Effect Of Deoxycholic Acid Intervention On C57BL/6 Mouse Ileum Organoids Growth And Intestinal Barrier Function

Objective:To establish the culture technique of mouse intestinal organoids(Enteroids)and demonstrate the effect of deoxycholic acid on Enteroids growth and intestinal barrier function.Further to investigate the mechanism in detail.Methods:The C57BL/6 mouse terminal ileum was collected and treated with EDTA.The crypts were collected and embedded in Matrigel Matrix.Enteroids growth was observed in the normal,anhydrous alcohol control,short-term(2 days)100 ?mol/L DC A treatment,and long-term(10 days)10 ?mol/L DC A treatment group.Enteroids were further cultured for 10 days after removal of DC A from the medium.Enteroids growth was observed by light microscope,Enteroids permeability by FD-4 test,and the morphology of intestinal epithelial cells and intercellular junctions by transmission electron microscope.Gene microarray surveyed the change of Enteroids gene expression profiles under long-term 10 ?mol/L DCA treatment.Dynamic expression of tight junctions associated protein Occludin and MLCK,ERK,and Tcf4 were monitored by Real time PCR and Western Blot.Results:(1)We successfully established mouse intestinal organoids culture system.The morphological characteristics were the same as document description.(2)Short-term treatment with high-concentration DCA resulted in significantly reduced Enterospheres formation,Enteroids formation,progression rate from Enterospheres to Enteroids and number of crypt buds per Enteroid(P<0.05),which remained unchanged even after removal of DCA for a short time(P<0.05).After DCA removal for long time,the Enteroids formation rate and number of the crypt buds still remained lower than those in normal Enteroids(P<0.05).Short-term treatment with low-concentration DCA only resulted in reduced Enteroids formation rate and number of crypt buds(P<0.05),and prolonged treatment caused reduced Enterospheres formation,Enteroids formation rate and number of crypt buds(P<0.05).After DCA removal,Enteroids formation rate and number of crypt buds still remained lower than those in the normal group(P<0.05).Short-term treatment with high-concentration DCA induced significantly increasing relative fluorescent gray of FD-4,which meant higher Enteroids permeability(P<0.05).It remained unchanged even after removal of DCA for long time(P<0.05).Long-term treatment with low-concentration DCA also resulted in high relative fluorescent gray of FD-4 and Enteroids permeability(P<0.05).After DCA removal,Enteroids permeability partly recovered.Low-concentration DCA for long time led to decreasing mucinous granules of Goblet cells,damage of tight junctions and Paneth cells function.(3)Gene microarray analysis was summarized 565 differential gene in Enteroids under long-term low-concentration DCA treatment,with 340 up-regulated genes and 225 down-regulated genes.Pathway and GO analysis showed many gene sets were related to biological metabolism function.We chose 2 meaningful differential genes,ERK and Tcf4,which were verified ERK2 and Tcf4 mRNA up-regulation and ERK1/2 and p-ERK1/2 protein overexpression in Enteroids under low-concentration DCA treatment for long term(P<0.05).Low expression of Occludin and overexpression of MLCK and p-MLCK in Enteroids were found in DCA treated Enteroids.Conclusions:DCA damaged Enteroids growth and intestinal barrier function,but the injury could partly be relieved after DCA removal for long time.Low-concentration DCA treatment for long term affected a lot of gene pathways.And part of them was related to biological metabolism.ERK and Wnt pathway involved in DCA adjustion of abnormal expression of Occludin and MLCK,which led to tight junctions damage and Enteroids barrier dysfunction.