| Background Diarrhea is a common clinical feature of Ulcerative Colitis and results from imbalance absorption and secretion of fluid and salt in intestinal. The intestinal absorption and secretion of Cl-, HCO3-and water partly mediated by SLC26A3, an enterocytes apical membrane C1-HCO3-exchanger. It has been confirmed that Lysophosphatidic acid (LPA) can upregulate SLC26A3function and expression in vitro, however, whether LPA increases the SLC26A3expression in the damaged colonic mucosa and relieves the diarrhea caused by dysfunctional ion in vivo is not yet clear. So, firstly, our studies were designed to investigate the effect of LPA on SLC26A3expression and phenotypic feature associated with diarrhea in colitis animal model, In addition, previous studies only hint that LPA can improve the membranal expression and the ion transport function of SLC26A3by increaseing promoter activity, whether LPA can improve the stability and the sustainability of SLC26A3membranal expression has not been reported. Given the function of glycosylation in maintaining stability intestinal mucosal expression of SLC26A3and the effect of NHERF4, a PDZ-containing protein, on the sustainability of SLC26A3intestinal mucosal expression, and in order to further understand the mechanism of LPA manage SLC26A3membranal expression, our next studies were designed to observe the effect of LPA on SLC26A3glycosylation and on the interaction between NHERF4and SLC26A3in Caco2cells.Methods Firstly, for evaluating the acute colitis mouse model induced by Dextran sodium sulfate (DSS) in C57BL/6J mice, the C57BL/6J female mice that were randomly divided into model group were fed4%DSS solution for7days to induce acute colitis. Mice body weight, stool moisture, stool consistency and the degree of hematochezia were recorded. The histopathological changes of mice colon specimens were observed visually and microcosmically, and the ion channel SLC26A3protein was detected by conducting Western Blot experiment. And then, for investigating the effects of LPA on SLC26A3expression and the diarrheal phenotype in colitis model, the mice were assigned to LPA treatment DSS group, phosphate-buffered saline (PBS) treatment DSS group, DSS only group and untreated mice with a completely randomized design. We observed the changes of phenotypic feature associated with diarrhea after LPA enema treatment, such as weight loss, DAI score, the degree of losserstools and mucosa inflammation, by measuring mice weight, situation of intestinal bleeding, stool moisture content and macroscopic inflammation score of colonic damage in the C57BL/6J mice with DSS-induced colitis, and investigated the effect of LPA on SLC26A3mRNA level and protein expression in the inflamed mid-distal colon by Real-time quantitative PCR and Western Blot. Next, for investigated the effect of LPA on the protein expression, the gene level, the glycosylation and the intracellular traffick of SLC26A3, the SLC26A3expression in Caco2cell incubated with LPA for0hour,6hours,12hours,18hours were respectively observed by Real-time quantitative PCR, Western Blot. At last, for observing the effect of LPA on the stability of SLC26A3membranal expression, the Caco2cells were assigned to LPA treatment cell group and untreated cell group, the SLC26A3protein cleavage of trypsin was used, cells were respectively incubated with pancreatic enzyme for0min,5min,10min,30min before the protein was extracted and investigated. The constructed recombinant pIRES2-ZsGreen1plasmid that contain sequence of NHERF4gene and the pIRES2-ZsGreenl plasmid were transfected into Caco2cells, named pIRES2-ZsGreenl-NHERF4-Caco2and pIRES2-ZsGreen1-NP-Caco2, respectively. The Caco2cells were assigned to pIRES2-ZsGreenl-NP-Caco2group(NP-Caco2group), pIRES2-ZsGreenl-NHERF4-Caco2group(NHERF4-Caco2group), LPA treatment pIRES2-ZsGreenl-NP-Caco2group(LPA+NP-Caco2group), LPA treatment pIRES2-ZsGreenl-NHERF4-Caco2group(LPA+NHERF4-Caco2group) and Caco2group, the effect of NHERF4on the influence of LPA on the expression of SLC26A3was preliminary investigated, as well as, the impact of LPA on NHERF4protein expression.Results During the experimental study of the acute colitis rat model, all experimental mice survived, and the model group mice appeared stool moisture increased, bloody diarrhea, weight lose, and acute colitis’s disease activity index (DAI) increased rapidly. At the end of this experiment, compared with control group mice, the model group mice appeared higher colonic damage score (p=0.00), significantly shortened colon (p=0.00), decreased expression of SLC26A3.During the experimental study of investigating the effects of LPA on SLC26A3expression and the diarrheal phenotype in colitis model. All mice treated with DSS lost weight, but the onset and severity of weight loss was attenuated in the LPA treatment DSS group. The increases in stool water content and the macroscopic inflammation score in LPA treatment DSS group were significantly lower compared to untreated control group or PBS treatment DSS group (18.89±8.67%vs.28.97±6.95%or29.48±6.71%,p=0.049, p=0.041, respectively and2.67±0.81vs.4.5±0.83or4.5±0.54,p=0.020, p=0.006, respectively), as well as the increase in DAI (p=0.004,p=0.008, respectively). LPA enema resulted in higher Slc26a3mRNA and protein expression levels compared to PBS-treated and untreated DSS colitis mice. Experimental results of observing the effect of LPA in SLC26A3glycosylation showed in Caco2cells incubated12hours with LPA, compared to untreated Caco2cells, the SLC26A3gene expression were increased1.67±0.03fold, the SLC26A3protein expression were increased (the relative expression quantity of SLC26A3protein, cells treated with LPA12hour vs. untreated cells:0.92±0.10vs.0.46±0.05, p<0.05), and the ratio of membrane protein and plasma protein were increased also (cells treated with LPA12hour vs. untreated cells:2.17±0.17vs.1.72±0.12, p=0.023), suggested LPA prompt SLC26A3protein expression and conduce to membrane localization of SLC26A3. Experimental results of observing the effect of LPA in the cleavage resistance of SLC26A3to trypsin showed that after incubated l0min with trypsin, the SLC26A3protein expression were significantly reduced in untreated Caco2cells, and no obvious reduction in LPA treatment Caco2cells (12h), suggested LPA can improve the cleavage resistance of SLC26A3to trypsin. The pIRES2-ZsGreenl-NHERF4eukaryotic expression plasmid transfected into Caco2cells successfully. When NP-Caco2and NHERF4-Caco2both incubated with LPA, the SLC26A3protein expression were less in NHERF4-Caco2(the relative expression quantity of SLC26A3protein, NP-Caco2vs. NHERF4-Caco2:0.56±0.022vs.0.27±0.042,p=0.003),suggested NHERF4weaken the promoting effect of LPA on SLC26A3protein expression. No change in NHERF4protein expression after NHERF4-Caco2incubated with LPA12hours.Conclusions The disease progression and phenotypic features of4%DSS-induced acute colitis animal model are very similar to human ulcerative colitis, and this model can be used to carry out the mechanism research about intestinal mucosal ion transport of IBD. LPA increases Slc26a3expression in the inflamed intestine and reduces diarrhea severity in DSS-induced colitis, suggesting LPA might be a therapeutic strategy in the treatment of colitis associated diarrhea. LPA promote SLC26A3glycosylation and protect SLC26A3from cleavage by trypsin. NHERF4lessens the promotion of LPA on SLC26A3protein expression in Caco2, and LPA doesn’t have enough influence on the expression of NHERF4. All of these tips LPA can improve the stability of SLC26A3in membrane. |
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