1,25（OH）₂D₃ Deficiency Promotes Colon Inflammation Via Influence Of Intestinal Barrier

Emerging epidemiological evidence suggests that vitamin D deficiency（usually defined as serum 25（OH）D<20 ng/m L）or insufficiency（serum 25（OH）D<30ng/m L）is associated with an increased risk of inflammatory bowel disease（IBD）.Increasing the intake of vitamin D will reduce the risk of IBD.It is suggested that vitamin D deficiency play an important role in the pathogenesis of IBD,while its mechanism is not clear.Studies have shown that intestinal barrier dysfunction,including intestinal flora imbalance、intestinal epithelial tight junction damage,plays an important role in IBD.The imbalance of the intestinal flora induced by environment,diet,age and host gene defects can promote the increase of harmful bacteria,causing a large number of endotoxin and/or pathogenic microorganism to enter through the damaged intestinal mucosal into the body circulation,inducing and aggravating intestinal inflammation.The purpose of this study was that we used the Cyp27b1 gene knockout mice（KO mice）model with active vitamin D（1,25（OH）₂D₃）deficiency,to analyze the effect of 1,25（OH）₂D₃deficiency on the intestinal microflora by 16S r RNA sequencing technology and to detect the expression of tight junction proteins in intestinal epithelial cells by immunofluorescence and Western Blotting,finally,to explore the protective mechanism of 1,25（OH）₂D₃on IBD through the maintenance of intestinal barrier.To determine whether the 1,25（OH）₂D₃deficiency could lead to colitis,we compared the colon length,the spleen weight,and the colon tissue inflammation type of 10 months old wild type（WT）mice,Cyp27b1 gene knockout（KO）mice and1,25（OH）₂D₃supplemented KO mice（KO+VD）.The results showed that:compared with WT mice,KO mice showed obvious colitis phenotype,including weight loss,shorter colon length,increased spleen weight,increased inflammatory cell infiltration in the colon tissue,increased expression of proinflammatory cytokines and increased intestinal permeability.However,the above symptoms were significantly rescused after the supplementation of 1,25（OH）₂D₃in the KO mice.These results indicated that1,25（OH）₂D₃deficiency can induce colonic tissue inflammation in mice,and1,25（OH）₂D₃can partly relieve the inflammation.In order to determine whether the 1,25（OH）₂D₃deficiency could lead to intestinal flora imbalance,we collected 5 pairs of feces from WT and KO mice,extracted the bacterial DNA from the feces and was followed by 16S r RNA sequencing,and compared the difference in the bacterial composition between the WT and KO mice.The results showed that there was no significant difference between the WT and KO mice on alpha-diversity.On the phylum level,Firmicutes and Bacteroidetes together accounted for a major part of the bacterial population in all samples.Beta-diversity analysis found that Akkermansia muciniphila（A.muciniphila）,a mucus-degrading bacteria was more abundant in KO mice than in WT mice.However,Bacteroides-acidifaciens bacteria which is considered to produce short chain fatty acids,was more abundant in WT mice.Quantitative PCR with primers specific to A.muciniphila confirmed the increased relative abundance of this bacterium in KO compared to WT mice.1,25（OH）₂D₃supplement greatly reduced the abundance of A.muciniphila bacteria.We found that the content of butyrate from feces in KO mice was significantly lower than that of WT mice,while1,25（OH）₂D₃supplementation could partly restore the content of butyrate.These results indicated that 1,25（OH）₂D₃deficiency did change the composition of the intestinal flora,especially the abundance of A.muciniphila.To determine whether the richness of A.muciniphila caused by 1,25（OH）₂D₃deficiency affects the thickness of the mucous layer of the colon mucosa,and thus allows the bacteria to easily access the intestinal mucosa,thereby exacerbating the colonic inflammation,we detected the thickness of the colonic mucus layer,calculated the number of colonic goblet cells,and detected the expression of Muc1,Muc2,Muc3 and Muc4 by q PCR.The results showed that the thickness of the colonic mucous layer in KO mice was significantly lower than that in WT mice,while1,25（OH）₂D₃supplementation partially restored the thickness of the mucous layer.However,there was no difference in the number of colonic goblet cells in WT,KO and KO+VD mice.At the same time,the q PCR data showed that the m RNA expression of Muc2 and Muc4 in the colon tissue of KO mice was higher than that of WT mice,but there was no significant difference in the expression of Muc1 and Muc3.In addition,1,25（OH）₂D₃deficiency significantly increased the translocation of bacterial to the mesenteric lymph nodes（MLNs）in KO mice by q PCR with the universal 16S r RNA gene primers（EUB）.These results indicated that 1,25（OH）₂D₃deficiency induced the enrichment of A.muciniphila which degraded the gut mucus layer leading to bacterial penetration into the gut mucosa to induce inflammation.In order to exclude the possibility that the change of A.muciniphila in KO mice was resulted from age not from gene,we checked the colon phenotype and A.muciniphila abundance between the young KO and WT mice of 10-12 weeks.We found that higher A.muciniphila abundance in fecal sample and increased the translocation of bacterial to MLNs of young KO mice.Consistently,the thickness of colon mucus layer of KO mice was thinner than that of WT,but was still thicker than KO mice with age of 10 month.This indicated that 1,25（OH）₂D₃deficiency increased the A.muciniphila abundance.In order to determine whether the 1,25（OH）₂D₃deficiency affected the tight junction of the mouse colonic epithelial cells,we detected the expression of the main tight junction proteins by immunofluorescence and Western Blotting.The results showed that compared with WT mice,the expression of tight junction proteins such as ZO-1,Occludin,and Claudin4 in KO mice decreased significantly,while1,25（OH）₂D₃supplementation partly restored the expression of tight junction proteins.These results indicated that 1,25（OH）₂D₃deficiency can affect the intestinal barrier function by reducing the expression of tight junction proteinsThe above results showed that 1,25（OH）₂D₃deficiency can increase the enrichment of Akkermansia muciniphila which destroyed the intestinal mucus layer,and reduce the expression of tight junction proteins,finally lead to the damaged intestinal barrier,the increased intestinal permeability and promoting the colitis.The results of this study will help to discover a potential mechanism of 1,25（OH）₂D₃in protection against colitis and to provide a new research direction and theoretical basis for the prevention of IBD.