Effect And Mechanism Of Butyric Acid On The Colonic Epithelial Barrier Regulation Mediated By GPR109A In Susscrofa

Diarrhea is a special stage in the growth and development of weaning piglets.Due to the fact that the intestinal microflora and intestinal epithelial barrier have not yet been established,the environmental influences will cause greater stress and lead to diarrhea.Sodium butyrate is a non-toxic short chain fatty acid produced by cellulosic fermentation under the action of bacteria and alleviate diarrhea rate in weaned piglets.G protein coupled receptor 109A(GPR109A,HM74A(human))is not only expressed in immune cells and adipocytes other than lymphocytes,also expressed in epithelial cells,mammary gland cells,nerve cells.GPR109 A is a Gi coupled receptor,its endogenous ligand is β-hydroxybutyric acid(BHBA),nicotinic acid,butyric acid and other exogenous ligands can also activate GPR109 A.Recent studies showed that the activation of GPR109 A can significantly inhibit the inflammatory response in various diseases such as neurodegenerative disease,diabetes,septicemia,obesity,colitis,atherosclerosis and so on,so that it can be used as an effective target for treatment or intervention of the diseases.In the view of above,the experiment is divided into two parts.In vivo,the performance of piglets were observed after adding 2000 mg/kg sodium butyrate for day 21,the ratio of lactulose and mannitol was detected by HPLC,and the expression of tight junction proteins in intestine was detected by RT-PCR,western-blot and immunofluorescence.In vitro,the human colon cancer epithelial cells(Caco-2)were used as a colon cell model,RT-PCR,Western-blot and other techniques were used to detect the expression of tight junction proteins under the treatment of sodium butyrate.Compared to the control group,the diarrhoea rate,diarrhoea frequency and diarrhoea index of the piglets were significantly decreased after adding 2000 mg/kg sodium butyrate.The sodium butyrate group showed a higher average daily gain(ADG)and lower feed gain ratio than the control group after the first week.At the same time,the ratio of lactulose and mannitol in urine was significantly lower than that in the control group,which showed that sodium butyrate could reduce intestinal permeability and improve intestinal integrity.Remarkably,the expression of tight junction Claudin-3,Occludin and ZO-1 in the colon was higher in the sodium butyrate group.1.5mM Sodium butyrate was selected to stimulate Caco-2 cells,silencing GPR109 A by GPR109A-shRNA or blocking Akt by MK2206(an allosteric Akt inhibitor)suppressed the sodium butyrate-induced expression of Claudin-3 in Caco-2 cells.However,10 μM U0126(ERK specific inhibitor)and 10 μM SB203580(P38 specific inhibitor)had no significant effect on the expression of tight junction protein Claudin-3 in Caco-2 cells.Taken together,sodium butyrate enhance the expression of Claudin-3 by activating the GPR109 A and Akt signaling pathway in Caco-2 cells,thereby maintain the colonic barrier function and relieve diarrhea of the weaned piglets.