Study On The Mechanism Of PER2 Affecting The Absorption Of Volatile Fatty Acids In The Digestive Tract

Volatile fatty acids(VFA)have a regulatory effect on rumen fermentation and is the main energy supply to the host in ruminants.Our previous study with rumen epithelial cells found that there was a certain correlation between PER2 factor and VFA absorption.However,the specific regulatory mechanism is still unclear.Therefore,this study was carried out to investigate the regulatory mechanism of PER2 on VFA uptake in the digestive tract epithelium by using both the in vitro silencing and the in vivo knocking out of PER2 gene.Experiment 1:Relationship between PER2 expression and VFA uptake-related genes in rumen epithelial tissue cultured in vitroIn this part,3-month-old Boer goats were selected for rumen tissue culture in vitro.The study included 4 groups:Control group.Acetic acid group.Propionic acid group and Butyric acid group.The culture medium inside was mixed with buffer liquid and rumen liquid(1:1),then,15 mmol/L acetic acid,propionic acid and butyric acid were added to Acetic acid group,Propionic acid group and Butyric acid group respectively.The culture medium outside is a conventional culture solution.The culture medium inside and outside was collected for Oh,3h,6h and 24h to measure the values of pH,OD,the ammonia nitrogen concentration and the VFA concentration.Tissue samples were collected at Oh and 24h to measure the expression of circadian clock genes such as PER2 and VFA related genes.The results showed that the concentration of ammonia nitrogen increased with time going(P<0.05),but there was no significant difference between treatment groups(P>0.05).The concentrations of acetic acid,propionic acid and butyric acid were significantly higher in the acetic acid group,propionic acid group and butyric acid group than in the other three groups(P<0.05).The relative expression of PER2 in propionic acid group was significantly higher than that in other groups(P<0.05).There was no significant difference in PER2 expression among acetic acid group,butyric acid group and control group(P>0.05).Furthermore,different VFA treatments significantly inhibited the expression of CLOCK(.P<0.05),but had no effects on the relative expressions of PPAR-a and PPAR-y(P>0.05).The correlation analysis indicated that the relative expression of biorhynomic PER2 is positively correlated with AE2,but negatively correlated with the relative expression of MCT1(P<0.05).In the study of culture of rumen epithelial tissue,it was confirmed that PER2 has a certain correlation with VFA-related genes by positively regulating the relative expression of AE2 and negatively regulating the relative expression of MCT1.Experiment 2:Effects of PER2 silencing on the proliferation of rumen epithelial cells and the protein expression of VFA uptake factorIn this part,three groups of siRNA interference sequences(iRNA-PER2-a/b/c)were used to transfect porcine goat rumen epithelial cells.Real-time PCR was used to determine the expression of PER2 mRNA in different sequences of interference fragments at 36h,48h and 60h to compare the silencing efficiency respectively.On this basis,the interference fragment with the best silencing effect and the time point of transfection were screened for the silencing of PER2 in rumen epithelial cells.The treatments were divided into control group and experimental group(silencing group).Cell viability was measured by CCK8 method(Cell counting 8),and apoptosis was measured by Annexin V-FTC kit.At the same time,the mRNA levels and related protein levels of the circadian clock and VFA uptake-related genes were determined by Real-time PCR and Western-bolt methods.The results showed that the interference fragment siRNA-PER2-b had the best silencing effect at 48h,and the silencing efficiency of PER2 reached 72.07%.The mRNA expression level and protein level of the PER2 in the experimental group were significantly lower than those in the control group(P<0.05),indicating that the selected siRNA fragment silenced the PER2.The cell viability in the experimental group decreased significantly with time going and then increased significantly(F<0.05),but the early apoptosis and late apoptosis in the experimental group were significantly lower than those in the control(P<0.05).In addition,the mRNA expression levels of CLOCK,PPAR-a and PPAR-y were significantly down-regulated in the experimental group(P<0.05),and the mRNA expression levels of PPAR-?,MCT1 and MCT4 were significantly up-regulated(P<0.05).The level of PPAR-a protein in the experimental group was significantly lower than that in the control group,but the level of NHE1 protein the experimental group was significantly higher than that in the control group(P<0.05).In the study of PER2 silencing in rumen epithelial cells,it was confirmed that PER2 positively regulates CLOCK,PPAR-? and PPAR-? genes,and negatively regulates PPAR-?,MCT1 and MCT4 genes.Experiment 3:Effects of PER2 knockout on the protein expression of VFA uptake factor and TLR in posterior digestive tract of miceIn this part,eight homozygous mice(4 males and 4 females)were used as the experimental group,and 8 wild-type mice(4 males and 4 females)were used as the control group,and each group had 4 pairs of mice,half male and half female.And these mice were provided by Beijing Biotech Biotechnology Co.,Ltd.Different genomic mice were managed and fed together with 8h light;16h dark per day for 2 weeks(8L:16D).The contents and tissue samples of the colon and cecum of the mice were collected to measure VFA concentration,Toll factor content,VFA absorption related genes and protein content.The results showed that in the cecal contents,PER2 knockout significantly down-regulated the molar concentration of propionate and significantly increased the molar concentration of butyric acid(P<0.05).PER2 knockout significantly down-regulated the content of TLR2 in colon tissue and up-regulated the content of MyD88 in cecal tissue(P<0.05).PER2 knockout significantly up-regulated MCT1 in colon tissue.PER2 knockout significantly up-regulated the relative expression of PPAR-? and NHE1 in colon tissue(P<0.05).In colon and cecal tissues,knockout of PER2 gene significantly down-regulated PER2 protein content and significantly up-regulated NHE1 and PPAR-a protein content(P<0.05).Knockout mouse PER2 gene study found that PER2 gene may regulate the intestinal VFA uptake in mice by up-regulating MCTl gene in colonic epithelial tissue and PPAR-? and NHE1 genes in cecal epithelial tissues.In conclusion,Low expression or knockout of PER2 promotes the metabolism of monocarboxylic acids from intestinal epithelial cells to tissues by up-regulating MCT proteins.The specific pathway relationship needs further study.