The Mechanism Of Ruminal Epithelial Adaptation To High-grain Diet In Sheep

High-grain(HG)diet is usually used to improve the production performance in the modern intensive farming.However,HG diet is fermented quickly in rumen with accumulation of VFA and decrease of pH.It can cause rumianl acidosis and impair ruminal stability.In response to the impairments,rumen physiological structure will be changed,and long-term feeding will adapt to the stress caused by HG diets.However,little is known about the molecular basis of epithelial proliferation in sheep during HG adaptation.Therefore,the objective of this study was to investigate the ruminal epithelial adaptation to HG diet at the tissue,molecular and cellular levels and to explore the mechanism of cellular signaling pathways involved in ruminal epithelial growth in sheep.1.Morphological and functional adaptation of sheep's rumen epithelium to HG diet.The objective of this study was to investigate the morphological and functional adaptation of sheep's rumen epithelium to HG diet.Twenty sheep(Hu Sheep,BW of 25.60±0.41 kg,mean±SD,approximately 180 days of age)were assigned to four groups with five animals in each.These animals were assigned to different periods of HG diet(containing 40%forage and 60%concentrate mix)feeding.The HG groups received an HG diet for 7(G7,n=5),14(G14,n=5)and 28 d(G28,n=5),respectively.In contrast,the control group(CON,n=5)was fed with the forage-based diet for 28 days.The results showed that the daily gain weight of G28 group was higher than that in G7 group(P=0.015).However,there was no difference of the dry matter intake within groups(P=0.367).The empty weight of rumen/body weight(%)in G14 group increased than that in CON group(P=0.018).HG feeding linearly decreased(P<0.001)ruminal pH,and increased the concentrations of ruminal total volatile fatty acid(linear,P=0.001),butyrate(linear,P<0.001)and valerate(quadratic,P=0.029).The length(quadratic,P=0.004),width(cubic,P=0.015)and surface of the ruminal papillae(linear,P=0.003)were all enlarged after 14 d of HG diet feeding.HG feeding cubically increased the number of cell layers forming the stratum corneum(SC,P<0.001)and the thickness of the SC(P<0.001)and stratum basale(P<0.001).The present study showed that HG feeding changed morphology of rumen epithelium,which adapted transitional diet and mitigated the damage of rumen.2.The mechanism of ruminal epithelium functional adaption to HG diet in sheep by the proteomic analysis.The objective of this study was to evaluate the mechanism of RE fuctional adaption to HG diet in sheep with LC-MS/MS label-free methods.The animal experimental design was the same as the first chapter.Results showed that totally 146 significant differential proteins were identified with<0.05 FDR and>1.5 VIP-value.These proteins were classified into three clusters according to their expression trends.Interestingly,56 significant differential proteins were selected based on biological process and KEGG pathway,which were classified into four categories:cell growth regulation,actin cytoskeleton regulation,cell junction or adhesion regulation,and metabolic regulation.Furthermore,29 candidate proteins corresponding genes were further verified by real-time PCR.Results showed that the relative transcript level of PTGIS,CRYAB,ITGA5,ITGB1,FLNA,PPP1R12A,ILK,VCP and STAT3 in the function 1 matched well with the comparative proteomic data.These proteins were regulated by HG feeding,indicating that the rumen epithelium growth associated with cell cycle regulation,apoptosis block and proliferation increase.The relative transcript level of ARPC4,APRC5L,PFN2,ACTN1 and ACTN4 in the function 2 and 3 was also matched well with the comparative proteomic data.These five proteins might protect the rumen epithelium barrier function and restore the integrity of the cell junctions.The expression of genes in function 4 including OXCT1,UGT1A1,PFKM,DDOST and P4HB were consistent with the proteomics data as well.In conclusion,HG feeding affected the functional adaption in RE involved in the interaction of cell proliferation,cell apoptosis,cell cycle progress,actin cytoskeleton regulation,cell junction or adhesion regulation and metabolic regulation.3.The effect of HG diet long term feeding on cell cycle and apoptosis of rumen epithelium in sheep.The objective of this study was to evaluate alterations of cell cycle and cell apoptosis in the RE of sheep.The animal experimental design was the same as the first chapter.Results showed that the proportion of basal layer cells in the RE decreased(linear,P<0.001)in G0/G1-phase,but increased linearly(P=0.006)in S-phase and cubically(P=0.004)in G2/M-phases.The mRNA expression of Cyclin El(P=0.021)and CDK-2(P=0.001)and the protein expression of Cyclin E1(P=0.027)were linearly decreased with HG diet feeding.The proportion of apoptosis cells in G7,G14 and G28 was reduced compared to that in CON(quadratic,P<0.001).The expression of cell apoptosis gene Caspase8 decreased(quadratic,P=0.012),while Bad mRNA expression tended to decrease(cubic,P=0.053)after HG feeding.Thus,long term HG feeding enhanced expression of CyclinE1 and CDK2,which accelerated cell cycle and blocked apoptosis.4.The effect of HG diet long term feeding on IGF-1 signal pathway of rumen epithelium in sheep.The objective of this study was to explore the molecular mechanism of the rumen epithelial proliferation and apoptosis during adaptation of HG feeding.The animal experimental design was the same as the first chapter.The results showed that the IGF-1 level in plasma increased quadratically(P=0.002)with long term HG feeding.IGF-1 mRNA expression increased quadratically(P<0.001).IGF-1R mRNA expression was increased linearly(P<0.001)and the IRS-1 gene expression was increased cubically(P=0.029)after HG feeding.Rafl(P<0.001)and MAPK(P=0.006)gene expression were increased linearly.IGF binding protein relative genes,IGFBP2 and IGFBP5 gene expression were increased linearly(P<0.001),while IGFBP3 gene expression was decreased linearly(P=0.002)with HG feeding.Additionally,the results of protein expression involved in IGF-1 signaling pathway showed that p-p38 was increased(linear,P=0.003)according to time series while no difference of p-ERK and p-AKT was observed within groups.Correlation analysis revealed that changes of butyrate concentration in rumen fluid and IGF-1 in plasma were coupled with changes in genes involved in proliferation and apoptosis.These results demonstrated that HG feeding increased butyrate concentration in rumen and IGF-1 concentration in plasma,accelerated cell cycle and blocked cell apoptosis by IGF-1 signaling pathway,which can accelerate cells proliferation of rumen epithelium and maximize the rumen papillae sizes.5.The mechanism of rumen epithelium cell growth in sheep's based on IGF-1 and sodium butyrate in vitroThe objective of this study was to explore the effects and mechanism of IGF-1 and butyrate on rumen epithelial cell growth in vitro.The primary ruminal epithelium cells were isolated from caudal blind sacs of rumen in sheep(180-day male).After the rumen epithelial cells were isolated and digested,the rumen epithelial cells were cultured to adhering,and then treated with different concentrations of IGF-1 or sodium butyrate.This study included three sub-experiments:5.1.Effects of different levels of IGF-1 on cell cycle and apoptosis of ruminal epithelium cells.Isolated ruminal epithelia from caudal blind sacs of sheep were incubated with IGF-1 at different concentrations.The IGF-1 concentrations were 0,25,50,75 ?g/L.Proportions of ruminal epithelium in different phases of the cell division cycle and apoptosis were determined by flow cytometric analysis,and the gene expression of cell cycle and apoptosis were determined by Real-time PCR.The proportion of epithelial cells in GOG1 phase(P=0.001)was increased quadratically(P=0.012)and G2M phase(P=0.002)was increased linearly(P=0.003)whereas the proportion of epithelial cells in S phase(P=0.001)was decreased linearly after incubated with increasing concentration of IGF-1.The proportion of apoptosis was increased quadratically and was the highest proportion in 50 ?g/L IGF-1.Cyclin E1(P=0.002)gene expression was decreased linearly(P=0.005).While CDK2,Caspase3,Caspase8 and Bad gene expression were decreased quadratically(P<0.001).The gene expression levels of CDK-4 and CDK-6 cubically changed with increasing IGF-1 concentration(P<0.001).The results indicated that IGF-1 promoted the cell cycle progression of rumen epithelial cells by shortening the S phase,and enhanced the apoptosis of rumen epithelial cells with the increase of IGF-1 concentration.5.2.Effects of different levels of sodium butyrate on cell cycle and apoptosis of RE.The objective of this study was to investigate the effects of different levels of sodium butyrate on rumen epithelial cell cycle and apoptosis.The rumen epithelial cells were treated with sodium butyrate at concentration levels of 0,2,4,6 and 8 mmol/L respectively.After 24 h of cell culture,the cells were collected and cell cycle and apoptosis were detected by flow cytometry.Gene expression related with cell cycle and apoptosis was detected by Real-time PCR.The results showed that the proportion of cells in G0G1 phase of rumen epithelial cells was increased linearly(P=0.006)with the increase of sodium butyrate concentration.The proportion of rumen epithelial cells in S phase was linearly decreased(P=0.001),and the proportion of G2M ruminal epithelial cells changed quadratically(P=0.012).The proportion of apoptosis in ruminal epithelial cells was changed quadratically(P<0.001)with the increase of sodium butyrate concentration.Gene expression levels of CyclinDl and CyclinA2 in rumen epithelial cells of sheep were changed quadratically(P<0.001)with the increase of sodium butyrate concentration.The gene expression levels of CyclinE1(P<0.001),CDK-2(P<0.001),CDK-4(P=0.004)and CDK-6(P<0.001)in rumen epithelial cells of sheep decreased linearly with increasing concentration of sodium butyrate.The gene expression of Caspase3 in ruminal epithelial cells changed quadratically(P<0.001),and the genes expression levels of Caspase8 and Bad in rumen epithelial cells of sheep were changed cubically(P<0.001)with the increasing concentration of sodium butyrate.The results showed that sodium butyrate also promoted the cell cycle progression of rumen epithelial cells by shortening the S phase.2 mM sodium butyrate can block cell apoptosis while 8 mM sodium butyrate didn't work.5.3.The mechanism of combined IGF-1 and sodium butyrate on cell proliferation and apoptosis of RE.The aim of this study was to know the effect and mechanism of combined IGF-1 and sodium butyrate on rumen epithelial cell proliferation and apoptosis.Ruminal epithelial cells were treated with DMEM(0 ?g/L IGF-1+0 mM sodium butyrate),25 ?g/L IGF-1+2 mM sodium butyrate,50 ?g/L IGF-1+4 mM sodium butyrate,75 ?g/L IGF-1+6 mM sodium butyrate and 100 ?g/L IGF-1+8 mM sodium butyrate.After 24 hours of culturing,cells were collected and then the genes of cell cycle and apoptosis were quantified.The results showed that the gene expression levels of CyclinDl(P=0.003),CyclinA2(P=0.011)and CyclinEl(P=0.033)in the rumen epithelial cells of the sheep changed quadratically with the increasing concentration of the combined IGF-1 and sodium butyrate.The gene expression levels of CyclinDl,CyclinA1 and CyclinE1 were the highest in the 50?g/L IGF-1 and 4 mM sodium butyrate treatment groups.The gene expression level of CDK-2(P=0.003)in rumen epithelial cells was linearly increased with the increasing concentration of the combined IGF-1 and sodium butyrate,and the gene expression levels of CDK-2 were the highest in the 50 ?g/L IGF-1 and 4 mM sodium butyrate treatment group.The gene expression level of CDK-6(P=0.001)in sheep rumen epithelial cells was changed cubically,and the genes expression levels of Caspase3(P=0.002)and Bcl2(P=0.022)in the rumen epithelial cells of sheep changed quadratically with the increasing concentration of the combined IGF-1 and sodium butyrate.Since the effect of combined 50?g/L IGF-1 and 4 mM sodium butyrate worked on rumen epithelial cells significantly.On this basis,the effects of combined 50 ?g/L IGF-1 and 4 mM sodium butyrate on the proliferation of rumen epithelial cells were further explored.The cells were treated with a combination of 50 ?g/L IGF-1 and 4 mM sodium butyrate.The cells were divided into five groups:negative control group(added DMEM)and positive control group(added 50 ?g/L IGF-1 and 4 mM sodium butyrate),ERK inhibitor group(added 50 ?g/L IGF-1,4 mM sodium butyrate and ERK inhibitor),AKT inhibitor group(added 50 ?g/L IGF-1,4 mM sodium butyrate and AKT inhibitors),p38 inhibitor group(added 50 ?g/L IGF-1,4 mM sodium butyrate and p38 inhibitor)and ERK inhibitor AKT inhibitor p38 inhibitor group(added 50 ?g/L IGF-1,4 mM sodium butyrate,ERK inhibitor,AKT inhibitor and p38 inhibitor).After 24 hours of treatment cells,the cells were collected,and the key proteins on the cell proliferation signaling pathway were detected by Western Blot.The results showed that 50 ?g/L IGF-1 and 4 mM sodium butyrate could up-regulate the proteins expression of pERK,pAKT and pP38 in rumen epithelial cells,while ERK inhibitors,AKT inhibitors and p38 inhibitors may affect the corresponding phosphorylated proteins in rumen epithelium cells.The results indicated that the biological processes of cell proliferation and apoptosis were achieved by Ras/Raf/MEK/ERK signaling pathway,PI3K/AKT signaling pathway and p38 MAPK signaling pathway in the presence of IGF-1 and butyrate.In conclusion,high-grain diet increased the butyrate concentration in rumen and IGF-1 concentration in plasma,accelerated cell cycle and blocked cell apoptosis,and promoted cell proliferation by Ras/Raf/MEK/ERK signaling pathway,PI3K/AKT signaling pathway and p38/MAPK signaling pathway,which can maximize the absorption area of rumen epithelium in order to keep ruminal homeostasis and the morphology adaptation.