Effects And Regulation Mechanism Of Sodium Valproate On IE-DAP Induced Inflammatory Response In The Bovine Mammary Epithelial Cells

Mastitis is a major problem plaguing the pasture.It is an inflammatory disease of mammary gland caused by pathogenic microorganisms.It occurs widely in our country and even in the world.Innate immunity is the first line of defense.The pathogen invades the epithelium and endothelial cells and colonizes the mammary gland in the breast tissue,causing an immune response followed by more severe infection and inflammation.Gram-negative bacteria that cause breast infection can produce a large amount of iE-DAP,which can bind to the intracellular receptor NOD1,activate the downstream NFκB and MAPK signaling pathways,regulate the expression of inflammation-related genes and trigger the inflammatory response in the breast.The inflammation of mammary gland not only causes the mammary gland damage and systemic inflammatory response,but also affects the milk yield and milk quality.Sodium valproate is widely used in the clinical treatment of epilepsy,bipolar disorder,migraine and other diseases.Recent studies have shown that sodium valproate plays an anti-inflammatory role both in vivo and in vitro.It can regulate NF-κB and MAPK signaling pathways,alleviate inflammatory response and regulate cell apoptosis and autophagy to defend against pathogens and protect the body.In this study,high-throughput sequencing was performed on the bacteria in milk samples of cows with mastitis to analyze the main bacterial types.Bovine mammary epithelial cells was stimulate by iE-DAP with iE-DAP to induce inflammatory response,and sodium valproate was used to investigate whether it can alleviate inflammatory response caused by iE-DAP and its regulatory mechanism.1 Detection of composition and diversity of bacteria in milk samples of cows with mastitis by 16S rRNA high-throughput sequencingMastitis is a mammary disease mainly caused by pathogenic microorganisms,which harms the health of cows and milk production and brings huge economic losses to the farms.The purpose of this experiment is to understand the composition and diversity of mammary gland flora of infected cows by analyzing milk samples collected from cows with mastitis.In this study,40 milk samples were collected from cows with mastitis in a farm in Wuhan.Bacterial DNA was extracted,amplified,purified and sequenced by 16S rRNA highthroughput sequencing on Illumina HiSeq2500 sequencing platform.The sequencing results were analyzed by Alpha diversity and bacterial community composition at different classification levels.The results showed that the number of OTU in the milksamples at the similar level of 97%was 12954.The analysis of the bacterial phylum level showed that Proteobacteria.Actinobacteria and Firmicutes were the main bacterial.The analysis of the bacterial family level showed that Moraxellaceae.Enterobacteriaceae and Xanthomonadaceae were the main bacterial.The analysis of the bacterial genus level showed that Acinebacteria,Klebsiella and Stenotrophomonas were the dominant bacteria.This study showed that the bacterial diversity in the mammary glands of cows with mastitis was widely distributed and the bacterial abundance was high,and gram-negative bacteria were the main pathogenic bacteria in the mammary glands of cows with mastitis.The result provided a basis for the prevention and control and research of mastitis for cattle farms.2 Effects of sodium valproate on iE-DAP induced inflammatory response in bovine mammary epithelial cellsStudies have shown that sodium valproate(VPA)can inhibit inflammatory responses in vitro and in vivo.This study was to investigate whether sodium valproate can modulate the inflammatory response in bovine mammary epithelial cells(BMECs)stimulated by y-Dglutamyl-meso-diaminopimelic acid(iE-DAP).The concentration and treatment points of iEDAP and VPA were optimized by detecting the gene expression of IL-6,IL-8 and HDAC3.Then,BMECs were cultured in complete media and separated into four groups:untreated control cells(CON group),cells stimulated by 10 μg/mL iE-DAP for 6 h(DAP group),cells stimulated by 0.5 mmol/L VPA for 6 h(VPA group),and cells treated with VPA(0.5 mmol/L)for 6 h followed by 10 μg/mL of iE-DAP for 6 h(VD group).Cell supernatant or cell lysates were collected after different treatments for the detection of related gene and protein expression.The results showed that the level of IL-6 and TNF-α in the culture medium increased in the iE-DAP-treated cells and the pretreatment with VPA reversed this increase.In contrast,VPA supplementation increased the level of anti-inflammatory cytokine IL-10 which was increased by iE-DAP treatment.iE-DAP could increase the expression of the proinflammatory cytokines IL-6,IL-8 and IL-1β and β-defensin LAP and VPA pretreatment decreased the expression of IL-6,BNBD5 and the acute phase protein SAA3.Compared with CON group,the level of apoptosis-related genes Bax and caspase-3 were significantly increased in DAP group,however,the VPA pretreatment increased the expression levels of Bax,caspase-3 and caspase-9.Morever,the autophagy-related expression of LC3B-II in VD group was significantly higher than that in DAP group,and the level of p62 was significantly lower than that in DAP group.This study showed that sodium valproate can reduce the expression level of inflammation-related genes,reduced apoptosis and induced autophagy in iE-DAP-induced BMECs.In conclusion,VPA treatment can attenuate the inflammatory response induced by iE-DAP so as to protect cells.3 Regulation mechanism of sodium valproate on inflammatory response in iE-DAPinduced bovine mammary epithelial cellsAs an inhibitor of histone deacetylase,sodium valproate can regulate inflammatory responses.This study was to investigate the potential regulatory mechanism of sodium valproate on inflammatory response in iE-DAP-induced bovine mammary epithelial cells.Bovine mammary epithelial cells were cultured in complete medium and divided into four groups:control group(CON),iE-DAP group(DAP),sodium valproate group(VPA),and sodium valproate+iE-DAP group(VD).After different treatments,cell supernatants or cell lysates were collected for detection of related gene and protein expression levels.The results showed that the mRNA levels of NOD1,RIPK2,NF-κB,and IκB and the protein expressions of NOD1,RIPK2,phosphorylated p65 and IκB protein in the DAP group increased significantly increased compared with the CON group.NF-κB p65 also transferred into the nucleus.However,VPA pretreatment can inhibit the activation of the NOD1/NF-κB pathway by down-regulating the expression of these genes and proteins and inhibiting the nuclear entry of NF-κB p65.Similarly,the mRNA levels of p38,ERK,and JNK and the relative expression of the phosphorylated p38,ERK,and JNK proteins significantly increased in the DAP group,while VPA pretreatment could significantly reduce the mRNA expression and the phosphorylated protein degree of p38,ERK,and JNK to attenuate the MAPK pathway.In addition,the mRNA levels of HDAC1-3 and the protein levels of STAT1,HDAC2 and HDAC3 significantly increased in the DAP group,while VPA pretreatment significantly increased the expression of STAT1 and down-regulated the mRNA expression of HDAC1 and HDAC3,the protein levels of HDAC2 and HDAC3 and the acetylation degree of histone H3.We also found the mRNA levels of MMP2,MMP3,MMP9,and MMP14 and the protein level of MMP9 significantly increased in the DAP group,while the mRNA levels of MMP2,MMP9 and MMP14 and the protein amount of MMP9 significantly decreased and the mRNA levels of TIMP1-3 increased significantly in the DAP group.In conclusion,sodium valproate increase the acetylation level of histone by inhibiting the activity of histone deacetylase and regulate gene transcription through post-translational modifications.By attenuating the activation of NOD1/NF-κB and MAPK signaling pathways and the activity of matrix metalloproteinases,sodium valproate regulated the expression of inflammation-related genes and inhibits the inflammatory response caused by iE-DAP stimulation.