| Liver is a crucial organ of organism,which is aptly called 'Processing Factory' of the body owing to be associated with the precess of digestion,absorption,detoxification,synthesis and metabolism of nutrients,and immune defense.The hepatic metabolism to nutrients derived from digeative tract is always the focus of sentific reaserch.However,the hepatic immune defense to hazardous substances derived from gastal intestinal tract remains unclear.Goats and cows installed rumen cannula and multiple liver vascular catheters were employed to observe the removal percentage of LPS translocated from digestive tract,and the effect of these LPS on hepatic innate immune respons and its involed epigenetic mechanism during high concentrate diet induced sub-acute ruminal acidosis(SARA).The experiment of LPS infusion in mammary gland of cows has been conduceted to ultimately elucidate epigenetic mechanisms involed in the expression of hepatic innate immune relevant genes.1.The removal percentage of LPS derived from digestive tract in goat liver and its effect on hepatic innate immune function The effects of feeding a high-concentration diet on lipopolysaccharide(LPS)clearance and innate immune defence responses in the liver remain unclear.Therefore,we conducted the present study in which twelve female goats installed ruman cannula were randomly assigned to either a treatment group fed a high concentrate diet(HC,60%grain,n = 6)or a control group fed a low concentrate diet(LC,40%grain,n = 6).Catheters were installed in the mesenteric,portal and hepatic veins,as well as one femoral artery of the goats.The feeding of HC significantly declined rume pH after 3 weeks and onwards,since the holding time of rumen pH<5.6 was more than 180 min/d indicating SARA was successfully induced.After 6 week feeding,blood flow and LPS concentration were significantly increased in the portal vein,hepatic vein and artery.Hepatic net LPS clearance showed that the feeding of HC elevated the rate of hepatic LPS clearance,but decreased the percentage of removed LPS accounting for the total entry of LPS into the liver.This result indicated that SARA induced by HC feeding resulted in the accumulation of active LPS in the liver.In addition,our results demonstrated that SARA increased plasma concentrations of pro-inflammatory cytokines and acute phase proteins and triggered a systemic inflammatory response.Peripheral blood plasma concentrations of alanine aminotransferase,alkaline phosphatase and total bilirubin were increased in treatment group compared to control group.This indicated that the impairment of hepatocytes occurred duiring SARA.The expression of genes involved in immune response and Toll-like receptor(TLR)4protein in the liver was up-regulated in treatment group compared to control group,indicating that increased entry of LPS enhanced hepatic immune defence responses.Collectively,these results indicated that SARA induced by HC feeding resulted in the accumulation of active LPS translocated from digestive tract,and caused the increase of pro-inflammatory cytokines and acute phase proteins concentration in peripheral blood and the enhancement of immune relevant genes and TLR4 protein expression in liver,thereby elevated the rate of LPS clearance.However,the reduction of the percentage of hepatic LPS clearance could be due to the formation of hepatocyte lesion elicited by the accumulation of active LPS and the production of pro-inflammatory cytokines in the liver..2.The effect of LPS translocated from digestive tract on TLR4 signaling pathway and the expression of its downstream immune relevant genes in the liver of lactating goats SARA caused by the feeding of high concentrate diet is known to trigger a systemic inflammatory response,possibly caused by Lipopolysaccharide(LPS)being translocated from the gastrointestinal tract into the bloodstream.We examined this suggested causal relationship between systemically increased levels of circulating LPS and inflammation of the liver.Therefore,eight lactating goats were randomly assigned to either a control group fed low concentrate diet(LC,40%concentrate,n=4,)or a treatment group fed high concentrate diet(HC,60%concentrate,n=4,).The HC diet significantly decreased the rumen pH after 4 weeks and onwards,since the holding time of rumen pH below 5.6 was more than 180 min per day indicating the induction of SARA was successed.These goats featured after 8 weeks significantly decreased milk yield,milk fat percentage and milk lactose percentage,and increased LPS concentrations in both,rumen and portal vein.Their livers featured increased mRNA concentrations of a panel of pro-inflammatory genes indicating inflammation.This was very likely mediated by enforced TLR4 signaling,since SARA increased the concentrations of TLR4 mRNA and protein in liver and also increased the abundance of both,the NF-kB-p65 factor and its active phosphorylated variant.We also validated that enhanced TLR4 expression was accompanied by chromatin decompaction of the proximal TLR4 promoter and its demethylation.Hence,epigenetic mechanisms are involved in enforced expression of immune genes during SARA opening innovative routes for intervention through modulating their activity.3.The effect of LPS translocated from digestive tract on expression of innate immune genes in the liver of cows and its invoved epigenetic mechanisms Epigenetic modifications critically regulate the expression of immune-related genes in response to inflammatory stimuli.It has been extensively reported that a high concentrate(HC)diet can trigger systemic inflammation in dairy cows,yet it is unclear whether epigenetic regulation is involved in the expression of immune genes in the livers of dairy cows.In present study,eight mid-lactating cows were installed a rumen cannula and catheters of the portal and hepatic veins.Cows were randomly assigned to either the treatment group fed a high concentrate diet(HC,60%concentrate,n=4)or a control group fed a low concentrate diet(LC,40%concentrate,n=4).The HC significantly decreased the rumen pH after 4 weeks and onwards,since the holding time of rumen pH below 5.6 was more than 180 min per day indicating the induction of SARA was successed.After 10weeks of feeding,levels of lipopolysaccharide(LPS)in the rumen,and portal and hepatic veins were notably increased in treatment group compared to the control group.The expression levels of detected immune response-related genes were significantly up-regulated in the livers of cows during induced SARA.Chromatin loosening at the promoter region of four candidate immune-related genes(TLR4,LPS-binding protein,haptoglobin,and serum amyloid A3)was elicited,and was strongly correlated with enhanced expression of these genes in the treatment group compared to control group.Demethylation at the promoter region of all four candidate immune-related genes was accompanied by chromatin decompaction.Collectively,SARA induced by the feeding of HC caused the translocation of large number of LPS from the digestive tract into the liver via the portal vein,enhancing hepatic immune gene expression.The up-regulation of these immune genes was mediated by epigenetic mechanisms,which involve chromatin remodeling and DNA methylation.Our findings suggest that modulating epigenetic mechanisms could provide novel ways to treat systemic inflammatory responses elicited by SARA.4.The effect of intramammary E.coli infusion on expression of innate immune genes in the liver of cows and its invoved in epigenetic mechanisms Endotoxins,such as lipopolysaccharide are liberated during bacterial infections and this may result in excessive activation of Toll-like-receptor(TLR)signaling,eventually causing severe systemic reactions.We examined if epigenetic mechanisms are involved in controlling their onset and process.In present study,sixteen mid-lactating cows were randomly assigned into three group,cows were inoculated with 500CFU of E.coli1303 in the front right,hind right and hind left quarter of mammary gland in first group called heavily infected group(HI,n=4);cows were inoculated with 500CFU of E.coli1303 in the front right quarter in second group called gently infected group(GI,n=7);cows were inoculated with0.9%sterile pyrogen free saline in the front right quarter in third group called control group(n=5).All cows were culled at 24 h post infection and liver samples were collected.Using Chromatin Accessibility PCR(CHART),we found in livers of cows from HI group that the chromatin at promoters of immune genes(TLR2,-4,lipopolysaccharide binding protein[LBP],haptogloblin[Hp])was opened up at 24 h after intramammary inoculation,accompanying their enhanced expression.However,such a modulation did not occur in the same samples at the aSl-casein promoter,serving as a control.Demethylation of the TLR4promoter accompanied its chromatin decompaction.Our data imply that intramammary E.coli inoculation enhaneced the expression of hepatic innate immune relevant gene,and their enforced expression were associated with epigenetic mechanisms.This may offer novel strategies to treat systemic reactions elicited by E.coli infection in mammary gland. |
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