Mechanism Research On Rumen Epithelial Permeability By Subacute Ruminal Acidosis In Dairy Goats

Subacute ruminal acidosis(SARA)is a common nutritional metabolic disease in rumiants,but it is unclear that the main cause and its molecular mechanism of epithelial damage.In this study,we used in vivo and in vitro methods to evaluate the influence of SARA on ruminal epithelial permeability in dairy goats,and explore its the molecular mechanism.1.Effect of subacute ruminal acidosis on rumen fermentation parameters and abnormal metabolites in dairy goatsEight healthy Sannan dairy goats in lactating period with permanent ruminal cannula weight about 43.58 ± 2.77 kg,2?3 years old,were randomLy divided into two groups:control group and SARA group,dairy goats in control group were fed basal deit that NFC/NDF ratio was 1.40,these in SARA group were fed successively four diets with different NFC/NDF ratios(1.40?1.79?2.31?3.32 respectively)to induce subacute ruminal acidosis,experimental period lasted 60 d.The results showed that:(1)Compared with control group,duration time of ruminal pH below 5.5 kept 3.98 hours per day in SARA group.And acetic acid content and A/P ratio decreased significantly(P<0.05),propionie acid,butyric acid and TVFA contents significantly increased(P<0.05).(2)LPS and HIS contents of ruminal and plasma in SARA group were increased significantly(P<0.05)These results indicated that SARA changed rumen fermentation pattern and increased abnormal metabolites.2.Effect of subacute ruminal acidosis on ruminal epithelial permeability in dairy goatsThe animals and experiment design were the same as Experiment 1.Before these animals were slaughtered,Cr-EDTA were perfusioned through rumen cannula,collected within 48 h of urine,urinary Cr content was determined;Rumen epithelium was taken from rumen ventral sac of slaughtered animals to determine electrophysiological properties and molecular markers(FITC,HRP)flows rates using Ussing chamber instrument.The results showed that:(1)Compared with control group,PD of rumen epithelium was decreased significantly(P<0.05),Isc and Gt were increased significantly(P<0.05),FITC and HRP flows rate were also increased significantly(P<0.05)in SARA group.(2)Compared with control group,Cr-EDTA flow rate were increased significantly(P<0.05)in SARA group.These results indicated that SARA disrupted the integrity of rumen epithelium,leading to ruminal epithelial permeability increased.3.Interactional effect of pH,LPS and HIS on ruminal epithelium permeability.Eight dairy goats in lactating period with similar lactation,weight and body condition.Experimental design ussing 3×3 second factors,rumen epithelium was collected from rumen ventral sac of the slaughtered animals,adding different pH,LPS and HIS to Ussing chamber mucosal side to determine electrical physiological indexes,molecular markers(FITC,HRP)flows rate and HIS and LPS contens in serosal side,The results showed that:Multiple factors of pHxLPS,pH×HIS treatments,rumen epithelial Isc,Gt and FITC flow were the highest(P<0.05),and the PD was lower under pH=5.2×LPS treatment(P<0.05);Interaction effect on LPS concentration in the serosal side had no significant effect(P>0.05).The interaction between pH and HIS showed that rumen epithelial I sc was significantly higher than that of other treatment(P<0.05)under pH=5.2×HIS 10 treatment,Gt was significantly higher than that of other treatments(P<0.05)under pH=5.5×HIS 10 treatment,PD was significantly lower than that of other treatments(P<0.05)under pH=5.5×HIS 10 treatment,HRP flow under pH=5.2×HIS 10 treatment was higher than that of under pH=5.5×HIS 10 treatment(P<0.05)and pH-7.4xHIS 10 treatment(P>0.05),HIS concentration in the serosal side was higher under pH=7.4xHIS 0.5 treatment(P<0.05).Rumen epithelial Isc,Gt,PD and FITC flow rate were significantly affected by different pHxHISxLPS treatments(P<0.05).When the pH×LPSxHIS treatment was 5.5×60×10,rumen epithelial Isc was highest and PD was lowest(P<0.05),rumen epithelial Gt was highest under pH=5.2xHIS 60×LPS 10 treatment,and the difference between treatments were significant(P<0.05).FITC flow rate of rumen epithelium under pH=5.5×LPS 60×HIS 10 treatment was significantly higher than that of other treatments(P<0.05),and there was no significant difference between the two treatments(P>0.05),HRP flow rate in pH=5.2 treatment was higher than the other treatment(P>0.05).The effects of different pHxHISxLPS treatments on concentrations of LPS and HIS in serosal side were not siginificant.The results showed that pH=5.2 had most serious effect on rumen epithelial permeability,andt wo factors pH=5.2xHIS10 and pH=5.2xLPS 60 and three factors pH=5.5xLPS 60×HIS 10 had siginificant effect on rumen epithelial permeability4.Interactional effect of pH,LPS and HIS on the express of tight junction proteins in rumen epitheliumExperimental animals and experiment designs were similar as experiment 3,adding different pH,LPS and HIS to Ussing chamber mucosal side to culture and determine the mRNA express of tight junction proteins,such as Claudin-1,Claudin-4,Claudin-7,Occludin and ZO-1 genes.The results showed that:(1)the interaction between pH and LPS significantly decreased the mRNA express of Claudin-1(P<0.05),the mRNA express of Claudin-4 and Occludin did not change significantly(P>0.05),express of ZO-1 tended to increase and was highest under pH=5.2×LPS 60 treatment(P<0.05)which was significantly higher than that in the control group.(2)Interaction of pH and HIS.The mRNA express of Claudin-1 was the lowest under pH?5.5×HIS 0.5 treatment.Claudin-7 genen express under pH=7.4×HIS 10 treatment was significantly lower than that under control treatment(P<0.05).The mRNA express of ZO-1 was increased significantly under pH=5.2×HIS 10 treatment.(3)The mRNA express of Claudin-1 under pH=5.2×LPS 60×HIS 10 treatment was decreased significantly than that under pH5.5×LPS60×HIS 10 and pH=7.4×LPS 60×HIS 10 treatments(P<0.05).Claudin-4 gene express had no significantly change(P>0.05),but had decline tendency compared with control treatment.Claudin-7 gene express was lower than that of control treatment,and the lowest and decreased by 99%under pH=5.2xLPS 60×HIS 10 treatment(P<0.05).The mRNA express of Occludin under pH=5.2×LPS 60×HIS 10 was significantly higher than that under control treatment(P<005).ZO-1 gene express under pH=7.4×LPS 60×HIS 10 treatment was increased significantly than that under pH=5.5×LPS 60×HIS 10 and pH=5.2×LPS 60×HIS 10 treatments.These results suggested that SARA increased rumen epithelial permeability was associated with decrease of tight junction protein expression.The above results showed that:(1)SARA increased rumen epithelial permeability,which was associated with decrease of the tight junction protein expression;(2)SARA induced increase of rumen epithelial permeability was pH,LPS and HIS together to play a role.