The Relationship Between The Expressions Of Volatile Fatty Acid Absorption Genes And Circadian Clock Genes Of Ruminal Epithelium

Circadian clock genes widely exist in mammalian cells.Circadian clock genes in gastrointestinal tract could affect the absorption and transport of macronutrition and the expression of rerelated transporters.Our laboratory previously found the circadian clock genes were expressed in rumen epithelial tissue from Hu lambs with different week ages using RT-PCR method.As the main product of rumen fermentation and principal energy source for ruminants,volatile fatty acid could be absorbed by rumen epithelium tissue with related transporters.To this end,this paper was aimed to preliminarily study the internal relationship between the expressions of volatile fatty acid(VFA)absorption genes and circadian genes in ruminal epithelial cells.This research mainly includes the following parts:Experiment 1:Isolation,primary culture and immunofluorescent identification of circadian clock proteins of goat ruminal epithelial cell in vitroThe ruminal epithelial cell was isolated by serial trypsin digestion,then the cell morphology was observed by optical microscope.The expression of cytokeratin 18(CK18)in these cells were determined by immunofluoresent assay and the growth curve was determined by Cell counting 8(CCK8)method.We also added 15 mmol/L sodium butyrate in the culture mediem to identified the biological function of the primary cells.The Results analysis:Cells displayed monolayer cobblestone morphous and growth curve of these cell presented typical"S" type.These cells had strong expression of cytokeratin 18(CK 18),which is a specific intermediate filament protein in epithelial cells.The culture medium with 15 mmol/L sodium butyrate could significantly increase the β-hydroxybutyric acid production and this result conformed to the biological function of ruminal epithelial cells.Furthermore,CLOCK protein and PERIOD2 protein had positive expression in ruminal epithelial cells in vitro.Experiment 2:Effects of different VFAs on the proliferation activity and absorption rate of ruminal epithelial cells in vitroIn vitro primary-cultured ruminal epithelial cells were used and the test groups were divided into 4 groups,respectively are control group,acetate group,propionate group and butyrate group,to analyze the effects of VFAs on cell growth,distribution and related gene expression of cell cycle,and the absorption rate of different VFAs.VFA concentration in treated group was 15 mmol/L.Cell vitality was determined using CCK8 and cell cycle was tested by flow cytometry analysis.In addition,the expression of cell cycle gene was determined by RT-PCR method and VFA concentrations in culture medium at different sample points were tested by high performance liquid chromatography(HPLC).Results analysis:1)the cell vitality in control group was significantly or extremely significantly greater than VFA-treated groups(P<0.05 or P<0.01).Propionate group was significantly greater than acetate and butyrate group at 12 h and 24 h(P<0.01).2)For the percentage of cells in G1 phase,VFA-treated groups were significantly greater than control group and propionate group was less than acetate and butyrate groups at 24 h(P<0.01).3)VFAs also affect the pyruvate concentration in culture medium with the maximum in propionate group(P<0.01).4)VFAs extremely significantly downregulated the relative expression of CCND1,CCNB1,CDK1 and CDK2(P<0.01).VFAs could also downregulated the relative expression of CDK4 with no significance(P>0.05).5)For the absorption rate of different VFAs,butyrate was the highest,propionate took the second place,while the absorption rate of actate was lowest(P<0.01).The VFA concentration in cultured medium was significantly decreased with the extension of incubated time as well(P<0.01).Experiment 3:The relationship between the expressions of volatile fatty acid transporter genes and circadian clock genes of primary ruminal epithelial cell in vitroIn vitro primary-cultured ruminal epithelial cells were used and 15 mmol/L VFAs including acetate,propionate and butyrate were added into culture medium to treat the primary cells for 6 h,12 h and 24 h.This part was aimed to study the effects of VFAs on relative gene expressions of circadian clock and VFA transporters,VFA absorption rate and correlation of these gene expression in primary-cultured ruminal epithelial cells.The expression of cell cycle gene was determined by RT-PCR method and correlation analysis was conducted with Pearson correlation analysis.Results analysis:1)VFAs could affect the gene expression of VFA transporters and circadian clock in varying degrees.VFA groups significantly inhibited Clock gene expression,and significantly improve the expression of Bmal1 and Per2 gene(P<0.01).Per3 expression in butyrate group was significantly greater than other groups(P<0.01).Cry2 gene expression in propionate group and butyrate group are significantly higher than it in control group and acetate group(P<0.01).VFAs could also improve NR1D1 and RORa,reduce FBXW11 and NR2F2(P<0.05)with no significant influence on NCOR1(P>0.05).2)VFAs could affect the gene expression of PPAR family.Propionate and butyrate groups could significantly upregulated Ppara expression(P<0.01)and the relative expressions of Ppard and Pparg in propionate group were greater than control group(P<0.05).3)VFA-treated groups could significant improve PAT1,reduce MCT1 and MCT4(P<0.01),while no significance was found on NHE1(P>0.05).In addition,VFAs had different effects on the gene expression of AE2 and VH+ ATPase.4)The gene expression of MCT1 and MCT4 had significant correlation with circadian clock factors(P<0.01)and expression of Bmall and PAT1 had significant positive correlation(P<0.05).Expression of circadian clock factors had significant positive or negative correlation with CCND1,CCNB1,CDK1 and CDK2(P<0.05).In conclusion,1)Serial trypsin digestion was used to isolate ruminal epithelial cells and primary culture.Cultured cells conformed to cytomorphology,immunochemistry,growth curve and biological function of goat ruminal epithelial cells.Goat ruminal epithelial cell had positive expression of CLOCK protein and PERIOD2 protein in vitro.2)VFAs could downregulated expression of cell cycle genes and CDK,and affect inhibit the DNA synthesis of ruminal epithelial cell to reduce the cell vitality.The absorption rates existed differences among VFA types.3)VFA could affect the expression of circadian clock and VFA related transporter genes,expression of circadian clock genes may have correlations with gene expressions of cell cycle gene and VFA transporters.