| Mastitis is a complex,multi-causal disease,with bacterial infection being the main cause.The disruption of the integrity of the blood-milk barrier by mammary inflammation affects the function of lactation.Taurine,as a natural physiological immunomodulator,has anti-inflammatory,antioxidant and metabolic regulating functions.Previous research by our group has shown that taurine can alleviate mammary inflammation in model animals and inflammatory damage to mammary epithelial cells in Streptococcus uberis（S.uberis）infection,suggesting its potential application in the prevention and control of bovine mastitis.Based on this,this study investigated the effects of taurine on performance,blood parameters and mastitis in lactating cows,and the protective effect of taurine on blood-milk barrier after S.uberis infection and its mechanism of action.The study provides a scientific basis and theoretical support for the application of taurine in healthy dairy farming and offers new ideas and methods for the prevention and control of S.uberis and other bacterial mastitis.Details of the research and results are as follows.1.Effect of taurine on performance,blood parameters and incidence of subclinical mastitis in lactating cowsIn order to investigate the effects of taurine on performance,blood parameters and incidence of subclinical mastitis of lactating dairy cows,32 healthy holstein dairy cows were randomly divided into 4 groups with 8 cows in each group,which were control group and each cow was fed with 30 g（low）,60 g（medium）and 90 g（high）taurine groups for 60 days,respectively.At the 15th,30th,45th and 60th days of the experiment,milk yield and the prevalence of mastitis was recorded and milk samples were collected for milk composition analysis.Venous blood was collected at the 30th and 60th days to determine blood routine,serum biochemical indices and antioxidant indices.The results showed as follows:The prevalence of subclinical mastitis was 37.5%（3/8）in the control group of cows during the trial and decreased to 12.5%（1/8）in both the 60 and 90 g/head taurine supplemented groups.Compared to the control group,milk somatic cell count（SCC）and serum levels ofβ-HB,non-esterified fatty acids（NEFA）and malondialdehyde（MDA）were significantly reduced in the 60 g/head taurine supplemented group,while the number of blood lymphocyte（Lym）,total protein（TP）,albumin（ALB）and globulin（GLB）,total antioxidant capacity（T-AOC）,glutathione（GSH）levels and superoxide dismutase（SOD）activity in serum were significantly increased（P<0.05）;in the 90 g/head taurine supplemented group,SCC and levels ofβ-HB,NEFA and MDA in serum were significantly decreased,while the blood Lym count,haemoglobin and red blood cell specific volume,TP,GLB,T-AOC,GSH levels and SOD activity were significantly increased（P<0.05）.In addition,milk production increased to some extent in all taurine supplemented groups.These results suggest that dietary supplementation of taurine can significantly reduce milk somatic cell count,regulate metabolic balance and play an antioxidant role in lactation,and thus reduce the incidence of subclinical mastitis.2.Effect of taurine on the blood-milk barrier in mice mastitis caused by S.uberisThe integrity of the blood-milk barrier is closely related to mammary gland health.The inflammatory response caused by infection can damage the blood-milk barrier.In order to explore the effect of taurine on the blood-milk barrier in mammary glands of mice infected with S.uberis 0140J,C57BL/6 mice were studied by gavage of 200 mg/kg of taurine or the corresponding volume of sterile saline daily after 14 days of pregnancy.The female mice were gavaged with S.uberis（S.uberis,S.uberis+Taurine）or the corresponding volume of sterile saline（Control,Taurine）at the fourth pair of teats 48 h after delivery,and then decollated and executed after 24 h.The mammary tissue was collected.Related indexes such as inflammatory response,oxidative stress,NF-κB and MAPK signaling pathways,blood-milk barrier permeability and tight junction were observed or detected by pathological tissue sections,bacterial plate culture,biochemical kits,fluorescent quantitative PCR,Western blot and immunofluorescence sections.The results showed that taurine could alleviate mammary gland congestion and edema,as well as inflammatory cell infiltration and local necrosis in mammary tissues of mice caused by S.uberis infection;and taurine also curbed the infection-induced increase in MPO protein expression and activity,increase in MDA levels and decrease in T-AOC and GSH levels（P<0.05）.Taurine had a down-regulatory effect on the m RNA expression levels of TNF-α,IL-1β,IL-6,NOS2,COX2,CXCL1 and CXCL2,as well as the phosphorylation levels of IκB,p65,p38,ERK and JNK proteins（P<0.05）.Taurine also reduced FITC-albumin levels and neutrophil density in mammary gland follicles（P<0.05）and prevented S.uberis-induced decreases in levels of the tight junction proteins claudin3 and occludin（P<0.05）.In addition,albumin content in mammary gland follicles was positively correlated with neutrophil density（P<0.05）.The results suggest that taurine may inhibit the mammary inflammatory response and oxidative stress induced by S.uberis infection and the disruption of blood-milk barrier integrity and tight junctions by modulating NF-κB and MAPK signalling pathways.3.Effect of macrophages on neutrophil chemotaxis in S.uberis infection and the regulatory role of taurineNeutrophils are the main players in the inflammatory response,and their recruitment affects the integrity of the blood-milk barrier and the progression of mastitis.To investigate the role and mechanism of taurine in regulating mammary gland alveolar chemokine expression and neutrophil chemotaxis in S.uberis infection,this study was conducted with mouse mammary epithelial cell line（Ep H4-Ev cells）,mouse monocyte macrophage line（RAW264.7 cells）and neutrophils.The cells were treated accordingly by S.uberis infection as well as by taurine or BAY 11-70825（NF-κB inhibitor）,Losmapimod（p38 inhibitor）,SCH772945（ERK inhibitor）,SP600125（JNK inhibitor）or NG25（TAK1 inhibitor）.Transwell chemotaxis model,fluorescence quantitative PCR,western blot,flow cytometry and other methods were used to observe or measure the relevant indexes.The results showed that S.uberis infection induced a significant increase in CXCL2 m RNA expression in macrophages compared to the control group（P<0.05）.CXCL2 neutralizing antibody significantly inhibited the mobility,F-actin activation and phosphorylation of p38,ERK and AKT in neutrophils induced by supernatant of S.uberis and macrophages（P<0.05）.Taurine pretreatment significantly reduced the m RNA and protein expression levels of CXCL2 in S.uberis infected macrophage compared to the infected group（P<0.05）.The m RNA expression of CXCL2 was significantly downregulated by pretreatment with BAY 11-70825,Losmapimod,SCH772945 or SP600125（P<0.05）,and taurine significantly inhibited the phosphorylation of p65 as well as p38,ERK and JNK（P<0.05）.TLR2-containing antibody treatment significantly reduced the phosphorylation levels of p65,p38,ERK and JNK proteins in the infection（P<0.05）.Taurine was able to inhibit TAK1 activation（P<0.05）.NG25 had a similar inhibitory effect on phosphorylation levels of IKK,IκB,p65,p38,ERK and JNK proteins as taurine（P<0.05）.The results suggest that CXCL2 expression in macrophages induced by S.uberis drives neutrophil chemotaxis and that taurine is able to reduce CXCL2 expression in macrophages in infection by regulating NF-κB and MAPK signalling through inhibition of TAK1 activation.4.Effect of taurine on S.uberis-induced release of neutrophils extracellular trap networkExtracellular trap networks（NETs）is produced by neutrophils upon stimulation which is a double-edged sword of host infection defense system and can damage mammary epithelial cells.To investigate the mechanism by which S.uberis infection induces NETs release from neutrophils and the role of taurine.In this experiment,neutrophils were studied after S.uberis infection and treatment with taurine,NAC（ROS scavengers）,mito TEMPO（mitochondrial ROS scavengers）,DPI（NADPH oxidase inhibitors）,TLR2-containing antibody or BAY 11-70825（NF-κB inhibitor）,Losmapimod（p38 inhibitor）,SCH772945（ERK inhibitor）or SP600125（JNK inhibitor）.NETs morphology was then observed by SYTOX GREEN staining,and NETs release was detected by luminescence zymography.Intracellular ROS levels were detected using flow cytometry,and TLR2 protein expression levels and protein phosphorylation levels of TAK1,p38,ERK,JNK and p47phox were detected using Western blot.The results showed that S.uberis infection for 2 h resulted in a significant increase in the release of neutrophil NETs（P<0.05）.NETs treatment at 200 ng/m L and 400 ng/m L significantly reduced the viability of mammary epithelial cells and resulted in increased LDH activity in cell supernatants compared to control（P<0.05）,while DNase?treatment increased cell viability and reduced LDH activity levels in cell supernatants compared to the400 ng/m L NETs treatment group（P<0.05）.400 ng/m L NETs had no significant effect on the proliferation of S.uberis（P>0.05）.S.uberis infection significantly increased ROS levels in neutrophils compared to controls.Pretreatment with NAC,mito TEMPO and DPI significantly inhibited the increase in intracellular ROS and NETs levels in the supernatant caused by infection（P<0.05）,and DPI was significantly more effective than mito TEMPO（P<0.05）.S.uberis infection caused a significant increase in the phosphorylation levels of p38,ERK and JNK in neutrophils compared to the control group（P<0.05）.Pretreatment with BAY 11-70825,Losmapimod,SCH772945 or SP600125 significantly reduced cellular p47phox phosphorylation levels and ROS levels compared to the S.uberis-infected group（P<0.05）.TLR2-containing antibodies treatment significantly downregulated cellular p38,ERK,JNK and p47phox protein phosphorylation levels and the levels of NETs in the supernatant（P<0.05）.15 m M and 45 m M taurine pretreatment significantly reduced cellular p38,ERK and JNK protein phosphorylation levels and NETs content in supernatant in the infections（P<0.05）.TAK1 phosphorylation levels were significantly increased in the S.uberis-infected group compared to the control group.Compared with the S.uberis-infected group,taurine pretreatment significantly inhibited the phosphorylation level of TAK1（P<0.05）.The effect of NG25 was consistent with that of taurine which reduced p38,ERK,JNK and p47phox protein phosphorylation levels.These results suggest that taurine inhibits NADPH oxidase activation and ROS production in neutrophils caused by S.uberis infection by modulating the TAK1/MAPK signaling pathway,thereby reducing the release of NETs and contributing to the protection of mammary epithelial cells and barriers from NETs damage.5.Effect of taurine on hypochlorous acid-induced mammary epithelial cells and barrier damageHypochlorous acid（HCl O）is the main production of the MPO-halide system of neutrophils,and its strong oxidative capacity is one of the most important factors contributing to tissue damage.In order to investigate the protective effect of taurine on mammary epithelial cells under HCl O stimulation,this experiment was conducted on mouse mammary epithelial cell line（Ep H4-Ev cells）.After pretreatment with HCl O stimulation and taurine,Compound C（AMPK inhibitor）or ML385（Nrf2 inhibitor）,cell activity was measured by CCK8,supernatant LDH and cellular MDA levels by kits,and AMPK protein phosphorylation levels and antioxidant-related proteins Nrf2,HO-1,NQO1 and the expression levels of the tight junction proteins ZO-1,ocludin and claudin3 were measured by Western blot.The mammary epithelial barrier was constructed using Transwell and the permeability of FITC-dextran was measured using a fluorescent enzyme marker.The results showed that HCl O above 200μM led to a significant decrease in mammary epithelial cell viability,a significant increase in supernatant LDH activity and in cellular MDA levels compared to the control group（P<0.05）.Taurine pretreatment significantly reduced LDH activity in cell supernatant and cellular MDA levels compared to the HCl O-treated group（P<0.05）.Compared with the control group,taurine pretreatment increased the protein phosphorylation level of AMPK and protein expression of Nrf2 in mammary epithelial cells（P<0.05）.Compared with the 45 m M taurine treatment group,Compound C pretreatment significantly decreased the protein phosphorylation level of AMPK and protein expression of Nrf2,and either Compound C or ML385 pretreatment significantly down-regulated the expression of antioxidant-related proteins HO-1 and NQO1（P<0.05）.Compared to the control group,HCl O stimulation significantly increased MDA content and supernatant LDH activity in cells（P<0.05）and led to a significant increase in mammary epithelial barrier permeability and a significant decrease in the expression of the tight junction proteins ZO-1,occludin and claudin3（P<0.05）.Compared with HCl O treatment group,taurine pretreatment reduced MDA content and LDH activity of supernatant,and restored the permeability of mammary epithelial barrier and the expression of ZO-1,occludin and claudin3 proteins（P<0.05）.Compound C or ML385 had antagonistic effect on taurine（P<0.05）.The above results suggest that the mitigating effects of taurine on HCl O-stimulated mammary epithelial cell injury,oxidative stress,and disruption of the tight junctions and increased permeability of the mammary epithelial barrier are associated with activation of AMPK/Nrf2 antioxidant signalling.In summary,taurine supplementation significantly reduced SCC in lactating cows,modulated immunity and metabolic homeostasis and improved host antioxidant capacity,reducing the incidence of cryptogenic mastitis.Taurine may inhibit macrophage-induced neutrophil chemotaxis,reduce neutrophil ROS and NETs production,and activate AMPK/Nrf2 antioxidant signalling in mammary epithelial cells to alleviate the inflammatory response and oxidative stress caused by S.uberis,thereby maintaining the integrity of the blood-milk barrier and tight junctions.The study suggests that taurine has good potential for clinical application in dairy cattle. |
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