The Study Of ABCB1 Gene Expression And The Changes Of Intestinal Flora In Patients With Ulcerative Colitis

BackgroundUlcerative colitis is a common digestive disease,the incidence of which is increasing year by year.It significantly increases the medical payment burden and reduces the quality of life in UC patients.The pathogenesis of ulcerative colitis may include heredity,diet allergies,infection,autoimmunity,depression and anxiety mood.It was reported that ABCB1 gene knockout mouse tended to develop colitis spontaneously with an accidence rate of 21%.However,the mice in control group did not develop into colitis.The pathogenesis and pathological changes in colitis occurred at the ABCB1 gene knockout mouse was similar to that in ulcerative colitis patients.The expression and activity of ABCB1 gene in inflamed mucosa were downregulated in the drug-induced mouse model with colitis.These animal experiments indicated that the susceptibility of UC is related with ABCB1 gene expression,but which remains controversial.This study aims to explore the relationship between human ABCB1 gene expression and the pathogenesis of ulcerative colitis.Human ABCB1 gene(also known as multidrug resistance gene)is located at the 7q21.1 locus of human chromosome,containing 28 introns,26 introns of which play roles in blocking the liner expression of P glycoprotein.The length of the mRNA encoded by ABCB1 is 4.7kDa,and the code area is less than 5%of the whole chain of it.Some studies suggested that the ABCB1 gene is related to susceptibility of inflammatory bowel disease.P-glycoprotein(P-glycoprotein,P-gp)is encoded by multidrug resistance gene,also known as a member of adenosine triphosphate(ATP)-binding cassette transporter family(ABC transporter family).The gene encoding Pgp is also called ABCB1 gene.Pgp as a transmembrane efflux transporter has protective functions include discharge drugs,potentially toxic xenobiotics and metabolic wastes from enterocytes.Pgp can combine and interact with the drug molecules in the lipid bimolecular layer of the cell membrane;Besides,ATP hydrolysis can assist Pgp promote transmembrane transport of drugs against a concentration gradient.The substrates of Pgp are usually lipophilic and amphipathic molecula.P-gp is expressed in many human normal organs and tissues,such as blood brain barrier,testicular capillary epithelial cells,liver cells,adrenal glands.The brush border membrane of large intestine and small intestine mucosa epithelial cells,mononuclear cells,NK cells,T lymphocytes and B lymphocytes in peripheral blood are also rich in Pgp.Pgp is a transmembrane transporter which can expell potentially toxic substances from the body through secreting bile,urine and intestinal juice.Therefore Pgp is closely related to the secretory function of normal tissue cells.Pgp is an important component of the intestinal barrier which play roles in reducing the absorption of toxins from the intestine and eliminating the accumulation of endogenous and exogenous toxins from some important organs.In the normal human digestive tract,the columnar epithelial cells of colon and distal small intestine is rich in Pgp,which can limit the absorption of endogenous and exogenous toxins.Pgp encoded by ABCB1 gene is highly expressed in the liver,lung,intestine and kidney,which are most frequently exposed to toxic substances.Pgp pathway is one of the important mechanisms to protect enterocytes from toxic substances.ABCB1 gene knockout mouse tend to idiopathic colitis in the SPF feeding environment,which can be blocked or reversed by antibiotics.Therefore,intestinal flora maybe necessary for initiating and maintaining an inflammatory response in the intestine.However,the loss of excretion function of exogenous toxins can promote the initiation of bowel inflammation.Intestinal micro-ecology may play a role in the development of inflammatory bowel disease.Some results have indicated that,compared with healthy controls,the diversity of intestinal flora in patients with inflammatory bowel disease was significantly impaired,of which some probiotics decreased and some "pathogenic bacteria" increased.However,the results of the studies on the changes of intestinal bacterial species and strains in patients with ulcerative colitis are significantly different.Therefore,it is necessary for us to further study the intestinal microflora changes of patients with ulcerative colitis.In addition,the study on the relationship between the expression of Pgp and intestinal microflora changes in patients with ulcerative colitis has not been reported.Thus we will make a preliminary study on this field.AimsThe aim of this study is to explore the etiology of ulcerative colitis via evaluating ABCB1 gene expression in inflamed mucosa of patients with ulcerative colitis before and after treatment.We will compare the ABCB1 gene expression level with that of health controls and illustrate the effect of ABCB1 gene expression on intestinal barrier.We will also analyze changes of intestinal bacteria species and strains in patients with ulcerative colitis by detecting intestinal bacterial 16S rRNA expression.We will further to analyze the effect of Pgp expression on intestinal microflora.At the end,we want to explore the relationship between ABCB1 gene expression and the intestinal microflora changes.Methods1.Through the immunohistochemistry assay,real-time PCR and Western blot assay,we detected the changes of ABCB1 gene expression in patients with ulcerative colitis,and compared the results with that of healthy controls.2.The changes of serum endotoxin and D-lactic acid levels,which indirectly reflects the function of intestinal barrier,were detected in this study.We analyzed the correlation between ABCB1 gene expression and the changes of serum endotoxin and D-lactic acid to explore the relationship of ABCB1 gene expression and intestinal barrier.3.By detecting the changes of fecal bacteroides,bifidobacterium,lactobacillus,winding coli,enterococcus,clostridium and fragile bacillus,the intestinal microecological changes in patients with ulcerative colitis were analyzed.The experiments above were conducted by detecting 16S rRNA of fecal bacteria.4.By analyzing the differences of fecal bacteroides,bifidobacterium,lactobacillus,winding coli,enterococcus,clostridium and fragile bacillus between healthy controls with Pgp positive expression and the ones with Pgp negative expression,we made a preliminary analysis of the effect of Pgp expression on intestinal microflora.5.By analyzing the differences of fecal bacteroides,bifidobacterium,lactobacillus,winding coli,enterococcus,clostridium and fragile bacillus between the patients with ulcerative colitis with Pgp positive expression and the ones with Pgp negative expression,we make a preliminary analysis about the effect of Pgp expression on intestinal microflora in patients with ulcerative colitis.Results1.The immunohistochemistry stain results showed that,the positive rate of Pgp expression in patients with ulcerative colitis was significantly lower than that of healthy controls.After treatment,the level of Pgp expression in inflamed mucosa of patients with ulcerative colitis was significantly higher than that before treatment,but it was still lower than that in healthy controls.2.The real-time PCR and western blot assay results showed that,the level of ABCB1 gene expression(mRNA and Pgp)in inflamed mucosa of patients with ulcerative colitis was significantly lower than that of healthy controls.After treatment,the level of ABCB1 gene expression in patients with ulcerative colitis was significantly higher than that of before treatment,but it was still lower than healthy controls.3.The levels of serum endotoxin and D-lactic acid in patients with ulcerative colitis were significantly higher than that of healthy controls.After treatment,the levels of serum endotoxin and D-lactic acid in patients with ulcerative colitis was decreased,but they were still higher than those of healthy controls,which indicated that the intestinal barrier was impaired.The level of Pgp expression in patients with ulcerative colitis was negatively correlated with the level of serum endotoxin and D-lactic acid.4.Compared to healthy controls,the amount of fecal bacteroides,bifidobacterium,lactobacillus,winding coli,enterococcus and fragile bacillus was significantly decreased in patients with new diagnosed ulcerative colitis,however,the amount of fecal clostridium difficile was significantly higher than that in healthy controls,which was statistically significant.5.Compared with the health controls with Pgp negative expression,the amount of fecal bacteroides,bifidobacterium,lactobacillus,enterococcus,and fragile bacillus in Pgp positive expression controls were significantly elevated;however,the amount of clostridium difficile was decreased,which was statistically significant.The amount of winding coli was elevated without statistically significant difference.6.Comparing patients with ulcerative colitis with Pgp positive expression and the ones with Pgp negative expression,the amount of fecal bacteroides,bifidobacterium,lactobacillus,winding coli,enterococcus and fragile bacillus was significantly elevated,however,the amount of clostridium difficile was decreased,which was statistically significant.ConclusionOur study shows a significant change in patients with ulcerative colitis in the distribution of seven main bacterial flora,including fecal bacteroides,bifidobacterium,lactobacillus,winding coli,enterococcus,clostridium and fragile bacillus,which is related to Pgp expression level.Combined with previous studies,it is suggested that the changes of intestinal microflora were involved in the pathogenesis of ulcerative colitis.Overall,the results of our study show a relative increase of pathogenic flora and a decrease of probiotics and normal flora.In this study,the expression of ABCB1 gene in inflamed mucosa of patients with new diagnosed ulcerative colitis was down-regulated,both in the mRNA and protein expression levels.Pgp expression in patients with ulcerative colitis was down-regulated,followed by the impaire and high permeability of intestinal barrier,which induced the inflammation of intestinal mucosa.Therefore,low Pgp expression is one of the causes of ulcerative colitis,and Pgp may play an important role in the pathogenesis of ulcerative colitis.After the treatment by prednisone,Pgp expression in patients with ulcerative colitis was up-regulated,accompanied by the improvement of intestinal barrier and clinical remission in some patients.Therefore,the up-regulation of Pgp expression was induced by the treatment by prednisone,which was not spontaneously.This study is helpful to further reveal the etiology of ulcerative colitis,and to find new approach and drug targets to prevent and treat ulcerative colitis,which has a certain clinical significance.After the treatment by prednisone,with Pgp expression up-regulated and intestinal barrier elevated,there are still some patients failed to achieve clinical remission,which suggests that Pgp expression level is only one of the many causes involved in the pathogenesis of ulcerative colitis.