Effects Of IE-DAP On Inflammatory Response And Tight Junction In The Mammary Gland Of Ruminants And Regulation Study Of Sodium Butyrate

In modern animal husbandry,ruminants are often fed diets with high concentrate(HC)diet with high nutritional density in order to maximize milk yield and growth performance efficiency.However,HC diet lacks effective crude fiber and contains a high proportion of decomposable carbohydrates,which can easily induce subacute ruminant acidosis(SARA).During SARA,bacterial endotoxins produced in the rumen,such as γ-D-glutamyl-mesodiaminopimelic acid(iE-DAP),enter the blood circulation through the damaged rumen epithelium,reach the mammary gland,induce inflammatory reaction,and damage the health of the mammary gland as well as production performance.Nucleotide oligomerization binding domain protein 1(NOD1)is an important pattern recognition receptor(PRR)in innate immunity.iE-DAP is the smallest and core structure recognized by NOD1,which plays an important role in innate immunity and inflammatory response.As the most predominant cell type in the mammary gland,mammary epithelial cells can not only secrete milk,but also participate in the innate immune response of the mammary gland.Secondly,the tight junction(TJ)in the epithelial cells participates in the formation of the blood-milk barrier and plays an important role in maintaining the normal physiological function of the mammary gland.However,the occurrence of inflammation response of mammary gland can affect the expression and distribution of TJ proteins,destroy TJ structure and damage the health of mammary gland.Sodium butyrate(SB),as a green,safe and inexpensive feed additive,has attracted more and more attention because of its advantages such as anti-inflammation,protective barrier,enhancing immunity,regulating gastrointestinal microecological balance and so on.Under the condition of HC diet,the addition of SB can also play a protective role.In this paper,in vivo and in vitro experiments were conducted to explore the effects of HC diet and iE-DAP direct stimulation on the inflammatory response and tight junction of mammary gland and mammary epithelial cells,as well as the regulatory effects and involved mechanism of SB.Experiment Ⅰ.Effects of high concentrate diets on inflammatory response and tight junction in the mammary gland of dairy cowsHigh concentrate(HC)diet can induce subacute ruminant acidosis(SARA).During SARA,increased bacterial endotoxins,such as γ-D-glutamyl-meso-diaminopimelic acid(i EDAP),translocated into mammary gland via blood circulation,which triggers inflammatory response.The occurrence of mammary inflammatory response,causes tight junction(TJ)structure injury and damages mammary health and lactation performance.However,studies on the relationship between inflammatory response of the mammary gland induced by HC diet and TJ are limited.In order to study the effects of HC diet on inflammatory response and TJ and its related mechanism,12 lactating Holstein cows were randomly divided into 2groups: low concentrate(LC,n = 6)group and high concentrate(HC,n = 6)group.They were fed with LC diet(concentrate : forage = 40 : 60)and HC diet(concentrate : forage =60 : 40)for 3 weeks.The results showed that HC diet could lead to SARA,and the time of rumen p H < 5.6 was more than 3 h.Under the condition of SARA,the contents of inflammatory cytokines and chemokines in the plasma of lacteal vein was significantly increased,the relative mRNA expression of inflammation-related genes and the activity of myeloperoxidase(MPO)in mammary gland were significantly up-regulated.HE staining results showed increase inflammatory cells infiltration in the mammary gland of dairy cows fed HC diets.All these results indicated HC diet induced inflammatory response in the mammary gland of dairy cows.Western blotting and epigenetic analysis results showed the activation of nucleotide oligomerization binding domain protein 1(NOD1)/NF-κB signaling pathway,decreased DNA methylation ratio and chromatin compaction level at the promoter region of inflammatory cytokines,which contributed to the upregulation of inflammatory cytokines transcription.In addition,the effect of HC diet on TJ was further studied by analyzing the expression and distribution of TJ proteins.The results showed that the gene and protein expression levels of ZO-1,Occludin and Claudin-4 were significantly decreased,while the expression level of Claudin-1 was significantly increased in the mammary gland of dairy cows fed with HC diet.Compared with LC group,immunohistochemical staining results showed that the continuously expression of TJ proteins in mammary epithelial cells were damaged accompanied with decreased expression of ZO-1,Occludin and Claudin-4 and increased expression of Claudin-1.To be conclusion,our studies showed that HC diet induced the activation NOD1/NF-κB signaling pathway and epigenetic modifications,enhanced the transcriptional expression of proinflammatory cytokines and chemokines,and finally induced inflammatory response in the mammary gland of dairy cows.HC diet can also cause changes in the expression and distribution of TJ proteins,destroy the structure of TJ and damage the health and function of mammary gland.This study is helpful to provide a theoretical basis for maintaining the health of the mammary gland and improving production performance of lactation animals.Experiment Ⅱ.Effect of iE-DAP on inflammatory response and tight junction of bovine mammary epithelial cellsγ-D-glutamyl-meso-diaminopimelic acid(iE-DAP),a bacterial cell wall component,as a pathogen associated molecular pattern(PAMP),can be recognized by intracellular molecular pattern recognition receptor(PRR)-nucleotide oligomerization binding domain protein 1(NOD1)and trigger inflammatory response.The occurrence of inflammatory response of the mammary gland will cause the change of the expression and distribution of tight junction(TJ)proteins and destroy integrity of TJ.In order to study the effect of iE-DAPinduced inflammation on TJ in bovine mammary epithelial cells(BMECs)and the involved mechanism,the following experiments were carried out.First of all,the time gradient test of iE-DAP treatment was carried out.According to the changes of the relative mRNA expression of inflammatory cytokines and TJ proteins in BMECs after iE-DAP treatment,selected the treatment time 12 h.After that,the concentration gradient test of iE-DAP treatment was carried out.After being treated with different concentrations of iE-DAP for 12 h,the samples were collected for further detection.At the same time different inhibitors and short hairpin RNA(sh RNA)interference technique were used to explore the potential molecular mechanism.The content of inflammatory cytokines in the culture supernatant was detected by ELISA,the relative mRNA expression of inflammatory cytokines and TJ proteins in BMECs was detected by real-time quantitative PCR(RT-q PCR),the activation of NOD1/NF-κB and MLCK signaling pathway and the expression of TJ proteins in BMECs were detected by western blotting(WB),and NF-κB p65 translocation and TJ proteins expression and distribution were detected by cellular immunofluorescence.The results showed that iE-DAP could promote the expression of NOD1,increase the phosphorylation level of IκB and NF-κB p65,promote the translocation of NF-κB p65 into the nucleus,activate NF-κB signaling pathway,and up-regulate the expression of inflammatory cytokines IL-1β,IL-6 and IL-8 in BMECs.It also increased the protein expression of myosin light chain kinase(MLCK)and the phosphorylation level of myosin light chain 2(MLC2),activated MLCK pathway,downregulated the expression of ZO-1,Occludin and Claudin-4,up-regulated the expression of Claudin-1,changed the distribution of ZO-1 and Occludin in BMECs,destroyed the structure of TJ.ML-7,an inhibitor of MLCK,reversed the activation of MLCK signaling pathway,the changes of expression and distribution of TJ proteins induced by iE-DAP stimulation.BAY11-7085,an inhibitor of NF-κB,inhibited the activation of NF-κB and MLCK signaling pathways,the up-regulation of inflammatory cytokines,and the changes of expression and distribution of TJ proteins induced by iE-DAP stimulation.After transfection of NOD1-specific sh RNA,the gene and protein expression of NOD1 was significantly down-regulated.At the same time,the activation of NOD1/NF-κB and MLCK signaling pathway,the upregulation of inflammatory cytokines and the changes of expression and distribution of TJ proteins induced by iE-DAP stimulation were inhibited.These results suggest that iE-DAP can activate the NOD1-dependent NF-κB signaling pathway,promote the translocation of NF-κB p65 into the nucleus,enhance the transcriptional expression of inflammatory cytokines and induce inflammatory response.At the same time,NF-κB promotes the expression of MLCK and activates MLCK signaling pathway.The elevated inflammatory cytokines and the activation of MLCK signaling pathway changes the expression and distribution of TJ proteins in BMECs and destroys the structure of TJ.This study provides a research basis for further regulation of the inflammatory response in mammary gland.Experiment Ⅲ.Regulatory effect and mechanism of sodium butyrate on iE-DAPinduced inflammation and tight junction injury in bovine mammary epithelial cellsSodium butyrate(SB),as a bioactive substance,has become a research hotspot because of its anti-inflammatory and barrier protection properties.In order to explore the regulatory effects and mechanism of SB on inflammatory response and tight junction(TJ)injury of bovine mammary epithelial cells(BMECs)induced by γ-D-glutamyl-meso-diaminopimelic acid(iE-DAP)stimulation,we carried out the following experiments.First of all,according to the CCK-8 detection results of SB,0.1,0.5,1 and 2 m M SB were used for the follow-up test.During iE-DAP stimulation,different concentrations of SB were used for protection.The overexpression plasmid of histone deacetylase 3(HDAC3)was transfected to explore the role of HDAC3 in the regulation effects of SB during iE-DAP stimulation.The results showed that,compared with DAP group,SB decreased the gene and protein expression of HDAC3,increased the acetylation level of histone H3,decreased the phosphorylation level of IκB and NF-κB in BMECs stimulated by iE-DAP and the translocation of NF-κB p65 into nucleus,down-regulated the mRNA relative expression of inflammatory cytokines and the content in supernatant.At the same time,SB significantly decreased the level of myosin light chain kinase(MLCK)protein and the phosphorylation ratio of myosin light chain 2(MLC2)in BMECs induced by iE-DAP stimulation,up-regulated the expression of ZO-1,Occludin and Claudin-4,down-regulated the expression of Claudin-1,and restored the changes of fluorescence expression and distribution of ZO-1 and Occludin induced by iE-DAP stimulation.After transfection of HDAC3 overexpressed plasmid,the gene and protein expression of HDAC3 in BMECs were significantly increased,indicating that the HDAC3 overexpression plasmid was successfully constructed and can be used in follow-up experiments.The overexpression of HDAC3 significantly decreased the acetylation level of histone H3 induced by SB,reversed the inhibitory effect of SB on NF-κB signaling pathway,and hindered the down-regulation of SB on the expression of inflammatory cytokines.At the same time,transfection of HDAC3 overexpression plasmid reversed the inhibitory effect of SB on MLCK and eliminated the protective effect of SB on the change of the expression and distribution of ZO-1 and Occludin induced by iE-DAP stimulation.Taken above results into consideration,we can draw the conclusion that SB inhibits the phosphorylation of IκB and NF-κB p65 by inhibiting HDAC3,thus inhibiting the activation of NF-κB signaling pathway,reducing the translocation of NF-κB p65 into the nucleus,downregulating the transcriptional expression of inflammatory cytokines,and alleviating the inflammatory response induced by iE-DAP stimulation.At the same time,SB reversed the changes of TJ proteins expression and distribution induced by iE-DAP stimulation by reducing inflammatory cytokines and inhibiting MLCK/p-MLC2 signaling pathway.This study provides a theoretical basis for further exploring the regulation of the inflammation of mammary gland by SB in vitro.Experiment Ⅳ.Regulatory effects of sodium butyrate on inflammation and tight junction injury in the mammary gland of dairy goats fed with high concentrate dietHigh concentrate(HC)diet feeding will induce subacute ruminant acidosis(SARA),resulting in increased harmful bacterial endotoxins that can be transported to mammary gland through damaged rumen walls,induce inflammatory response,destroy the integrity of tight junction(TJ),and damage production performance and the health of mammary gland.Dietary supplementation of sodium butyrate(SB)can alleviate inflammation and improve animal health and production.In order to study the effects of dietary supplementation of SB on mammary inflammation and TJ damage induced by HC diet feeding,12 healthy mid-lactation Saanen dairy goats were randomly divided into HC group(concentrate : forage = 60 : 40)and sodium butyrate regulated group(BHC)(1% SB was added to HC diet,n = 6).The results showed that dietary supplementation of SB could enhance the buffering capacity of rumen and hinder the decrease of rumen p H value of dairy goats induced by HC diet.Compared with HC group,SB could inhibit the gene and protein expression of histone deacetylase 3(HDAC3),up-regulate the acetylation level of histone H3,decrease the protein abundance of NOD1,phosphorylated IκB,phosphorylated NF-κB p65 and the phosphorylated level of IκB,NF-κB p65 in the mammary gland of dairy goat.Dietary supplementation of SB could significantly reduce the concentration of inflammatory cytokines in lacteal vein plasma and the activity of myeloperoxidase(MPO)in mammary gland induced by HC diet,and downregulate the mRNA relative expression of nucleotide oligomerization binding domain protein1(NOD1)and other inflammation-related genes.At the same time,compared with HC group,dietary supplementation of SB could significantly reduce the protein expression of myosin light chain kinase(MLCK),phosphorylated myosin light chain 2(MLC2)and the phosphorylated ratio of MLC2 in the mammary gland of dairy goats,significantly up-regulate the gene and protein expression of ZO-1,Occludin and Claudin-4,and down-regulate the expression of Claudin-1.We concluded that during the period of SARA induced by HC diet,dietary supplementation of SB could enhanced the buffer ability of rumen,down-regulated the HDAC3 expression in mammary gland,inhibited the activation of NF-κB signaling pathway,decreased the MPO activity in mammary gland and down-regulated the concentration of inflammatory cytokines in the plasma of lacteal vein,finally relieved the inflammatory response in mammary gland.At the same time,decreased inflammatory cytokines concentration and inhibited MLCK signaling pathway can help to reverse the changes in TJ proteins expression induced by HC diet and protect the health of mammary gland.This study is helpful to provide theoretical basis for the application of SB in maintaining the health of mammary gland,improving production performance and its applications in production practice.