NF-?B Is Involved In The LPS-mediated Proliferation And Apoptosis Of MAC-T Epithelial Cells

Subacute rumen acidosis(SARA)is a common metabolic disease.When the lactation dairy cows suffering from SARA,the concentration of lipopolysaccharide(LPS)in rumen and peripheral blood will increase and make the inflammatory reaction of body,and to decrease milk production.LPS can activate nuclear transcription factor-kappa B(NF-?B).Activated NF-?B is not only play an important role in inflammatory,but also regulate in cell proliferation and apoptosis.Proliferation and apoptosis of MAC-T cells play an important role in the maintenance of milk production performance.Whether NF-?B regulates the proliferation and apoptosis of MAC-T cells in SARA,and the decline in milk production has not been reported.In this paper,we used MAC-T cells as the research object to reveale the effect of LPS-mediated NF-?B in proliferation and apoptosis of MAC-T cells which from the cellular level.MTT assay and LDH were used to detect the effect of LPS on the proliferation,and DNA ladder and RT-PCR were used to detect changes in apoptosis of MAC-T cells.LPS and NF-?B inhibitor(PDTC)in combination with MAC-T cells.According to changes in inflammatory molecules and apoptotic signals,to investigate the role of NF-?B in LPS-mediated proliferation and apoptosis of MAC-T cells The results of this study will provide theoretical basis for milk productivity decline in SARA.Our results show:1.LPS on the proliferation of MAC-T cells have a obviously inhibitory effect and show a concentration-and time-dependent responseThe results of MTT assay showed that the MAC-T cell viability didn't change significantly when it was treated with low concentration of LPS,but the cell growth was significantly inhibited by high concentration,and decreased linearly.MAC-T cells exposed to high concentration of LPS also showed a time-dependent.that is,LPS can promote cell proliferation and show a concentration-and time-dependent response.2.LPS promoted MAC-T cells injury,the degree of injury was positively correlated showed a concentration-dependent.The results of LDH assay showed that the exposure of the cells to various concentrations(50 to 200 ?g/m L)of LPS for 24 h had little effect on the release of LDH.In contrast,when we used the concentration(250 to 450 ?g/m L)of LPS to treat MAC-T cells,LPS increased LDH enzyme activity.Suggesting that LPS can promote MAC-T cell damage,the degree of injury dependented with concentration of LPS.3.LPS has a pro-apoptotic effect on MAC-T cellsDNA ladder results showed that MAC-T cells' DNA electrophoresis appeared "ladder" band after LPS-treated,and the DNA of cell cultures presented classic Laddering pattern by PDTC pretreatment;RT-PCR results showed that LPS could significantly up-regulate the expression of Bax and caspase-4 m RNA and down-regulate the expression of Bcl-2,but had little effect on caspase-3 in MAC-T cells;PDTC pretreatment reduced the effect of LPS on apoptotic molecules.4.LPS activates the inflammatory response of MAC-T cells.RT-PCR and Western blot results showed that the expression of the TLR4/NF-?B signaling pathway key genes(NF-?Bp50,NF-?Bp65,TLR4,IRAK4,My D88,IRF3)and downstream signaling molecules(TNF?,IL-6,IL-8)increased with the addition of LPS.Indicating that LPS can activate the TLR4/NF-?B signaling pathway and cause cellular inflammatory response.5.The role of NF-?B in LPS-induced inflammatory response and apoptosisLPS-induced signaling pathway key genes(NF-?Bp50,NF-?Bp65,TLR4,IRAK4,My D88,IRF3)and downstream gene expression were significantly altered by PDTC(NF-?B inhibitor)pretreatment.Compared with LPS group,the transcriptional levels of NF-?B,TLR4,IRAK4,My D88,IRF3,TNF? and IL-6 in PDTC group were decreased,and the expression of Bax and Bcl-2 were opposite,but IL-8 and caspase-3transcription levels rised further.Protein expression levels also validated changes in transcription levels.IRF3 protein appeared two immunoblottings(47 k Da and 54 k Da).The 54 k Da band for IRF3 protein was increased in MAC-T cells after LPS treatment and decreased by PDTC pretreatment.However,a 47 k Da-sized band for IRF3 protein was decreased after LPS treatment and further decreased more by using PDTC preprocessing.