| Diarrhea is the key factor for the survival rate of piglets.Bacterial diarrhea is mainly caused by Enterotoxigenic Escherichia coli(ETEC).The heat-stable enterotoxin(STp)produced by E.coli is the main pathogenic factor of diarrhea in piglets.STp can destroy the structure and function of intestine,but the underlying mechanism is still unclear.In order to study the mechanism of STp destroying intestinal epithelial integrity,Twelve four-week old male C57BL/6 mice were selected and randomly divided into control group(CON)and STp group,with 6 replicates in each group and 1 mouse in each replicate.The mice in the STp group were administered with 5.0 mg/kg BW STp,while the the mice in the CON group were treated with the same volume of PBS.The mice were sacrificed after 6 hours of administration.The intestinal tissue was collected and intestinal structure and function were detected.The crypt was isolated and cultured in vitro and enteroid expansion and budding efficiency was counted.Jejunum morphology was observed by HE staining and scanning electron microscopy.Diamine oxidase(DAO)of jejunum was detected by ELISA.Tight junction protein expression in jejunum and crypt was detected by Western blotting.Wnt/beta-catenin signaling pathway-related protein expression in enteroid was detected by Wes.MTT,Ed U and counting assays were performed to detected the proliferation of IPEC-J2(porcine jejunal epithelial cell)and CMT-93(mouse colonic epithelial cell line),the cell apoptosis,cell barrier and Wnt/?-catenin signaling pathway were detected by Western blotting,cell immunofluorescence,flow cytometry and transwell at time point of difference.In addition,25 ng/m L R-spondin1 was used to alleviate STp-induced intestinal epithelial cell injury.Furthermore,three dimensional(3-D)model of enteroid,cultured from crypts which are isolated from 7-day-old crossbred boars(Yorkshire×Landrace),were used to explore the effect of STp exposure on the expansion of intestinal stem cells(ISCs)and Wnt/?-catenin signaling.The results were as follows:1.STp destroyed the intestinal structure of mice,significantly decreased the weight and villus height of jejunum(P<0.05),and the number of Mucin2~+and Lysozyme~+cells on jejunal villi;STp impaired intestinal barrier function,significantly reduced the value of jejunal transmembrane resistance and ZO-1,Occludin and Claudin-1 protein expression in jejunum and crypt(P<0.05);STp inhibited Wnt/?-catenin signaling pathway and decreased the protein expression of?-catenin,PCNA and Lgr5 in enteroid(P<0.05).2.400 ng/m L STp treatment for 48 hours significantly inhibited the proliferation of IPEC-J2 and CMT-93 cells,decreased the proportion of Ed U~+and Ki67~+cells and the expression of PCNA protein(P<0.05);induced apoptosis,increased the expression of Caspase-3 protein(P<0.05);destroyed the barrier of intestinal epithelial cells and decreased TEER and expression of tight junction proteins ZO-1,Occludin and Claudin-1(P<0.05);decreased Wnt/?-catenin signaling pathway expression,up-regulated negative feedback factor GSK-3?protein expression(P<0.05),down-regulated positive regulatory factors?-catenin,Cyclin D1 and c-Myc protein(P<0.05);R-spondin1 treatment for 72 h reversed the inhibitory effect of STp on Wnt/?-catenin and the intestinal epithelial cell proliferation.3.Enteroid became spherical shape and the budding efficiency was significantly reduced after 400 ng/m L STp treatment for 24 hours(P<0.05).In addition,the expression of?-catenin,Lgr5?PCNA and tight junction protein ZO-1 and Claudin-1 in the STp group decreased significantly(P<0.05),while the proportion of apoptotic intestinal mass and the protein expression of Caspase-3 increased significantly(P<0.05).These results suggest that STp inhibits the ability of intestinal stem cell by down-regulating Wnt/?-catenin signaling pathway,reduces the expansion efficiency of stem cell into enteroid,hinders the intestinal epithelial cell proliferation,promotes cell apoptosis,and ultimately leads to impaired intestinal epithelial barrier function. |
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