Effects Of High-concentrate Diet On Hindgut Epithelial Barrier In Lactating Dairy Goats And The Mechanisms Involved

In current intensive production system,large amounts of cereal grains or easily degradable byproducts in diet are fed to lactating cows to meet energy requirement for supporting maintenance and high milk yields.It is well documented that feeding high amounts of concentrate diet to ruminants results in subacute ruminal acidosis(SARA),a common metabolic disease especially occurred in high-producing animals.Previous studies mainly focused on the effects of feeding high-grain diet on the histological structure and functions of ruminal epithelium in dairy cows.Compared to rumen epithelium with a stratum corneum layer and multicellular layers in the middle,the large intestine epithelium is much more "leaky" due to the monolayer structure.To date,information regarding the effect of feeding ruminants with diet enriched high level of concentrate on hindgut is not available.In this study,we use lactating dairy goats as models,to investigate the changes of fermentation,micro flora,metabolites of the gut lumen,and the status of epithelial barrier in lactating dairy goats fed a high concentrate diet for a long period.Additionally,we use intestinal goblet cells as models,to study the effects of quorum sensing(QS)signal molecule C12-HSL on the cellular state and mucous secretion,and to provide new ideas for the prevention and treatment of SARA.1 Effects of high-concentrate on microbiome and metabolome in hindgutWe used 454 pyrosequencing of 16S rDNA genes and gas chromatography-mass spectrometry to evaluate the effects of long-term feeding(HL,19 weeks)or short-term(HS,4 weeks)feeding of an HC diet(65%mixed concentrate and 35%forage)on changes in bacterial microbiota and their metabolites in the hindgut,with Guanzhong goat as a ruminant model.Compared with the LC group(65%forage and 35%mixed concentrate,19 weeks),the HS,and HL groups contained significantly higher free LPS concentrations in the hindgut digesta.Moreover,the total VFA and its components in the hindgut digesta were increased in different degrees.The compounds responsible for the respective difference between the LC and HS groups or between the LC and HL groups were identified in this study.Seven compounds were identified as major distinguishing substances in the colonic digesta compounds among the 3 groups.Three of these compounds(betaalanine,p-cresol,and glycylproline)were markedly increased,whereas 4(threonic acid,stigmasterol,erythronic acid,and quinic acid)were decreased by the HC diet relative to the control.Six compounds were recognized as the differentiating substances in the cecal digesta compounds among the 3 groups.Only one of these compounds(betaalanine)was increased,whereas the remaining 5 compounds(glyoxylic acid,stigmasterol,1-eicosanol,phytol,and quinic acid)were decreased by the HC diet relative to the control.Moreover,we found that HL goats showed markedly higher level of quorum sensing signal molecular C12-HSL in colonic digesta than LC goats.The ACE,Chao 1,and Shannon indices were significantly decreased in the hindgut digesta of the HL group relative to those of the LC group.Compared with the LC group,the HS goats showed a lower Shannon index in the colonic digesta,as well as ACE and Chao 1 indices in the cecal digesta.At the genus level,in the colonic digesta,as the proportion of concentrate increased,the percentages of Turicibacter,Clostridium,Oscillospira,Prevotella,and Bacteroides also increased.Bycontrast,the abundance of Ruminococcus,Bulleidia,5-7N15,Mogibacterium,and Blautia was reduced.In the cecal digesta,as the proportion of concentrate increased,the percentages of Clostridium,Turicibacter,SMB53,Mogibacterium,YRC22,and Pseudoramibacter was increased,whereas the proportions of Oscillospira,Coprococcus,CF231,and Parabacteroides decreased.Our results indicate that an HC diet increases fermentation parameter such as LPS,VFA and lactate concentration,decreases microbiota diversity and structure,eventually causes metabolic disorders in the hindgut of lactating dairy goats.2 Effects of high-concentrate on epithelial barrier in hindgutIn this study,twelve lactating dairy goats were randomly assigned to either a HC diet group(65%mixed concentrate and 35%forage;n=6)or a LC diet group(65%forage and 35%mixed concentrate;n=6)for 10 weeks.The-level of starch,acetate,propionate,butyrate and total VFA concentrations in colonic and caecal digesta of HC goats was significantly higher than that of LC goats.Additionally,HC goats showed markedly higher level of free LPS concentration in caecal digesta than LC goats,while there was no significant difference of free LPS concentration in colonic digesta between HC and LC goats.Hematoxylin and eosin(HE)staining showed that in colonic and caecal epithelium,desquamation and severe cellular damage was observed in HC goats.The ultrastructure of hindgut epithelium was detected by Transmission Electron Microscopy(TEM)method.TJs in the colonic and caecal epithelium of HC goats were damaged with wider intercellular space,while LC goats displayed integrity and normal TJs structure.Moreover,LC goats showed normal cell nucleus and mitochondria structure,whereas HC goats displayed apparent nuclear breakdown and mitochondrial swelling.The proportion of TUNEL-positive apoptotic cells in the hindgut epithelium of HC goats was markedly increased compared to LC goats.Consistently,HC goats showed a significant increase of caspase-3 and-8 gene expression and activity in colonic mucosa compared to LC.The expression of MKI67 and CCND2(proliferation markers)mRNA and protein was significantly decreased in the caecal mucosa of HC goats compared to LC.HC goats showed a significant decrease of local PGE2 content and the expression of PGE2 receptor in caecal mucosa.Moreover,the level of p-CREB and p-AKT protein abundance,was down-regulated in caecal mucosa of HC goats compared to LC.The relative mRNA expression levels of the genes functionally associated with inflammation were evaluated.The expression levels of the genes related to the inflarmnatory response were not significantly altered in the colonic mucosa by the HC diet.Compared with the control group,the HC group showed up-regulated expression of TLR4,MyD88 and IL-1β in the cecal mucosa.We also detected the gene and protein expression of tight junction protein.The gene expression of occludin,claudin-1 and claudin-4 were not markedly altered in the hindgut mucosa by the HC diet.The level of claudin-1 and claudin-4 protein expression in the colonic mucosa was significantly up-regulated in HC goats compared to LC.These results reveal that long-term feeding HC diet to lactating goats causes severe damages to the hindgut mucosa barrier associated with activating cells apoptosis and inhibiting cell proliferation.Moreover,HC diet fed induces the changes of local inflammation and tight junction status in hindgut of goats.3 Effects of quorum sensing molecule on intestinal goblet cells functionIn this study,we investigated the effects of two representative long-and short-chain AHLs,C12-HSL and C4-HSL,on cell viability and mucus secretion in LS174T cells.Our results show that treatment of LS174T cells with C12-HSL>50 μM significantly decreased cell viability,however,C4-HSL did not affect the viability of LS174T cells.Additionally,cells treated with 100 μM of C12-HSL for different time periods showed time-dependent decreases in cell viability.Cells treated with DMSO(control group)or C12-HSL at a low concentration(10 μM)displayed a normal subcellular structure.In contrast,cells treated with 100 μM C12-HSL for 4 h exhibited apparent mitochondrial swelling.Unsurprisingly,cells treated with 100μM C4-HSL showed similar mitochondrial structure as the control cells.We found that ΔΨm was significantly decreased in cells treated with 100μM C12-HSL.In agreement with observed mitochondrial dysfunction as indicated by loss of ΔΨm,treatment of LS174T cells with 100 μM C12-HSL also increased mitochondrial superoxide production-Additionally,compared to control cells,C12-HSL(100 μM)markedly increased active-caspase3 protein expression level.We also measured the mitochondrial respiration chain complex activity in LS174T cells exposed 100 μM C12-HSL for 4 h.Our results show that treatment with C12-HSL significantly increased complex Ⅳ and Ⅴactivity.The level of intracellular ATP and Ca2+ in the C12-HSL-treated LS174T cells was significantly higher than that in DMSO control group.Additionally,we found that the mitochondrial integrity was markedly decreased in LS174T cells treated with C12-HSL.C12-HSL significantly increased ROS production in dose-dependent manner.Intracellular ROS level was significantly increased in LS174T cells exposed 10 to 200 μM C12-HSLfor 4 h.Exposure time also showed a significant impact on cellular ROS production.When LS174T cells were treated with 100 μM C12-HSL for 1 h to 6 h,the level of cellular ROS was markedly increased and reached the maximum level at 4 h of exposure.Moreover,the level of cellular MDA and H2O2 was also greatly increased by 100 μM C12-HSL treated for 4 h in LS174T cells.C12-HSL at 100 μM significantly decreased MUC2 mRNA and protein expression levels compared to those of the DMSO control group.Additionally,we also tested the MUC2 expression in different time periods treated with 100μM of C12-HSL.100μM of C12-HSL at 4 h markedly decreased the level of MUC2 in mRNA expression and secreted to culture medium.Meanwhile,the PAS and alcian blue staining results showed that the mucin secretion and sulfation level were markedly decreased in C12-HSL treated LS174T cells.Our results show that treatment of HCT116 cells with C12-HSL>200 μM significantly decreased cell viability in a dose-dependent manner.C12-HSL at 400 μM significantly increased MUC2 levels of intracellular and secreted to culture medium compared to those of the DMSO control group.The PAS and alcian blue staining results showed that the mucous glycoprotein and sulfation level were obviously increased in C12-HSL(400 μM)-treated HCT116 cells.Our results indicate that C4-HSL and low concentrations of C12-HSL showed no effects on cell viability and mucin secretion in goblet LS174T cells,but C12-HSL at high concentration(100μM)triggers events associated with the intrinsic pathway leading to apoptosis:mitochondrial swelling,ΔΨm depolarization,enhanced mitochondrial ROS generation and activation of caspase3.Moreover,100 μM C12-HSL inhibits mucus secretion and synthesis and mature in LS174T cells.4 The mechanisms involved in intestinal goblet cells function damageTo determine the functional importance of membrane cholesterol in mediating C12-HSL-caused cell damage,LS174T cells were cholesterol-depleted using the lipid raft sequester MβCD.Our results showed that treatment of LS174T cells with varying concentrations of M(3CD(1-10μM)marginally but significantly rescued C12-HSL-induced cell death.However,treatment of LS174T cells with 10 μM MβCD did not influence C12-HSL-induced cell apoptosis,ΔΨm depolarization or mitochondrial superoxide generation.To investigate the functional relevance of PON2 in C12-HSL induction of cell damage,the PON2 inhibitor TQ416 was used to inhibit PON2 activity.1 μM TQ416 significantly reversed C12-HSL-induced apoptosis and restored the C12-HSL-caused mitochondrial dysfunction in LS174T cells.Moreover,1 μM TQ416 completely restored C12-HSL-induced MUC2 secretion and mature disorder.Additionally,when co-treatment with C12-HSL,TQ416 nearly completely blocked C12-HSL-induced cell death.However,this rescuing effect of TQ416 significantly decreased when TQ416 was administered 10-30 min post-C12-HSL challenge and was completely lost when TQ416 was administered 40 min later.Our study found that the level of cellular pro-caspase 1 protein was significantly decreased,whereas the activity of caspase-1 was markedly increased in LS174T cells treated with C12-HSL.There was no obvious change in cellular IL-1β and GSDMD protein expression in LS174T cells after C12-HSL treatment.Moreover,the concentration of IL-1β,TNF-α and IL12P70 in the supernatant was significantly decreased,while IL-8 concentration was greatly increased by C12-HSL treatment.Additionally,expression of TLRs mRNA and proteins was detected by real-time PCR and western blot analysis.The results demonstrated that TLR2,3 and 4 mRNA expression were significantly up-regulated by C12-HSL.In contrast,the level of TLR 2 and TLR4 protein showed a significant decrease by C12-HSL.Unfortunately,the level of phosphorylated NF-κB was not significantly changed by C12-HSL.NF-κB transcription activity inhibitor JSH-23 did not show an obvious effect on cell viability after co-treated with C12-HSL.PON2 inhibitor TQ416,caspase-1 inhibitor VX-765 and oxidative stress remover NAC can remarkably rescue cell damaged by C12-HSL treatment in LS174T cells.The level of cellular ROS and H2O2,as well as MDA can be reduced by TQ416.TQ416 and NAC can significantly inhibit the activation of caspase-1 activity induced by C12-HSL.Caspase-3 inhibitor Z-DEVD-FMK rescued cells viability and ROS level induced by C12-HSL.TQ416,NAC and VX-765 inhibit the activation of caspase-3 initiated by C12-HSL.Additionally,When C12-HSL co-treated with VX-765 and Z-DEVD-FMK,showed a significant rescuing effect on mucin secretion and sulfation.We also found that TQ416,VX-765 and Z-DEVD-FMK can significantly rescue the decreased of cell viability and mitochondrial membrane potential,inhibit the increased of apoptosis,mitochondrial and intracellular ROS generation in HCTll6 cells induced by C12-HSL.Our results indicate that C12-HSL exposure induced damages to cell viability and secretary function of LS174T goblet cells,which was mediated by PON2,oxidative stress,and caspase-1&3 cascade signal.The iulate inflammation is also involved in the adverse impacts of C12-HSL to LS174T cells,cytokines particularly IL-8 may be involved in this process.Taken together,the present study demonstrated that feeding an HC diet to lactating goats for a long period alters fermentation pattern,bacterial dysbiosis,and metabolic perturbations in the hindgut lumen.The negative effects of feeding an HC diet cause hindgut epithelial barrier dysfunction and threaten the health of the gut.