| In this study,taurine,Streptococcus uberis 0140J(S.uberis 0140J),mouse mammary epithelial cells(MECs)and bovine MECs were studied as the objects to explore the effects and mechanism of taurine on lipid metabolism and lipid droplet(LD)disorder caused by S.uberis infection.We observed changes in lipid metabolism-related indicators and related protein,enzyme and LD levels in signal pathways in two different cell lines to clarify that taurine regulates LD levels by influencing lipid metabolism pathways to relieve S.uberis infection.This study provides new cogitation and methods for the prevention and control of S.uberis and other similar pathogen infection from the perspective of immunometabolism.The results are as follows:1.The role of cellular LDs in MECs infected by S.uberisAs an important energy storage organelle,LDs participate in many biological events and play an important role in pathogenic infection.MECs are not only the most important cells in mammary gland,but also the first defensive line of protective barrier in mammary gland.In order to explore the role of cellular LDs in the infection of MECs by S.uberis,this study used mouse MEC line(Ep H4-Ev),bovine MEC line(MAC-T)and S.uberis as research targets.We used taurine(a small metabolic regulatory molecule),oleic acid(to promote cell LD synthesis),C75(FASN inhibitor),bacterial plate culture,transmission electron microscopy,flow cytometry and other techniques or methods to detect the relationship between the level of cellular LDs and the amount of intracellular bacteria.The results showed that transmission electron microscopy observed direct interaction between S.uberis and LDs,and treatment with taurine or C75 significantly reduced the LD increase,and they also effectively reduced the bacterial load(P<0.05).However,pretreatment with oleic acid significantly increased the level of intracellular LDs and resulted in a significant increase in bacterial load(P<0.05).These results indicate that the level of cellular LDs plays an important role in S.uberis infection,and taurine may relieve bacterial infection by reducing the level of LDs.2.The effect of taurine on the disorder of lipid metabolism and LD levels in MECs caused by S.uberis infectionIn order to explore the effect of taurine on the lipid metabolism and LD level disorders caused by S.uberis infection,taurine,mouse MEC line(Ep H4-Ev)and bovine MEC line(MAC-T)were used as the research objects by observing or measuring related indicators through biochemical kits,flow cytometry,fluorescent quantitative PCR,projection electron microscope and other methods.The results showed that transmission electron microscope observation revealed that S.uberis infection caused a sharp increase in the number of LDs in Ep H4-Ev and MAC-T cells,and the quantitative analysis of flow cytometry further showed that the level of LDs in the host increased significantly(P<0.05).Compared with the control group,S.uberis significantly increased the expression level of genes related to lipid synthesis pathway(Fasn,Scd1,Srebf1)and the ACC activity(P<0.05)in Ep H4-Ev cells,and significantly reduced the expression level of genes related to lipid oxidation(Acox1,Ehhadh)(P<0.05),but had no significant effect on genes involved in fatty acid absorption(Cd36)(P>0.05).In addition,taurine pretreatment significantly reduced the level of LDs in Ep H4-Ev and MAC-T cells,as well as Fasn,Scd1,Srebf1 gene expression levels and ACC activity in Ep H4-Ev cells(P<0.05).However,it had no significant effect on Acox1,Ehhadh and Cd36 expressions(P>0.05).Furthermore,45 m M taurine pretreatment for 24 h significantly reduced the enhancement of lipid synthesis pathways and the increase of LDs in MECs caused by S.uberis infection.3.Taurine inhibits IDOL signaling pathway to relieve the surge of LDs in MECsIn order to further explore the mechanism of taurine alleviating the enhancement of lipid metabolism and the increase of LDs caused by S.uberis infection,the experiment used mouse MEC line(Ep H4-Ev)and bovine MEC line(MAC-T)as the research objects.The cells were pretreated with 45 m M taurine for 24 h,5 μM Fatostatin(SREBPs inhibitor)for 24 h,1 μM GW3965(LXRs agonist)for 24 h;IDOL-specific interfering RNA pretreated the cells for 48 h,and then infected with S.uberis for 3 h to collect samples.Western blot,RT-PCR,biochemical kit,immunofluorescence,confocal microscopy,flow cytometry and other methods were used to determine the indicators related to fatty acid metabolism pathway and inflammation level.The corresponding inhibitors or activators were used to evaluate the LD level changes,so as to explore the target protein of taurine.Finally,the expression of Idol was specifically interfered to detect its effect on the protein expression of lipid metabolism pathway and the content of LDs.The results showed that S.uberis infection significantly increased the expression levels of SREBP1(closely related to fatty acid synthesis)and LDLR(low density lipoprotein receptor)proteins in Ep H4-Ev cells,and significantly decreased the expression levels of LXR(upstream protein of SREBP1),ABCA1(downstream protein of LXR)and IDOL(intermediate linking SREBP1 with LXR)proteins(P<0.05).Besides,S.uberis significantly increased the m RNA expression levels of related inflammatory genes(Cxcl9,Nos2,Tnf,Il1 b,Il6)and the content of inflammatory factors(TNF-α,IL-1β,IL-6)(P<0.05).Compared with S.uberis infection group,taurine pretreatment further significantly reduced SREBP1 and IDOL protein expression levels in Ep H4-Ev cells(P<0.05);significantly increased LXR and ABCA1 protein expression levels(P<0.05).Taurine significantly reduced the m RNA expression of genes related inflammation and the content of inflammatory factors(P<0.05).Compared with the control group,si Idol treatment significantly reduced the expression of SREBP1 protein and its downstream proteins in Ep H4-Ev cells infected with S.uberis(P<0.05),significantly increased the protein expression of LDLR,LXR and ABCA1(P<0.05).Moreover,si Idol reduced the increase in LDs caused by S.uberis,and the amount of intracellular bacteria also decreased(P<0.05).Altogether,these results indicate that taurine inhibits the expression of proteins related to downstream lipid synthesis pathways by regulating the IDOL signaling pathway,and alleviates the surge of LDs caused by S.uberis infection in MECs.In summary,taurine inhibits the expression of IDOL,reduces the expression of lipid synthesis-related proteins,and alleviates the increase in LDs caused by S.uberis infection,thereby reducing the amount of intracellular bacteria.This study provides a theoretical basis for applying nutritional elements to regulate the host metabolic immunity to resist bacterial infection,and provide new ideology and methods for the prevention and control of mastitis in animals. |
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