Effect And Regulation Mechanism Of Sodium Butyrate On The NOD1/RIP2 Inflammatory Signaling Pathway In The Mammary Gland Of SARA Ruminants

Owing to pursue higher milk yield,people often fed the dairy cows with high-concentrate(HC)diet in the modern dairy industry.However,long-term HC diet can lead to abnormal fermentation and excessive accumulation of volatile fatty acids and lactic acid in the rumen,and then resulted in low rumen pH value,finally inducing subacute ruminal acidosis(SARA).Under the condition of SARA,rumen microecological balance destroyed,Gram-negative bacteria lysised and released lipopolysaccharide(LPS),histamine,diaminopimelic acid(DAP),etc.,excessive DAP accumulated and translocated into the mammary gland through bloodstream,which can activate the NOD1-NF-?B inflammatory signaling pathway to induce inflammatory response.Sodium butyrate is a green,efficient,safe feed additive,which has many functions,such as regulating the microecological balance of gastrointestinal tract,promoting the proliferation and maturation of gastrointestinal epithelial cells.At the same time,it can also down-regulate the activity of NF-?B to inhibit the release of inflammatory cytokines,finally relieving the inflammation.The experiment was divided into three parts to investigate the effect and regulation mechanism of sodium butyrate on the NOD1/RIP2 inflammatory signaling pathway in the mammary gland of dairy ruminants during SARA.1.Determination of iE-DAP in rumen fluid of dairy cows by Pre-column ChiralDerivatization-HPLCIn order to investigate the effect of sodium butyrate supplementation on the concentration of iE-DAP in rumen fluid of dairy cows when feeding high-concentrate diet,12 lactat:ing Holstein cows were randomly divided into low-concentrate group(LC)and high-concentrate group(HC).We established a high performance liquid chromatography(HPLC)method combining with pre-column chiral derivatization to detect the concentration of y-D-glutamic acid-meso-diaminopimelic acid(iE-DAP)in goat rumen fluid.iE-DAP was derivatized with o-phthaldehyde and N-acetyl-eysteine reagent and using a mobile phase consisting of 0.05 mol/mL ammonium acetate-acetonitrile(90:10),at the detection wavelength of 345 nm.The results showed that the linear range of iE-DAP was 5-500 ?g/mL,the RSDs of intra-day and inter-day were lower than 7%.The concentrations of iE-DAP in the rumen fluid of HC group were(9.82+0.48)?g/mL significantly increased than LC group,which was(5.45±0.83)?g/mL.In conclusion,high concentrate diet can up-regulate iE-DAP concentration in the rumen fluid.Meanwhile,the method is feasible and can be used to detect the concentration of iE-DAP.2.Effect of sodium butyrate on iE-DAP and milk quality of lactating goats duringSARAIn order to investigate the effect of sodium butyrate on iE-DAP and milk quality of lactating goats during SARA,12 Saanen lactating goats were randomly divided into two group.The high-concentrate group(HC)was fed with a HC diet which was composed of 40%forage and 60%concentrate,the sodium butyrate regulated group(BHC)was fed with a HC diet adding with sodium butyrate,the experiment lasted for 18 weeks.The lactating goats were fitted with a rumen fistula and vascular cannulae,and the experiment started after 2 weeks.The results showed that comparing with the HC group,dietary sodium butyrate addition can alleviate the decrease of rumen pH value.The concentration of iE-DAP in rumen fluid and blood were both significantly decreased in the BHC group comparing with HC group.Meanwhile,inflammatory cytokines IL-1??IL-6 and TNF-? in the peripheral blood of lactating goats were all significantly decreased in the BHC group.In conclusion,dietary sodium butyrate addition can reduced the concentration of iE-DAP in rumen fluid and blood,which finally improved milk production and milk quality.3.Effect and its involved epigenetic mechanisms of sodium butyrate on NODIinflammatory signaling pathway in the mammary gland of lactating goatsIn order to investigate the effect and its involved epigenetic mechanisms of sodium butyrate on NODI inflarumatory signaling pathway in the mammary gland of lactating goats,12 Saanen dairy goats were randomly divided into high-concentrate group(HC)and sodium butyrate regulated group(BHC),the experiment lasted for 18 weeks.The results of real-time quantitative PCR showed that comparing with HC group,the mRNA expression of NOD1 and other genes implicated in the idflammation were all significant decreased in BHC group.The results of western blotting showed that the protein expression of NODI?NF-?B p65 and NF-?B pp65 in BHC group were significant lower than HC group,which was consistant with the mRNA expression.The results of CHART?PCR and DNA methylation analysis showed that decreased NOD1 and RIP2 expression were accompanied by chromatin compaction in the promoter region of NOD1 and RIP2 gene and its DNA methylation.Collectively,sodium butyrate can down-regulate the expression of NODI,which was mediated by epigenetic mechanisms,involving chromatin remodeling and DNA methylation.Subsequently it can inhibited the activation of the NODI inflammatory signaling pathway,down-regulated the expression of inflammation related genes and alleviated the inflammation.