Effect Of Lipopolysaccharide On The Inflammatory Response In Rumen Epithelium And Mammary Epithtelial Cells Of Ruminants And Its Regulation

To meet high-energy need of dairy animals with high milk production,high-concentrate diet is always used in dairy industry while short of fiber.However,long-term high-concentrate diet feeding induced the depression of rumen pH and increase of LPS concentration in rumen fluid which triggered inflammatory response in rumen epithelium,apoptosis of rumen epithelial cells and the disruption of rumen epithelium.Subsequently,the permeability of rumen epithelium will be increased which will facilitate the translocation of toxic components e.g.LPS from rumen fluid to circulation system.Rumen derived LPS has been demonstrated to be associated with the inflammation of mammary gland,apoptosis of mammary epithelial cells and decrease of milk yield and quality.Our study showed that sodium butyrate alleviated the damage of rumen epithelium triggered by long-term highconcentrate diet feeding and enhanced the innate immune response in mammary epithelial cells induced by LPS.Additionally,increased methionine or arginine supply modified the mRNA or protein abundance related to innate immune response during LPS challenge.1.Long-Term High-Concentrate Diet Feeding Induced the Apoptosis of Rumen Epithelial cells and inflammation of rumen epithelium in Dairy CowsHigh-concentrate diet is commonly applied to dairy cows with high milk yield,but it is harmful to rumen epithelium and the health of dairy cows when fed with high-concentrate diet for long period of time.The aim of the present study was to evaluate the effect of longterm high-concentrate diet feeding on rumen epithelium of dairy cows.So,12 mid-lactating Holstein dairy cows were selected and fed either with high-concentrate diet or lowconcentrate diet.Results showed that mRNA abundance of IL-6,IL-8,CCL5,caspase-3,caspase-8 and caspase-9 were upregulated in HC compared with LC group(P<0.05).Greater protein abundance of phosphorylated NF-?B,IL-6 and TNF-? was observed in HC group compared with LC group(P<0.05).Additionally,protein abundance of Cyto c,BAX and caspase-3 in HC group was greater compared with that in LC group(P<0.05),while protein abundance of Bcl-2 was lower in HC group compared with LC(P<0.05).Therefore,the present study showed that long-term high-concentrate diet feeding induced the inflammation and apoptosis of rumen epithelial cells by enhancing the activation of NF-?B,expression of proinflammatory cytokines and chemokines and proapoptotic factors while suppressed the expression of anti-apoptotic factor,which subsequently caused the disruption of rumen epithelium.2.Sodium butyrate ameliorates high-concentrate diet-induced inflammation in the rumen epithelium of dairy goatsTo investigate the effect of sodium butyrate on high-concentrate diet-induced local inflammation of the rumen epithelium,18 mid-lactating dairy goats were randomly assigned to 3 groups:a low-concentrate diet group as the control(concentrate:forage=4:6),a highconcentrate(HC)diet group(concentrate:forage=6:4),and a sodium butyrate(SB)group(concentrate:forage=6:4,with 1%SB by weight).The results showed that,with the supplementation of sodium butyrate,the concentration of lipopoly saccharide(LPS)in rumen fluid was significantly lower than that in the HC group.The protein abundance of pp65,gene expression of proinflammatory cytokines,and activity of myeloperoxidase(MPO)and matrix metalloproteinase(MMP)-2,9 in the rumen epithelium were significantly downregulated by SB compared with those in the HC group.With sodium butyrate administration,the concentration of NH3-N(19.2 ± 0.890 mM)in the rumen fluid was significantly higher than that for the HC group(12.7 ± 1.38 mM).Severe disruption of the rumen epithelium induced by HC was also ameliorated by dietary SB.Therefore,local inflammation and disruption of the rumen epithelium induced by HC were alleviated with SB administration.3.Effect of sodium butyrate on the innate immune response initiated by LPS in bovine mammary epithelial cellsLong-term high-concentrate diet feeding induced the disruption of rumen epithelium which may increase the permeability of rumen epithelium enabling the translocation of LPS into the circulation system.Rumen derived LPS induced the inflammatory response of mammary gland and diminished milk yield and milk quality.To explore effective therapeutic strategies for mastitis,an infectious diseases dampening dairy industry,effect of sodium butyrate on the LPS-induced innate immune response was evaluated by analyzing the expression of proinflammatory cytokines,chemokines and ?-defensins and key signaling involved in innate immune response in immortalized bovine mammary epithelial cells(MAC-T).Transcription of TNF-?,IL 1B,CXCL2,CXCL8,BNBD5 and TAP was enhanced by NaB during LPS or deactivated E.coli challenge.Acetylated histone H3 was increased by NaB during LPS stimulation but transcription of TNF-?,IL1B,CXCL2,CXCL8,BNBD5 and TAP was greater in NaB+LPS compared with TSA+LPS.Although NaB attenuated NF-?B activity,it increased the phosphorylated p38 MAPK,JNK and Erk 1/2 protein abundance during LPS stimulation.Inhibitors of MAPK signaling attenuated the upregulatory effect of sodium butyrate on those genes related with innate immune response induced by LPS challenge.Overall,NaB boosted the LPS-induced innate immune response in MAC-T cells by upregulating the transcription of proinflammatory cytokines,chemokines and defensins through the enhancement of MAPK signaling and histone acetylation.4.Increased Met and Arg supplementation at constant ratios of Thr:Phe,Lys:Thr,Lys:His,and Lys:Val alters oxidative stress and proinflammatory responses during lipopolysaccharide challenge in bovine mammary cellsMethionine(Met)and arginine(Arg)are key nutrients with potential to regulate inflammation and oxidative stress.The aim of this study was to evaluate the effect of increased supply of Met and Arg on mRNA and protein abundance associated with innate immune response and redox balance during lipopolysaccharide(LPS)stimulation in primary bovine mammary epithelial cells(BMEC).Primary BMEC(n=4 replicates/treatment)were pre-incubated for 12 h in media with increased levels of Met or Arg alone or in combination.Subsequently,cells were challenged with or without LPS(1?g/mL)and incubated for 6 h.mRNA abundance of SLC7A5,SLC7A1 and SLC36A1 encoding proteins that facilitate the uptake of amino acids were downregulated by LPS stimulation.However,increased Arg supply attenuated the downregulation of SLC36A1 and SLC7A1 mRNA abundance induced by LPS.Abundance of phosphorylated p65(RELA)was greater after LPS stimulation,but the response was attenuated by supply of Met.Greater ratio of pRELA to total RELA in responses to LPS was also attenuated with increased supply of Met and Arg in combination.The greater mRNA expression of the proinflammatory cytokine IL-1? induced by LPS was attenuated by Met,and the same trend induced by LPS on CXCL2 was also alleviated with greater Arg supply.Although greater supply of Met and Arg could not rescue the inhibition of antioxidant mechanisms controlled by NFE2L2,the data suggest that these greater supply of Met and Arg alleviated the proinflammatory responses triggered by LPS.One of the underlying mechanisms was through the controlling of NF-?B activity and the abundance of proinflammatory cytokines and chemokines.Long-term high-concentrate diet feeding induced the inflammation in rumen epithelium and apoptosis of rumen epithelial cells causing the disruption of rumen epithelium.However,sodium butyrate alleviated the inflammatory damage induced by long-term high-concentrate diet feeding by attenuating NF-?B activation,expression of proinflammatory cytokines and metalloproteinase activity.Disruption of rumen epithelium will increase the permeability of rumen wall enabling the translocation of toxic components and pathogens in the rumen to circulation system.Rumen dirved LPS induced the damage of mammary gland and depression of milk yield and quality.Our study showed that sodium butyrate promoted the innate immune response by enhancing the activation of MAPK signaling and histone acetylation in MAC-T cells induced by LPS.Additionally,increased supply of methionine or arginine also modulated the inflammatory response in primary bovine mammary epithelial cells triggered by LPS.