Mechanistic Study Of Nod1 Signaling Pathway-mediated Inflammation In Liver Of Ruminants During Sara And Its Regulation Animesh Chandra Roy

Overfeeding of highly fermentable non-structural carbohydrate forage with low physically effective fiber produces large amounts of volatile fatty acids(VFAs)and lactic acid beyond the rumen's absorptive,buffering and outflow capacity.Excessive accumulation of VFAs decreases the rumen pH which causes the subacute ruminal acidosis(SARA)characterized by at least 3 hours per day pH values ranged from 5.2 to 5.6 or below5.8.Induction of SARA increases the accumulation of endotoxins such as lipopolysaccharide(LPS)and D-glutamyl-meso-diaminopimelic acid(iE-DAP)by the lysis of Gram-negative bacteria.iE-DAP and LPS are translocated into portal circulation to reach the liver and then in the systemic circulation through breaking the integrity of epithelial tight junction(TJs)of rumen-reticulum.The liver functions as a sentinel organ between gut contents and systemic circulation possessing a unique position in the host body.The liver not only contributes in metabolism,detoxification,and nutrient storage but also functions as an essential immune organ.The objectives of this study were to investigate the effect of long-term high-concentrate feeding on nucleotide-binding oligomerization domain-containing protein 1(NOD1)-mediated inflammation and injury in the liver as well as milk yield and milk quality of dairy cows,to evaluate the impact of sodium butyrate on iE-DAP-induced liver inflammation in dairy goats during subacute ruminal acidosis(SARA)caused by high-concentrate feeding,and to explore the contribution of NOD1 to autophagy and inflammation in response to iE-DAP treatment in bovine hepatocytes in vitro.1 SUBACUTE RUMINAL ACIDOSIS(SARA)INDUCES NOD1-MEDIATED INFLAMMATION AND INJURY IN THE LIVER OF DAIRY COWS FED HIGH-CONCENTRATE DIETHigh-grain feeding,a common practice to increase the milk yield has a negative impact on the body homeostasis and the milk quality.Therefore,we aimed to study the effect of long-term high-concentrate feeding on nucleotide-binding oligomerization domain-containing protein 1(NOD1)-mediated inflammation and injury in the liver as well as milk yield and milk quality of dairy cows.To achieve this goal,twelve healthy lactating Holstein Friesian cows weighing 455±28 kg were divided into two groups:a low-concentrate feeding group(n=6,concentrate:forage=4:6)as the control group and an high-concentrate feeding group(n=6,concentrate:forage=6:4)as the treatment group.We observed the induction of subacute ruminal acidosis(SARA),as rumen pH was less than 5.6 for 223 min per day during high-concentrate feeding.SARA increased the concentration of D-glutamyl-meso-diaminopimelic acid(iE-DAP)and lipopolysaccharide(LPS)in the rumen.iE-DAP and LPS can reach the liver via the hepatic portal circulation,as LPS was detected in the hepatic vein and jugular vein plasma.Liver enzymes ALT,AST,ALP,and LDH,as well as ammonia,were increased and liver proteins albumin and total protein were decreased post-high-grain feeding,indicating the injury in the liver.The mRNA expression of NOD1,RIPK2,TAK1,I?B?,NF-?B,ERK,JNK,IL-6,IL-1?,TNF-?,IL-8,CCL5,CCL20,FOS,SAA3,HP,NOS2,and HDAC3 was up-regulated in cows fed HC diet compared to cows fed LC diet.The up-regulation of NOD1,HDAC3,p-I?B?,p-p65/NF-?B,p-ERK1/2,and p-JNK proteins during the HC feeding indicates the activation of NOD1-mediated inflammatory molecules.However,both the mRNA and protein expression of FOXA2 were significantly down-regulated in the HC group.We also noticed that long-term high-grain feeding deteriorated the milk production and milk fat and increased the somatic cells.We conclude that the induction of SARA by the long-term high grain feeding can produce iE-DAP and LPS which can stimulate the NOD1-mediated inflammation and cause liver injury and reduce the milk production and milk quality.2 SODIUM BUTYRATE MITIGATES iE-DAP INDUCED INFLAMMATION CAUSED BY HIGH-CONCENTRATE FEEDING IN LIVER OF DAIRY GOATSThe aim of this study is to explore the impact of sodium butyrate on iE-DAP-induced liver inflammation in dairy goats during subacute ruminal acidosis(SARA)caused by high-concentrate feed.To achieve this aim,12 lactating dairy goats were randomly divided into 2 groups:a high-concentrate feed group(n=6,concentrate:forage=6:4)as the control group and a sodium butyrate with high-concentrate feed group(n=6,concentrate:forage=6:4,with 1%SB by wt.)as the treatment group.A rumen pH below 5.6 lasted for at least 4 h/d due to long-term HC feeding.The concentration of iE-DAP was significantly lower(11.67±3.85?g/mL,and 7.74 ± 1.46?g/mL,at the 4th h and 6th h of feeding,respectively)in the SB-treated group than that in the HC group(51.45±5.71?g/mL,and 18.31 ± 3.83?g/mL,at the 4th and 6th h of feeding,respectively).Meanwhile,SB significantly suppressed the mRNA expression of inflammatory genes(NOD1,RIPK2,TAK1,NF-?B/p65,ERK,JNK2,p38,IL-1?,TNF-?,CCL5,CCL20,CXCL12,FOS??-defensin/LAP).Moreover,the protein expression of NOD1,p-I?B?,p-NF-?B/p-p65,p-ERK1/2,p-JNK,p-p3 8?and HDAC3 was significantly downregulated in the HC+SB group.In conclusion,iE-DAP-induced inflammation and liver disruption generated by the HC diet was mitigated by SB treatment.3 SODIUM BUTYRATE SUPPRESSES NOD1-MEDIATED INFLAMMATORY MOLECULES EXPRESSED IN BOVINE HEPATOCYTES DURING iE-DAP AND LPS TREATMENTNucleotide oligomerization domain protein-1(NOD1),a cytosolic pattern recognition receptor for the y-D-glutamyl-meso-diaminopimelic acid(iE-DAP)is associated with the inflammatory diseases.Very little is known how bovine hepatocytes respond to specific ligands of NOD land sodium butyrate(SB).Therefore,the aim of our study was to investigate the role of bovine hepatocytes in NOD1-mediated inflammation during iE-DAP or LPS treatment or SB pre-treatment.To achieve this aim,hepatocytes separated from cows at?160 days in milk(DIM)were divided into 6 groups:the non-treated control group(CON),the iE-DAP-treated group(DAP),the lipopolysaccharide-treated group(LPS),iE-DAP with SB group(DSB),LPS with SB group(LSB),and the SB group.Both iE-DAP and LPS highly increased the expression of both NOD1 and RIPK2,the two key factors for the immune response in hepatocytes.I?B?,NF-?B/p65 and MAP kinases(ERK,JNK,and p38)were activated through phosphorylation.The activation of NF-?B and MAPK pathway consequently increased the pro-inflammatory cytokines,IL-6,TNF-?,IL-8,and IFN-? and the chemokines CCL5,CCL20,and CXCL-10.Both treatments improved iNOS/NOS2 expression.However,iE-DAP was failed to express acute phase protein SAA3,but HP and LPS HP but SAA3.These ligands also increased LRRK2,TAK1,TAB1,and ?-defensins expression.SB pre-treatment at lower dose restored the function of hepatocytes by suppressing these increased molecules,as HDAC3 was inhibited.The activated NOD1 negatively regulated the expression of FOXA2.Altogether these data suggest an important role of bovine hepatocytes to promote immune responses via NOD1 expression during infection in the liver and a key role of SB to attenuate inflammation.4 MECHANISTIC STUDY OF AUTOPHAGY IN BOVINE HEPATOCYTES DURING NOD1-MEDIATED INFLAMMATION INDUCED BY iE-DAP AND ITS INHIBITION BY 3-MAAutophagy is a crucial cellular homeostatic process and an important part of the host defense system.Dysfunction in autophagy enhances the tissue susceptibility to infection and multiple diseases.However,the role of nucleotide oligomerization domain 1(NOD1)in the autophagy in bovine hepatocytes is not well known.Therefore,our aim was to study the contribution of NOD1 to autophagy during inflammation in response to the specific ligand(iE-DAP).To achieve this aim,hepatocytes separated from cows at?160 days in milk(DIM)were divided into 6 groups:the non-treated control(CON)group,the rapamycin-treated(RAP)group as a positive control,the iE-DAP-treated(DAP)group,the 3-MA-treated(MA)group,the rapamycin with 3-MA(RM)group,and the iE-DAP with 3-MA(DM)group.iE-DAP treatment increased the mRNA expression of NOD1,ATG16L1,RIPK2,ULK1,AMBRA1,DFCP1,WIPI1,ATG5,ATG7,ATG10,ATG4A,I?B?,NF-?B,CXCL1,IL-8,and STAT6 and decreased PIK3C3.The protein expression of NOD1,p-I?B?,p-NF-?B/p-p65,LC3-?,ATG5,and Beclin 1 were up-regulated and that of SQSTM1/p62,p-mTOR,and FOXA2 were down-regulated in response to iE-DAP.iE-DAP also induced the formation of LC3-GFP autophagic puncta in bovine hepatocytes.We also knocked down the NOD1 with siRNA.NOD1 silencing suppressed the autophagy and inflammation-related genes and proteins.The application of the autophagy inhibitor increased the expression of inflammatory molecules and alleviated the autophagy-associated molecules.Taken together,these findings suggest that NOD1 is the key player to regulate both ATG16L1 and RIPK2-ULK1 directed autophagy during inflammation in response to iE-DAP in bovine hepatocytes.