Rhythm Of Volatile Fatty Acid Transporters In Rumen Epithelium And Its Regulation By Period2

Circadian rhythm is a kind of biorhythm with a cycle of approximately 24 h.The physiological and metabolic rhythms of mammals are controlled by circadian rhythm.The oscillations of circadian rhythm lead to rhythmic changes in the downstream molecular pathways and physiological processes,and play a certain regulatory role in the digestion,absorption and metabolism of nutrients.Previous studies on rumen epithelial cells and tissues in vitro culture,rumen epithelial cell silencing and PER2 gene knockout in mice showed that PER2 was correlated with VFA absorption and metabolism in rumen,which may have an important regulatory role,but the specific rules and mechanisms are still unclear.Therefore,in this study,the regulation mechanism of PER2 on volatile fatty acid transport in rumen epithelium was explored by in vivo perfusion of VFA and in vitro overexpression of PER2 gene.Trail 1:Rumen VFA Perfusion to study the relationship between rumen epithelial rhythm proteins and VFA absorption Based on the previous experimental results of rumen epithelial cells and tissues in vitro culture,this part further explored the mechanism of rumen epithelial rhythm proteins regulating VFA absorption in vivo.In this experiment,dairy goats were infused with 0.8 g/kg·d sodium acetate,0.8 g/kg·d sodium propionate and 0.5 g/kg·d sodium butyrate solution,which were acetic acid group,propionic acid and butyric acid group.In addition,the control group was infused with equal volume of normal saline.During the sampling period,50 ml rumen fluid was collected by gastric tube after perfusion to detect rumen fermentation indexes.After gavage,the rumen epithelial tissues of dairy goats were collected and the rumen epithelial histomorphology,the expression of biological clock genes and VFA absorption and metabolism related genes were detected.The results showed that the pH value fluctuated from 5.90 to 6.73,which was within the normal range of rumen pH,and there was no significant difference among the groups(P>0.05).The concentration of NH3-N showed a trend of curve variation,which varied from 3.43 mg/dL to 7.40 mg/dL,and there was no significant difference in NH3-N concentration among all groups(P>0.05).The molar concentrations of acetic acid,propionic acid and butyric acid in acetic acid group,propionic acid group and butyric acid group were significantly higher than those in the other three groups(P<0.05).In addition,the expression of PER1 and PER2 genes were the highest in the propionic acid group and the lowest in the butyric acid group,and the differences among all groups were significant(P<0.05).The expression of CLOCK gene was significantly affected by SCFAs(P<0.05).The expression level of NCOR1 gene in normal saline group was significantly higher than that in acetic acid and butyric acid groups(P<0.05),but there was no significant difference between normal saline group and propionic acid group(P>0.05).The expression of BMAL1,Cry1,CRY2,PPARA and FBXW11 gene were the highest in acetic acid group and the lowest in butyric acid group,but there was no significant difference among all groups(P>0.05).In the correlation between PER2 gene and VFA related absorption gene expression,PER2 gene was positively correlated with the relative expression of NHE1,NHE3,Na/KATPase and PAT1 gene(P>0.05),and negatively correlated with the relative expression of MCT1 and MCT4 gene(P>0.05).In conclusion,in vivo studies on rumen epithelial tissues verified that PER2 gene regulates the absorption and metabolism of rumen VFA through positive regulation of NHE1,NHE3,Na/K ATPase and PAT1 genes,and negative regulation of MCT1 and MCT4 genes,and confirmed the results found in the previous experiments in vitro culture of rumen epithelium and tissue.Trail 2:In vitro culture of rumen epithelial cells to investigate the rhythmic expression of VFA transport related geneIn order to further study the mechanism of VFA absorption regulated by the circadian proteins in rumen epithelium,this part of the study explored whether VFA transport-related factors were regulated by the rhythm factors and whether they are related to the expression of circadian genes.The primary goat rumen epithelial cells were selected to study the circadian rhythm of the above VFA transporters.The goat rumen epithelial cells were cultured in vitro for 48 h.The samples were collected at 4 h intervals and repeated in 6 Wells at each time point to determine the rhythmicity of Rhythm-related factors and VFA transport-related gene expression.The results showed that the expression of PER2 gene was significantly different at the time point(P<0.05),and the expression of PER2 gene was fitted by Nonlinear regression,which was in accordance with the daily rhythm variation of cosine function,the fitted cosine function equation is YPER2=1.497+1.086*cos(x+0.359),R2=0.896.In addition,the expression of PER1,PER3,BMAL1,Cry1,Cry2,NR1D1,NR2F2,NCOR1 and FBXW11 genes were significantly different at different time points(P<0.05).The gene expression of VFA transport-related factors was significantly different at the time point,and partly accorded with the daily rhythm of cosine function.The fitting curves were as follows,MCT4:Y=2.306+1.997*cos(x+0.743),R2=0.926;NHE1:Y=1.130+0.770*cos(x+19.383),R2=0.678;NHE3:Y=1.095+0.658*cos(x-0.678),R2=0.832;VH ATPase:Y=4.386-3.024*cos(x-2.173),R2=0.664;Na/KATPase:Y=1.095+0.658*cos(x-0.678),R2=0.832.The relative expression of PER2 gene and MCT4 gene was negatively correlated(P<0.05).The results of this part showed that the expression of PER2 gene,related circadian clock genes and the VFA-related transporter gene in goat rumen epithelial cells had a daily rhythm pattern of near cosine function,and there was a certain correlation between PER2 gene and the daily rhythm of VFA related genes.Trail 3:The effect of PER2 gene overexpression on the growth and proliferation of rumen epithelial cells and the expression of VFA absorption proteinBased on the results of perfusion test in vivo and the exploration of the rhythm of the expression of VFA transport-related factors,pcDNA3.1-PER2 plasmid was constructed in this part,and goat rumen epithelial cells were cultured in vitro to overexpress PER2 gene,and the treatment Control group(pcDNA3.1)and the overexpression group(pcDNA3.1-PER2)were set,with 4 biological replicates in each group.The levels of cell proliferation,apoptosis,cell cycle,antioxidant indexes,the expression of circadian clock related genes,VFA transporter genes and related regulatory factors were measured.The results showed that 48 h after pcDNA3.1-PER2 plasmid was transfected,the relative expression of PER2 gene was 4061.63,the overexpression of PER2 gene in the overexpression group was 3471.48 times of that in the control group(P<0.01),which indicated that the PER2 gene was overexpressed successfully,providing a reliable experimental basis for the follow-up experiments.After overexpression,the level of apoptosis was significantly increased(P<0.05),and the proliferative activity of overexpression group was significantly lower than that of control group at 24 h and 48 h after transfection(P<0.05),indicating that overexpression inhibited the proliferation of rumen epithelial cells.The overexpression of PER2 gene significantly increased the expression levels of CCND1,WEE1,P21 and P16(P<0.05),but significantly decreased the expression levels of CDK4 gene(P<0.05).There was no significant change in the expression of intracellular inflammatory genes after the overexpression of PER2 gene(P>0.05),but the expression of TNF-α was significantly increased.Compared with the control group,there were no significant changes in SOD,MDA,GSH-Px,CAT and T-AOC in rumen epithelial cells of goats in the overexpression group,indicating that the overexpression of PER2 gene did not cause oxidative damage to rumen epithelial cells of goats.Furthermore,PER2 overexpression significantly down-regulated the mRNA expressions of AE2,NHE1,MCT1,and MCT4,and up-regulated the mRNA expressions of PAT1,VH+ATPase.In this part,the results showed that overexpression of PER2 gene inhibited cell proliferation and promoted cell apoptosis,and confirmed the regulation of PER2 gene on VFA transport-related factors in rumen epithelium.To sum up,PER2 gene may regulate VFA absorption by regulating VFA absorption transporter in rumen epithelial cells,and its specific regulatory pathway needs to be further studied.