| In this study,the bovine mammary epithelial cell line(MAC-T)was studied as the object.Based on the co-incubation model of S.uberis 0140J and MAC-T,the effects of taurine on the inositol phospholipid-Ca2+ signaling pathway and inflammatory cell response induced by S.uberis 0140J infection were investigated.The results were as follows.1 Effects of taurine on related signaling pathway mediated by inositol phospholipid-Ca2+system and inflammatory responseTo investigate the effect of taurine on the signaling pathway mediated by the inositol phospholipid-Ca2+system and inflammatory response in MAC-T in S.uberis 0140J infection,β-alanine was used as a taurine uptake inhibitor.Cells were divided into 8 groups due to different treatments,which were control,S.uberis,taurine,S.uberis+taurine,β-alanine,β-alanine+taurine,βalanine+S.uberis,β-alanine+taurine+S.uberis,respectively.Fistly,flow cytometry was used to detect the change of Ca2+in MAC-T cells after S.uberis 0140J infection.Secondly,including enzyme linked immunosorbent assay(ELISA),western blot(WB),fluorescent technology were used for detecting the changes of inositol phospholipid content,Ca2+ signaling-related protein expression and nuclear transcription factor activity.Then,oxidant damage and cell injury were evaluated by flow cytometry and chemical kits.Finally,protein chip technology was used to detect the changes of downstream inflammatory cytokines and the level of inflammatory cytokines(TNF-α、IL-1β、IL-6 and IL-8)were determined by qRT-PCR and ELISA.The results showed that after two hours’ infection with S.uberis 0140J,the expression of PIP3 and IP3 in MAC-T were significantly increased(P<0.05)and the expression of PIP2 were significantly decreased(P<0.05).An opposite result took place when the above MAC-T was pre-treated with taurine(P<0.05).S.uberis 0140J could prompt the nuclear transcription factor(NF-κB and NFAT)entering the nucleus from the cytoplasm(P<0.05),and taurine greatly inhibited the occurrence of this situation(P<0.05).The mRNA levels of TNF-α,IL-1β and IL-6 were increased significantly(P<0.05)after the infection of S.uberis 0140J,however,pre-treatment with taurine significantly reduced their expression levels(P<0.05).Three hours after MAC-T infected with S.uberis 0140J,the intracellular Ca2+concentration was significantly increased(P<0.05),while the decline happened after the pre-treatment with taurine(P<0.05).Four hours after MAC-T infected with the S.uberis 0140J,protein expression level and the phosphorylation level of those related proteins(PLCyl,PI3K,PTEN,PKCα,IKKα,IκBα,GSK-3β)were obviously increased(P<0.05),and pre-treatment with taurine had a crosscurrent(P<0.05);the activity of CaN and the level of CaM had the same increasing tendency(P<0.05).In addition,the intracellular ROS content,the content of NO,the activity of iNOS,the activity of NAGase and the activity of LDH were increased significantly(P<0.05),which were obviously reduced after the pre-treatment with taurine(P<0.05).The protein levels of TNF-α,IL-1β and IL-6 were increased significantly(P<0.05)and pre-treatment with taurine significantly reduced their expression levels(P<0.05).40 cytokines in the cell culture supernatant were further detected,and the level of most cytokines(24/40)were higher than the blank control group(fold change>1).After pre-treatment with taurine(33/40),most of the cytokines were lower than the infected group(Fold change<1).The results showed that taurine could reduce the transition between the phospholipid inositol components,the intracellular Ca2+ concentration,the expression of Ca2+-related signaling protein,nuclear transcription factor activity,oxidation,injury and downstream secretion of related inflammatory factors in S.uberis 0140J infection.This suggested that the mechanism of taurine against S.uberis 0140J infection was closely related to the inflammatory pathways mediated by phospholipid inositol-Ca2+signaling.2 Anti-inflammatory effect of taurine after inhibition of Ca2+mobilization or NF-κB、NFAT transcriptional activity via RNAiTo investigate the role of taurine on S.uberis 0140J infection in MAC-T cells,small interference RNA(siRNA)interfereing with the key protein expression(siPLC,siCaN,siPKC)were used to inhibit Ca2+mobilization or NF-κB,NFAT transcriptional.Cells were divided into 18 groups due to different treatments,which were control,S.uberis,taurine,S.uberis+taurine,MOCK,NC,siPLC,siPLC+taurine,siPLC+S.uberis,siPLC+S.uberis+taurine,siCaN,siCaN+taurine,siCaN+S.uberis,siCaN+S.uberis+taurine,siPKC,siPKC+taurine,siPKC+S.uberis,siPKC+S.uberis+taurine,respectively.Firstly,qRT-PCR was used to determine the effective siRNA and flow cytometry was used to detect the changes of Ca2+and ROS in MAC-T cells after S.uberis0140J infection.Secondly,the level of pro-inflammation cytokines(TNF-α、IL-1β and IL-6)were detected by ELISA kits.Finally,the cell injury indicators were determined by chemical kits.We found that the siRNA with the best interference effects were siPLC3,siCaN2 and siPKC3,respectively.After disturbing the expression of PLC and CaN,the Ca’+concentration was decreased significantly(P<0.05)with S.uberis0140J for 3 h.Pre-treated with taurine further reduced it significantly(P<0.05)after disturbing the expression of PLC,CaN and PKC.After disturbing the expression of PLC,CaN and PKC,intracellular ROS content,the level of pro-inflammation cytokines(TNF-α、IL-1β and IL-6),the activity of NAGase and the activity of LDH were significantly decreased(P<0.05)with S.uberis 0140J infection for 4 h,which would further significantly decreased(P<0.05),if pre-treated with taurine.These results showed that interfering with key proteins expression to inhibit Ca2+mobilization or inhibition of NF-κB,NFAT transcriptional activity can obviously reduce the intracellular Ca2+ and ROS level,the secretion of pro-inflammatory cytokines,the activity of NAGase and the activity of LDH in S.uberis 0140J infection.Futhermore,pre-trearment with taurine can further reduce them.These results provided a supplement to the mechanism of S.uberis 0140J infection mediated by taurine through phospholipid inositol-Ca2+signaling.3 Anti-inflammatory effect of taurine after inhibition of Ca2+mobilization or NF-κB、NFAT transcriptional activity with inhibitorsInhibitors(U 73122,FK 506 and RO 31-8220)were used to inhibit Ca2+mobilization or NF-κB,NFAT transcriptional.Cells were divided into 18 groups due to different treatments,and it is control,S.uberis,taurine,S.uberis+taurine,DMF,U 73122,U 73122+taurine,U 73122+S.uberis,U 73122+S.uberis+taurine,FK 506,FK 506+taurine,FK 506+S.uberis,FK 506+S.uberis+taurine,RO 31-8220,RO 31-8220+taurine,RO 31-8220+S.uberis,RO 31-8220+S.uberis+taurine,respectively.Firstly,flow cytometry was used to detect the changes of Ca2+and ROS in MAC-T cells after S.uberis 0140J infection.Secondly,the level of pro-inflammation cytokines(TNF-α、IL-1β and IL-6)were detected by ELISA kits.Finally,the cell injury indicators were determined by chemical kits.These results showed that the addition of inhibitors to inhibit Ca2+mobilization or NF-κB,NFAT transcriptional activity can obviously reduce the intracellular Ca2+and ROS level,the secretion of pro-inflammatory cytokines,the activity of NAGase and the activity of LDH in S.uberis0l40J infection.Moreover,pre-trearment with taurine can further reduce them.These results which were consistent with the above results,further illustrated that the mechanism of taurine on anti-inflammatory was mediated by phospholipid inositol-Ca2+signaling pathway. |
