| This study was to explore the effect of taurine on the improvement of mammary gland metabolic disturbances induced by Streptococcus uberis（S.uberis）infection,and to further study the regulation of taurine on energy metabolism and its molecular mechanism during these processes in mouse mammary epithelial cells.We aimed to provide a new strategy for the prevention and control of S.uberis and similar bacterial infection targeting immunometabolism.1.The effect of taurine on mammary gland metabolism in S.uberis infectionTaking C57BL/6 mice as the research object,the metabolites of mammary gland were determined by metabonomics,and the expressions of key enzymes in related metabolic pathways were verified by fluorescence quantitative PCR.The results showed that compared with the control group,S.uberis infection significantly increased the contents of metabolites related glucose metabolism,amino acids or amino acid derivatives and other metabolites in the mammary gland（P<0.05）,and affected the pentose phosphate pathway,pantothenic acid and Co A biosynthesis,arginine and proline metabolism,glycine,serine and threonine metabolism,valine,leucine and isoleucine biosynthesis,the metabolic pathways of glutathione metabolism and aminoacyl-t RNA synthesis,as well as upregulated the gene expression of key enzymes of glycolysis（HK,PFK1）,key enzymes of pentose phosphate pathway（G6PDH）,key enzymes of tricarboxylic acid cycle（PDH,SDH）,key enzymes of fatty acid metabolism（（LIPA））and factors（PPAR-γ）.Taurine pretreatment changed the pathways of glycerol ester metabolism,fatty acid and unsaturated fatty acid synthesis during infection,down-regulated the gene expression of key enzymes of energy metabolism（HK,PFK1,PDH,SDH）,lipid oxidation key enzyme（LIPA）and factor（PPAR-γ）,and promoted the gene transcription of key enzyme of pentose phosphate pathway（G6PDH）.The results showed that 100 mg kg-1taurine could reduce the energy metabolism（glucose metabolism,fat metabolism）in infected tissues,promote the biosynthesis of unsaturated fatty acids and alleviate the metabolic disturbances of mammary gland induced by S.uberis.2.Taurine alleviates the metabolic disturbances in S.uberis challenged mammary gland epithelial cellsIn order to explore the metabolic changes induced by S.uberis infection of mammary epithelial cells and the alleviating effect of taurine on the metabolic disturbances of infected mammary epithelial cells,the mouse mammary epithelial cell line（Ep H4-Ev）was used as the research object.The cells were pretreated with 45 mmol·L-1taurine for24 h,then S.uberis was added to infect the cells for different time.The samples were collected for metabonomic detection.Then inactivated S.uberis was used to stimulate Ep H4-Ev cells,LPS and inactivated E.coli were used as controls,for further confirming the metabolic pattern through Seahorse analysis,and the metabolic responses of Ep H4-Ev cells and RAW264.7 cells under the same stimulation were compared.Finally,the effect of taurine on cell energy metabolism stimulated by S.uberis was detected.The results showed that the effect of S.uberis infection on the metabolism of Ep H4-Ev cells changed in a time-dependent manner.S.uberis infected for 1 h only increased several metabolites including glucose 6-phosphate in Ep H4-Ev cells（P<0.05）.2 h-or 3 h-S.uberis infection upregulated glycolysis-related metabolites,lipid-related metabolites,10amino acids and other carbohydrates in Ep H4-Ev cells,along with the production of itaconic acid（P<0.05）,while decreased the levels of citric acid andα-ketoglutaric acid（P<0.05）,resulting in metabolic imbalance in Ep H4-Ev cells.Pathway enrichment analysis showed that S.uberis enhanced amino acid and nucleic acid metabolism and promoted aminoacyl-t RNA biosynthesis in the early stage of infection.With the aggravation of infection,the pathways related to energy metabolism such as tricarboxylic acid cycle,pyruvate metabolism and glycolysis were affected.A lot of amino acids were metabolized,and biosynthesis reactions were enhanced,increased consumption of intracellular antioxidants.The glycolysis capacity,mitochondrial respiratory capacity and reserve respiratory capacity of Ep H4-Ev cells were significantly increased by inactivated S.uberis or E.coli stimulating,which was consistent with the energy metabolic response of RAW264.7 cells stimulated by inactivated S.uberis or E.coli.LPS stimulation inhibited the ability of mitochondrial respiration and reserve respiration of RAW264.7 cells,and increased the capacity of glycolysis,while in Ep H4-Ev cells,LPS stimulation led to the enhancement of glycolysis and mitochondrial respiration at the same time.These results suggested that the energy metabolism pattern of mammary epithelial cells stimulated by microorganisms was different from that of immune cells and was influenced by both host and pathogen.Compared with S.uberis infection,taurine can effectively improve the metabolic disturbances of S.uberis infection.Taurine pretreatment decreased the levels of glucose metabolism-related metabolites and amino acids in cells,and increased the synthesis of anti-infective lipids in infected cells.Metabolic pathway analysis showed that taurine pretreatment improved energy metabolism,amino acid metabolism,increased anti-infective metabolites production.The analysis of energy metabolism showed that taurine pretreatment could significantly reduce the glycolysis capacity,mitochondrial respiration capacity and reserve respiration capacity of Ep H4-Ev cells stimulated by inactivated S.uberis 0140J（P<0.05）.The activities of key enzymes of glycolysis and oxidative phosphorylation,and key enzymes of amino acid conversion to energy substances were down-regulated（P<0.05）.These results showed that S.uberis infection can lead to metabolic imbalance of mammary gland epithelial cells and show complex metabolic changes,which was the result of the interaction of many factors between the pathogen and the host.Taurine can alleviate the metabolic disturbances of mammary gland epithelial cells induced by S.uberis infection,reduce the pressure of energy metabolism and maintain the metabolic homeostasis of cells.3.Taurine alleviates inflammation induced by Streptococcus uberis infection by reducing energy metabolic fluxIn order to explore the role of energy metabolism in mammary gland epithelial cells against S.uberis infection,Ep H4-Ev cells were pretreated with taurine,2-DG（competitive inhibitor of hexokinase）and CPI-613（specific inhibitor of pyruvate dehydrogenase）.The levels of inflammatory factors,reactive oxygen species and cell damage were detected by ELISA,flow cytometry,biochemical kit and other techniques or methods.The results showed that taurine could regulate the key enzymes of energy metabolism（glycolysis and oxidative phosphorylation）,effectively reduce the content of TNF-αand IL-1βand the level of ROS during infection,and decreased the activity of NAGase in the supernatant of cell culture.2-DG treatment significantly decreased the contents of TNF-αand IL-1βsecreted by Ep H4-Ev cells infected with S.uberis 0140J,inhibited the production of ROS and decreased the activity of NAGase in the culture supernatant.The co-treatment of 2-DG and taurine showed the effect of further relieving inflammation and injury（P<0.05）.It was suggested that glycolysis activity was closely related to the inflammation induced by S.uberis 0140J infection.On the contrary,when Ep H4-Ev cells were treated with CPI-613,the secretion of TNF-αand IL-1βin S.uberis0140J infection was further increased（P<0.05）,the content of ROS and NAGase activity in the supernatant of S.uberis 0140J infection were further increased（P<0.05）.In addition,CPI-613 blocking oxidative phosphorylation could antagonize the anti-infective effect of taurine and reverse the relieving effect of taurine on inflammation and injury.It could be seen that the maintenance of oxidative phosphorylation limited the inflammation induced by S.uberis 0140J infection.Therefore,taurine can appropriately reduce the level of glycolysis and oxidative phosphorylation of cells,balance the whole energy metabolism in S.uberis infection and alleviate the inflammation induced by infection without destroying the oxidative phosphorylation of cells.4.Taurine attenuates inflammation induced by S.uberis infection by reducing energy metabolic flux through AMPK-mTOR panthwayIn order to explore the mechanism of taurine regulating energy metabolism in S.uberis infection,Ep H4-Ev cells were pretreated with 45 mmol·L-1taurine for 24 h,then pretreated with 100 nmol·L-1MHY1485（mTOR activator）for 24 h or 10μmol·L-1Compound C（AMPK inhibitor）for 1 h,followed by S.uberis 0140J infection for 3 h.The related indexes were determined by Western blot,Seahorse cell energy metabolism analysis,biochemical kit,ELISA,flow cytometry and so on.The results showed that taurine pretreatment inhibited the phosphorylation level of mTOR and its downstream target proteins p70S6K and 4E-BP1 in S.uberis 0140J infection.MHY1485 treatment further increased the phosphorylation level of mTOR signal related proteins during infection,and correspondingly increased the glycolysis ability,mitochondrial respiration ability and reserve respiration ability of S.uberis 0140J infected cells.The activities of key enzymes of glycolysis（HK,PFK,LDH）（P<0.05）and key enzymes of oxidative phosphorylation（PDH,SDH,Complex IV）（P<0.05）were enhanced.The co-treatment of MHY1485 and taurine reversed the regulatory effects of taurine on mTOR signal pathway and mTOR-mediated energy metabolism in infected cells.We found that taurine could promote the phosphorylation of AMPK,inhibit the phosphorylation of downstream mTOR and mTOR signal related proteins,and decrease the glycolysis capacity,mitochondrial respiratory capacity and reserve respiratory capacity in S.uberis infection.Inhibiting the activities of key enzymes of glycolysis and oxidative phosphorylation,thus reduce the secretion of TNF-αand IL-1β,the production of ROS and the activity of NAGase in the supernatant.However,Compound C treatment had a reverse effect on the above-mentioned indexes and had an antagonistic effect on taurine（P<0.05）.These results showed that taurine can activate AMPK,to promote the phosphorylation of AMPK,inhibit the mTOR pathway and its mediated inflammatory energy metabolism phenotype,reduce the secretion of inflammatory factors and alleviate cell damage.Overall,S.uberis infection led to metabolic disturbances in mammary gland and mammary epithelial cells.In order to against infection,mammary epithelial cells regulated their metabolism to meet the needs of innate immunity,but continuous metabolic intension also caused metabolic disorders and excessive inflammation.Taurine pretreatment alleviated the metabolic disturbances of mammary gland and mammary gland epithelial cells.Taurine can effectively inhibit the overall energy metabolism by increasing the level of AMPK phosphorylation and reducing the activity of mTOR pathway,thus significantly reducing mammary gland epithelial cells inflammation induced by S.uberis.The development of the study will provide a theoretical basis for the application of nutrient elements to regulate metabolic immunity to against S.uberis infection,and provide a new strategy for the prevention and control of mastitis in dairy cows. |
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