatory injury could induce EMT in goat mammary epithelial cells(GMECs)and secrete pro-fibrogenic cytokines,its molecular mechanism needs further study.Therefore,in this study,the mammary gland of Guanzhong dairy goat was used as the experimental material.Firstly,GMECs were isolated and identified,and then the LPS was used to induce inflammatory reaction to construct goat mastitis cell model.Finally,the model was used to explore the signal cross-linking between LPS induced signal transduction pathway and EMT induced transcription factors.The research contents and results are as follows(1)Epithelioid cells were isolated by tissue block method.The cells expressed Cytokeratin 18(CK18)and did not express Cytokeratin 14(CK14),indicating that the cells were GMECs.Futhermore,RT-PCR and Western blotting were used to detect the expression of β-casein.The results showed that the cells could continuously express β-casein during cell passage,which further proved that the isolated epithelioid cells were GMECs.Then,GMECs were treated with LPS of different concentrations and time gradients.The activation of TLR4/NF-κB signaling pathway and the expression of inflammatory cytokines were detected by qRT-PCR and Western blotting.The results showed that LPS could activate TLR4/NF-κB signaling pathway,promote the transcription of inflammatory cytokines,and up regulate the phosphorylation level of NF-κB p65,which proved that the establishment of goat mastitis cell model induced by LPS was successful.(2)To verify whether LPS induced EMT in this cell model,the expression of β-casein and α-smooth muscle actin(α-SMA),a molecular marker of myofibroblasts,were detected by qRT-PCR and Western blotting.The results showed that the expression of α-SMA was significantly up-regulated and the expression of β-casein was significantly down-regulated with the increase of LPS treatment time,which indicated that LPS induced GMECs to lose their epithelial differentiation phenotype.To verify the occurrence of EMT,TGF-β1 was used as a positive control for EMT induction.Western blotting,qRT-PCR and immunofluorescence staining techniques were used to detect the expression and localization of epithelial cells marker E-cadherin,mesenchymal cells marker N-cadherin and Vimentin,and extracellular matrix Collagen Ⅲafter LPS or TGF-β1 treatment.The results showed that the expression of molecular marker of epithelial cells was significantly down-regulated,and the expressions of molecular markers of mesenchymal cells and extracellular matrix were significantly up-regulated,and the results of mRNA and protein levels were consistent,proving that LPS promoted EMT of GMECs.(3)At the tissue level,the expression of NF-κB,Snail and Slug were detected by qRT-PCR,Western blotting and immunohistochemistry.The results showed that p65 and Snail were up-regulated in mastitis mammary gland compared with normal goat mammary gland,but there was no significant difference in Slug expression.In the cell model,the mRNA and protein expression levels of p65,Snail and EMT markers were detected by qRT-PCR,Western blotting and immunofluorescence staining.The results showed that LPS could significantly up-regulate the mRNA and protein expression levels of p65 and Snail.The expression and localization of p65 and Snail in nucleus and cytoplasm were detected by extracting nuclear protein and cytoplasmic protein respectively.The results showed that the expression of Snail depended on the nuclear translocation activity of p65.Furthermore,TLR4 inhibitor TAK-242 and NF-κB inhibitor QNZ were added before LPS treatment.The results showed that the up-regulation of p65 and Snail induced by LPS was significantly inhibited,and the EMT induced by LPS was partially reversed.The results showed that LPS could induce EMT in GMECs by activating p65/Snail signaling pathway.