Effects Of Butyrate On Nutrition Metabolism Of Rumen Epithelial Cells Of Dairy Based On Transcriptome And Metabolome Analysis

Butyrate can maintain the structural integrity of rumen epithelial cells,is an important regulator of cell metabolites,and is also an important stimulating factor for the development of rumen epithelial functions,and plays an important role in rumen epithelial cells proliferation and nutrition metabolism,but the specific regulation process needs further research.In order to establish a reliable and stable culture method for rumen epithelial cells,and further explore the effect of butyrate on cell nutrition metabolism and its regulation process,rumen epithelial cells of Holstein cows were selected as the research object,and the following three aspects were carried out:Research 1:Culture of rumen epithelial cells in vitro and the effect of butyrate on its growthThe rumen epithelium is an important regulatory site for the utilization of nutrients in animals,and plays an important role in nutrient absorption,ion transport and metabolism of Short chain fatty acids(SCFAs).Rumen epithelial cells cultured in vitro are good materials for the study of rumen metabolism.In this study,epithelial tissue was continuously digested with trypsin from three Holstein bull calves,and then cells with typical epithelial and pavement morphology were obtained.The cell growth curve was plotted.The isolated cells were identified as rumen epithelial cells by keratin 18(CK-18),and there was no senescence signal in the fourth generation,these cells could be used for subsequent experimental studies.Next,the experiment was conducted to investigate the effects of different concentrations of butyrate on the growth and apoptosis of rumen epithelial cells.CK,Ⅰ and Ⅱ groups were set in the study,respectively adding 0,10 and 20 mM butyrate to the rumen epithelial cells,6 repetitions per group,and cells were collected after a continuous culture of 6 h.The results showed that butyrate significantly decreased the cell activity(P<0.01);20 mM butyrate increased the proportion of late apoptotic cells and apoptotic index(P<0.05).Research 2:Metabonomics analysis of the effect of butyrate on nutrition metabolism of dairy cow rumen epithelial cellsThe purpose of this experiment was to study the differential metabolites and enrichment pathways after butyrate stimulation of rumen epithelial cells of dairy cows by metabome analysis.CK,Ⅰ and Ⅱ groups were set in the study,respectively adding 0,10 and 20 mM butyrate to the rumen epithelial cells,and supernatant were collected after a continuous culture of-6 h,this experiment was based on six duplicates in each groups,and metabonomics analysis was performed based on LC-MS platform.The results showed that the metabolome of cell supernatant was well separated by principal component analysis,we received 259 differential metabolites,KEGG pathway enrichment analysis found that purine metabolism,glycerol phospholipid metabolism,metabolism of phenylalanine and tyrosine metabolism were affected.Research 3:Transcriptomics analysis of the effect of butyrate on nutrition metabolism of dairy cow rumen epithelial cellsThe purpose of this experiment was to study the differential genes and their enrichment pathways after butyrate stimulation in rumen epithelial cells of dairy cows by transcriptome sequencing technology.CK,Ⅰ and Ⅱ groups were set in the study,respectively adding 0,10 and 20 mM butyrate to the rumen epithelial cells,and cells were collected after a continuous culture of 6 h,this experiment were based on three duplicates in each groups.2196 differential genes were screened,the expression of IL-1 and TNF cytokines were significantly upregulate while adding 10 mM butyrate compared to the other two groups(P<0.05).IL-17,MAPK and TNF signaling pathway were activated after stimulating with butyrate.The results showed that butyrate could activate the inflammatory response of rumen epithelial cells.Under the conditions of this experiment,the combined analysis of transcriptomics and metabolomics showed that the addition of butyrate affected the lipid metabolism,carbohydrate metabolism,energy metabolism and nucleotide metabolism of rumen epithelial cells,and accelerated material synthesis and metabolism to provide energy for cells.