SCFAs Regulate The Inflammatory Response Of Bovine Rumen Epithelial Cells By GPR41

The 70%of the energy requirement of bovine and sheep is provided by rumen short chain fatty acids(SCFAs)in ruminants,which is produced by rumen microbe to fermentate,carbohydrate from daily diet in the bovine rumen.Acetate,propionate,and butyrate constitute approximately 95%of total SCFAs.High concentration of SCFAs was produced by microbial fast fermentation due to feeding a high-concentrate diet,which provide the energy requirement and promote the milk yield.However,feeding a high-concentrate diet may resut in the fast inflammatory response.Past research had demonstrated G protein-coupled receptor 41(GPR41)as receptors for SCFAs.We therefore speculated that SCFAs which provides the main source of energy for the ruminants whether can regulate the inflammatory response of bovine rumen epithelial cells by GPR41 should be investigated.Before we study that GPR41 how to regulate the underlying mechanism of inflammatory response,we must establish an immortal BRECs lines.However,immortal BRECs lines remain not established.Therefore,the aim of study isfirstly to establish an immortal BRECs lines.Secondly,we will study that GPR41 how to regulate BRECs inflammatory response,and further study the underlying regulatory effects of GPR41 in inflammatory response.Experiment one:Establishment and characterization of immortalized bovine rumen epithelial cells lineBovine rumen epithelial cells(BRECs)play a critical role in short chain fatty acids(SCFAs)uptake and metabolism in ruminants.Availability of a continuous BRECs line will be a valuable tool to study the function of genes by CRISPR/Cas9 system.However,the immortal BRECs remain not reported.Therefore,the aim of this study was to establish an immortal BRECs line.BRECs were isolated using serial tryptic digestions from three newborn Holstein calves,and BRECs were transducted by lentiviruses expressing SV40 large T antigen(SV40T).The immortal BRECs maintained typical epithelial-like and "cobblestone" morphology and reached 100%confluence after 4 days of culture.These cells can have the ability to proliferate robustly.The immortal BRECs were characterized by the expression of cytokeratin 18,monocarboxylate transporter 1(MCT1),MCT4,Na+/H+ exchanger 1(NHE1),NHE2,NHE3,and G-protein coupled receptor 41(GPR41).The pH 5.5 significantly enhanced the absorption of propionate when compared to pH 7.4 in BRECs.In conclusion,we have established the immortal BRECs line for the study of molecular mechanisms of functional gene to regulate SCFAs absorption and metabolism,cell growth,and differentiation.Experiment two:Effect of SCFAs on transporters involved in SCFAs and GPR41The aim of study is to investigate the effect of different pH and SCFAs on the mRNA abundance of genes involved SCFAs transporters and GPR41.The experiment was divided into six groups:pH 6.2,pH 6.8,and pH 7.4(as control group)for 24 h;20 mM SCFA at pH 6.2,pH 6.8,and pH 7.4(as treatment group)for 24 h.The experiment was based on three independent experiments.After 24 h incubation,these cells were collected to extract total RNA.The results show that pH 6.2,pH 6.8,and pH 7.4 do not alter the mRNA level of MCT1 by adding SCFAs(p>0.05).MCT1 mRNA expression level was significantly enhanced by 20 mM SCFAs at pH 6.8 compared with treatment groups in absence of SCFAs at pH 6.8 and 7.4(p<0.05).The mRNA abundance of MCT4 was significantly elevated by SCFAs at pH 6.2 compared with treatment groups at pH 6.8 and 7.4(p<0.05).In comparison with the absence of SCFAs,the mRNA abundance of MCT4 was significantly elevated by SCFAs(p<0.01).NHE1,NHE2,and NHE3 mRNA expression level were significantly enhanced by adding SCFAs compared with the absence of SCFAs(p<0.01).A decrease in pH from 7.4 to 6.2 enhanced the NHE1,NHE2,and NHE3 mRNA expression level.GPR41 was not expressed in absence of SCFAs,but activated by adding SCFAs.In comparison with the pH 7.4,pH 6.8 enhanced the mRNA abundance of GPR41(p<0.05).These results demonstare that low pH and adding SCFAs markedly enhanced mRNA abundance of genes involved SCFAs transporters and GPR41.Experiment three:Transcriptome analysis of differential expression mRNA and signal pathway in BRECs induced by SCFAsThe aim of study is to investigate the effect of SCFAs on differential expression mRNA and signal pathway in BRECs induced by SCFAs.The experiment was divided into two groups:pH 6.2(as control group)for 24 h;20 mM SCFA at pH 6.2,(as treatment group)for 24 h.The experiment was based on three independent experiments.These results show that 4011 genes were markedly upregulated in BRECs induced by SCFAs(p<0.01),and 1431 genes were significantly attenuated(p<0.01).The proinflammatory cytokine IL-1? and TNF-? were significantly upregulated in BRECs induced by SCFAs compared to control group(p<0.01).Compared with control group,CCL20,CXCL2,CXCL3,CXCL5,CXCL8,and CXCL14 were markedly enhanced in BRECs induced by SCFAs(p<0.01).The PLC?2 and IPR31 key signal molecular involved in Ca2+ signal pathway were markedly increased in BRECs induced by SCFAs(p<0.01).MAPK signaling pathway,Cytokine-cytokine receptor interaction signaling pathway,and Calcium signaling pathway were activated after BRECs were stimxulated with SCFAs.The results demonstrate that SCFAs can activate the inflammatory response and Ca2+signal pathway.Experimeint four:Effect of SCFAs on the expression of proinflammatory cytokine,chemokine and tight junction proteinThe aim of study is to verify the results of transcriptome by qRT-PCR and Western blottiong and effect of different concentration of SCFAs and dififerent times point on proinflammatory reponse,chemokine and tight junction protein.The results show that qRT-PCR data are consistent with transcriptome analysis.SCFAs can enhance the mRNA abundance of genes involved in proinflammatory cytokine,chemokine,and Ca2+ signal pathway.In addition,SCFAs induces the inflammatory reponse in BRECs that is involved in the activation of p-Erkl/2 MAPK singal molecular,but not NF-?B signal pathway.High concentration of SCFAs enhanced the mRNA expression of proinflammatory IL-1? and TNF-?,but decreased the mRNA abundance of CXCL2,CXCL3,CXCL5,and CXCL8 chemokine and Claudin-1 and ZO-1 in volved in tight junction protein.Compared with 24 h and 48 h cultures,proinflammatory IL-1?was markedly increased in 72 h of cultures.However,the mRNA abundance of Occludin,Claudin-1,and ZO-1 was attenuated in 72 h compared to 48 h.A increase in time from 24 h to 72 h can trigger the proinflammatory response of BRECs,but,attenuated the expression level of tight junction protein.These results demonstrated that high concentration of SCFAs triggered the proinflammatory response and disrupted the function of immunologic chemotaxis and barrier.Experiment five:Regulation of the expression of proinflammatory cytokine,chemokine,and tight junction protein by GPR41The aim of study is to konck out GPR41 gene by CRISPR/Cas 9 system to further study whether GPR41 regulates the inflammatory reponse,chemokine,tight junction protein,and Ca2+signal pathway.The experiment was divided into three groups:culture medium+BRECs,culture medium containing 20 mM SCFAs+BRECs,and culture medium containing 20 mM SCFAs+BRECs konok out GPR41 for 24 h,respectively.The experiment was based on three independent experiments.The results show that the first exon of GPR41 was deleted 142 bp by the role of gRNAl and gRNA3.Compared to wild type,the mRNA abundance of GPR41 is mardedly attenuated by CRISPR/Cas 9 system(p<0.05).In comparsion to wild type,mRNA abundence of genes involved in proinflammatory cytokine IL-1? and TNF-a was markedly increased(p<0.05),but CCL20,CXCL2,CXCL3,CXCL5,CXCL8,and CXCL14 chemokine and Occludin and ZO-1 mRNA levels of gens involved in tight junction protein were significantly downregulated(p<0.05).In addition,the mRNA abundance of IP3R1 involved in Ca2+ signal pathway was attentuted(p<0.05).The resutes demonstrate that downregulation of GPR41 triggered the proinflammatory response of BRECs,but decreased the mRNA levels of chemokine and the mRNA abundance of genes involved in immunologic barrier.