Effect Of Dietary Concentarte Level On Rumen Epithelium Growth And Its Underlying Mechanism

Past research has demonstrated that diets could modulate SCFA absorption capacity of rumen via the changed expression of transporters,which are involved in SCFA absorption.Despite,the underlying mechanisms are not completely clear.The present study showed that the diet concentrate level affect the growth of rumen epithelium,but the mechanism remains unclear.This paper by ruminants rumen epithelium as the research object,concentrate to study level of rumen epithelial cell proliferation and apoptosis effect and its mechanism1.Effect of dietary concentrate level on mRNA expression of transporters related to cell proliferation and cell apoptosis in ruminal epithelium of goats and its underlying mechanism.Goats(n=12)were randomly allocated to 2 groups and fed either a low concentrate diet(LC,10%concentrate,n=6)or a medium concentrate diet(MC,35%concentrate,n=6)in 2 equal portions daily.Goats were fed separately with their respective diet for 3 wk.The goats were slaughtered 6 h after the morning feeding on d 22.By meteorological chromatography determination of rumen content of volatile fatty acids(SCFA),flow cytometry detection rumen epithelial cell cycle changes,Tunel method to detect rumen epithelial cell apoptosis,Real-time PCR method to detect gene expression.Results show that compared with LC group of goats,MC set is slaughtering tumor significantly increased the content of gastric juice in SCFA,the pH value of gastric juice and tumor significantly below LC group.In addition,the goat test results also showed that increasing diet concentrate content in promoting goat rumen epithelial cell proliferation related genes(cyclin A,cyclin B1,cyclin D1,cyclin E1,CDK1,CDK2,CDK4 and CDK6)and cell apoptosis related gene(Caspase 3,Caspase 9,p53 and Bax)gene expression(P<0.05).In vitro experiment results show that the genes involved in cell proliferation,compared with the control group(pH7.4 slightly),acid treatment(pH6.8)to promote the generation of culture goat rumen epithelial cell cyclin D1,CDK2 and CDK6 mRNA expression(P<0.05);At the same time,pH7.4 slightly+SCFA group can promote A,the generation of cyclin culture goat rumen epithelial cells,cyclin D1,cyclin E1,CDK2,CDK4 and CDK6 mRNA expression(P<0.05);Compared with group pH6.8 pH6.8+SCFA treatment can promote the original generation culture goat rumen epithelial cell cyclin B1,mRNA expression of cyclin E1,CDK1 and CDK4(P<0.05);Compared with pH7.4 slightly+SCFA group compared,pH6.8+SCFA group of primitive culture goat rumen epithelial cells can promote the cyclin B1,cyclin E1,CDK1,CDK2 and CDK6 mRNA expression(P<0.05),but decreased the expression of cyclin A mRNA(P<0.05).At the same time,in regulating cyclin A,cyclin B1,cyclin E1 and CDK6 mRNA expression in the process of acid and SCFA has synergy(interaction-the value P<0.05).In cell apoptosis related gene,compared with the control group(pH7.4 slightly),acid treatment(pH6.8)to promote the generation of Caspase 3 culture goat rumen epithelial cells,Caspase 8,Caspase 9 and p53 mRNA expression(P<0.05),but the Bel-2 and the ratio of Bax(Bel-2/Bax)lowered(P<0.05);At the same time,pH7.4 slightly+SCFA treatment group can promote the original generation culture goat rumen epithelial cells of Caspase 3,Caspase 9 and Bax mRNA expression(P<0.05),but the Caspase 8,p53 and Bel-2/Bax lower(P<0.05);Compared with group pH6.8 pH6.8+SCFA treatment can promote the original generation culture goat rumen epithelial cells of Caspase 3,the Bel-2 and Bax mRNA expression(P<0.05),Caspase 8 and mRNA expression of p53,lower(P<0.05);Compared with pH7.4 slightly+SCFA group compared,pH6.8+SCFA group of primitive culture goat rumen epithelial cells can promote the Caspase 3 mRNA expression(P<0.05).At the same time,the regulation of p53,Bcl-2 and in the process of the Bel-2/Bax,acid and SCFA has synergy(interaction-the value P<0.05).The results show that in vivo and in vitro diet concentrate level through the rumen SCFA and pH effects in goat rumen epithelial cell proliferation and apoptosis related genes mRNA expression.2.Effect of dietary concentrate level on mRNA expression of transporters related to cell proliferation and cell apoptosis in ruminal epithelium of cows and its underlying mechanism.Four non-lactating,farrow Holstein cows,fitted with ruminal cannulas,were used in this experiment.Test points and 2),each feeding concentrate content is 60%(high concentrate group,HC)and 20%(low concentrate group,LC)of two kinds of mixed diet.During the test,8:00 and 17:00 feeding the cows twice a day,and guarantee its free drinking water.The last day of each period(15 days)to collect the samples.In vitro experiment selected 4 healthy cow,slaughtering acquisition rumen epithelium,trypsin digestion after winning rumen epithelial cells cultured the original generation,after waiting for 24 h,cell adhesion,respectively,to accept the following processing:pH7.4 slightly,pH 6.8,pH7.4 slightly+SCFA(20 mM SCFA)with pH6.8+SCFA processing.24 h hours,collection cell waiting for inspection.By meteorological chromatography determination of rumen content of volatile fatty acids(SCFA),flow cytometry detection rumen epithelial cell cycle changes,Tunel method to detect rumen epithelial cell apoptosis,Real-time PCR method to detect gene expression.Results show that compared with LC group of goats,MC set is slaughtering tumor significantly increased the content of gastric juice in SCFA,the pH value of gastric juice and tumor significantly below LC group.In addition,the goat test results also showed that increasing diet concentrate content in promoting goat rumen epithelial cell proliferation related genes(cyclin A,cyclin Bl,cyclin Dl,cyclin E1,CDK1,CDK2,CDK4 and CDK6)and cell apoptosis related gene(Caspase 3,Caspase 9,p53 and Bax)gene expression(P<0.05).In vitro experiment results show that the genes involved in cell proliferation,compared with the control group(pH7.4 slightly),acid treatment(pH6.8)to promote the generation of culture goat rumen epithelial cell cyclin D1,CDK2 and CDK6 mRNA expression(P<0.05);At the same time,pH7.4 slightly+SCFA group can promote the generation of cyclin culture goat rumen epithelial cells,cyclin Dl,cyclin E1,CDK2,CDK4 and CDK6 mRNA expression(P<0.05);Compared with group pH6.8 pH6.8+SCFA treatment can promote the original generation culture goat rumen epithelial cell cyclin Bl,mRNA expression of cyclin E1,CDK1 and CDK4(P<0.05);Compared with pH7.4 slightly+SCFA group compared,pH6.8+SCFA group of primitive culture goat rumen epithelial cells can promote the cyclin B1,cyclin E1,CDK1,CDK2 and CDK6 mRNA expression(P<0.05),but decreased the expression of cyclin A mRNA(P<0.05).At the same time,in regulating cyclin A,cyclin Bl,cyclin El and CDK6 mRNA expression in the process of acid and SCFA has synergy(interaction-the value P<0.05).In cell apoptosis related gene,compared with the control group(pH7.4 slightly),acid treatment(pH6.8)to promote the generation of Caspase 3 culture goat rumen epithelial cells,Caspase 8,Caspase 9 and p53 mRNA expression(P<0.05),but the Bel-2 and the ratio of Bax(Bel-2/Bax)lowered(P<0.05);At the same time,pH7.4 slightly+SCFA treatment group can promote the original generation culture goat rumen epithelial cells of Caspase 3,Caspase 9 and Bax mRNA expression(P<0.05),but the Caspase 8,p53 and Bel-2/Bax lower(P<0.05);Compared with group pH6.8 pH6.8+SCFA treatment can promote the original generation culture goat rumen epithelial cells of Caspase 3,the Bel-2 and Bax mRNA expression(P<0.05),Caspase 8 and mRNA expression of p53,lower(P<0.05);Compared with pH7.4 slightly+SCFA group compared,pH6.8+SCFA group of primitive culture goat rumen epithelial cells can promote the Caspase 3 mRNA expression(P<0.05).At the same time,the regulation of p53,Bel-2 and in the process of the Bel-2/Bax,acid and SCFA has synergy(interaction-the value P<0.05).The results show that in vivo and in vitro diet concentrate level through the rumen SCFA and pH affects the cows in the rumen epithelial cell proliferation and apoptosis related genes mRNA expression.3.Effects of dietary concentrate level on cell proliferation and cell apoptosis in rumen epithelium of cows and its underlying mechanism.Four non-lactating,farrow Holstein cows,fitted with ruminal cannulas,were used in this experiment.The cows were fed in equal allotments at 0800 and 1700.The experiment was conducted over 1 month,during which time the animals were fed either a high concentrate diet(HC,60%concentrate)or a low concentrate diet(LC,20%concentrate)in two phases.Each experimental phase consisted of 15 days,and samples were collected on d 15 of each experimental stage.In vitro experiment selected 4 healthy cow,slaughtering acquisition rumen epithelium,trypsin digestion after winning rumen epithelial cells cultured the original generation,after waiting for 24 h,eell adhesion,respectively using TNF alpha(50 mu g/L),TNF alpha inhibitors(50 mu M/L),ERK inhibitors(20 mu M/L),JNK inhibitors and p38 lightning inhibitors(15 mu M/L)cells(0 and 1 h and 24 h).After the treatment,collecting cells waiting for inspection.By meteorological chromatography detection in the rumen SCFA levels;Real-time PCR and Western blot method respectively to detect the expression of genes and proteins;Ria method is used to detect the content of cow serum TNF alpha.Cows in vivo experiment results show that the high concentrate diet leads to the cow in the rumen SCFA levels rise,pH,concentration of serum TNF alpha increased(P<0.05).High concentrate diet promote cow rumen epithelial cycle D1,the expression of CDK4 and Caspase3 protein,enhance TNF alpha receptor mRNA expression,but also increase the rumen epithelium in phosphorylation of ERK and JNK and p38 lightning protein level(P<0.05).The in vivo experiment results show that high concentrate diet activate the cow rumen epithelium of ERK,p38 lightning and JNK pathway.In vitro test results show that compared with the control group(0 min),TNF alpha treatment(50 mu g/L)significantly increased the original generation of rumen epithelial cells ERK,JNK and p38 lightning protein phosphorylation level(P<0.05);TNF alpha on rumen epithelial cells ERK,JNK and p38 lightning protein phosphorylation of inhibition by TNF alpha inhibitors(Lenalidomide)(P<0.05);TNF alpha in the rumen epithelial cells cycle Dl,CDK4 and Caspase3 gene and protein expression(P<0.05),and the promoting effect of TNF alpha inhibitors,ERK inhibitors,JNK inhibitors and p38 lightning inhibitors inhibit(P<0.05).In vitro test results show that the cyclinD1 and CDK4 mRNA and protein expression of TNF alpha ERK and TNF alpha p38 lighining channel regulation,Caspase3 mRNA and protein expression of TNF alpha JNK and TNF alpha p38 lightning channel regulation.The results show that high concentrate of diet in vivo and in vitro by TNF alpha and its receptor pathway(ERK and p38 lightning)promote cow rumen epithelial cyclinD1 and CDK4 gene and protein expression;After through TNF alpha and its receptor pathway(JNK and p3 8 lightning)promote cow rumen epithelial Caspase3 gene and protein expression.To sum up,this thesis studies have shown that the diet of concentrate level by rumen cavity factor(SCFA and pH)and plasma TNF alpha influence in the rumen epithelial cell proliferation and apoptosis related gene expression.