Dysbiosis Contributes To Chronic Constipation Development In The Intestine

AimIt is well-known that large numbers of microorganisms are present in gastrointestinal tract.There are about 10 trillion micro-organisms existing in the human digestive system.These microorganisms distribute in the different segments in small intestine as well as large intestine,affecting intestinal physiological function and participating in the life activities which are crucial for the host,such as contributing the motility,regulating the immune function and energy metabolism,and inhibiting the development of pathogenic bacteria.However,specific mechanism and pathways were not clarified.Growing evidences showed that patients with chronic constipation accompanied with intestinal dysbiosis.Specifically,studies have found that Bacteroides were more abundant in colonic mucosa of patients with chronic constipation,and the populations of Lactobacillus and Bifidobacterium were significantly increased.Recent study showed that 5-hydroxytryptamine?5-HT?and serotonin transporter?SERT?play an important role in the mechanisms of intestinal motility disorders and visceral anomalies.Meanwhile,dysbiosis contributes to SERT abnormal expression and intestinal motility disorders.In our study,the suspensions from fecal sample were transplanted to the antibiotic depletion model by gavage.And then we evaluate the parameters of defecation function,the level of 5-HT and SERT and fecal microbiota in different groups.The aim is to investigate the possible mechanism of intestinal microbiota dysbiosis in the generation of chronic constipation.Material and MethodsPatients with chronic constipation and healthy controls were conducted according to Rome III criteria to recruit.Healthy controls are the health physical examination personnel without any diseases for fecal microbiota transplantation?FMT?.C57BL/6mice were purchased from Beijing Animal Research Center,China,weighting 16–18g,6 weeks of age.The mice were fed normal mouse-chow diet under specific pathogen free?SPF?condition.Mice were randomized into two groups:FMT-C group?transplant fecal microbiota of chronic constipation patients,n=10?and FMT-H group?transplant fecal microbiota of healthy controls,n=10?.The mice were given antibiotic cocktail?a mixture of 500 mg of ampicillin,250 mg of vancomycin,500 mg neomycin and 250 mg of metronidazole?daily for 3 days by gavage,considering as the antibiotic depletion model.The suspensions from fecal sample?0.2 mL/10 g body weight?were transplanted to the mice by gavage.According the previous reports,the mice were inoculated once daily for 3 consecutive days,then on alternate days for 4 times,with a total of 7 times during transfer experiments.On the 7th and 15th day,we detected defecation parameters of mice including defecation frequency,fecal weight,fecal dry weight and fecal water.All animals were killed after 15 days,and the colons of mice were removed for later use.The Caco-2 cells as the human intestinal epithelial cells,were grown in Dulbecco's Modified Eagle Medium?DMEM?media supplemented with 10%fetal bovine serum,1.0%nonessential amino acid and 1%solution of antimycotic mixture at 37°C and in air plus 5%CO₂.Cells were grown as standard monolayers on six-well plate untilreached approximately 70–80%confluency.Cells were serum starved?0.5%?at37°C for approximately 12 h before the experiments and then treated with fecal bacteria liquid from FMT-C and FMT-H groups?fecal bacteria liquid-to-cell media ratio 1:2000?for 3 h,and the blank serum was used as the blank control.We detected SERT mRNA and SERT protein levels using Realtime-PCR and Western blot,detected 5-HT levels using ELISA and immunofluorescence stain.Meanwhile,we detected the level of transgelin protein using immunofluorescence stain,detected the intestinal barrier function of the mice using immunohistochemistry and periodic acid schiff.And we used 16 sRNA pyrosequencing analysis to evaluate intestinal microbiota of mice.Results1.The mice receiving fecal microbiota from patients with constipation presented abnormal defecation parameters.We used the parameters of blank control as the baseline data which were treated with antibiotics?defecation pellets in 2 h:12.6±2.51;fecal weight:301.8±33.8 mg;fecal dry weight:119.3±37.2 mg;fecal water:60.5±10.1%?.There is no significance between blank control and FMT-H group in the 7th and 15th day.The parameters on the 15th day have significantly decreased in the FMT-C group compared with blank control.While there was no significant difference between blank control and FMT-C in the 7th day.Meanwhile,the defecation function was evaluated on the 7th day.The number of defecation pellets in2 h?13.65±3.11 vs 15.39±2.58,P?0.05?,the fecal weight?225.2±46.45 mg vs269.86±32.66 mg,P>0.05?,fecal dry weight?114.72±29.94 mg vs 134.89±25.15mg,P>0.05?and fecal water content?48.76%±6.12%vs 50.46%±5.38%,P>0.05?in the FMT-C group are decreased,but without significant differences comparing with the FMT-H group.The parameters on the 15th day showed that the number of defecation pellets in 2 h?8.48±1.78 vs 12.12±2.89,P<0.05?,the weight of feces?151.88±32.32 mg vs 246.68±63.89 mg,P<0.01?,fecal dry weight?65.49±11.74 mg vs 92.91±23.04 mg,P<0.05?and the fecal water content?56.61%±3.01%vs61.92%±3.68%,P<0.05?have significantly decreased in the FMT-C group compared with those in the FMT-H group.2.On the 15th day,the gastrointestinal transit time?GITT?was significantly prolonged in the FMT-C group compared with the FMT-H group?83.24±11.31 min vs 69.06±2.72 min,P<0.05?;Transgelin protein levels in the mice intestinal tissues in the present study were decreased in the FMT-C group compared with that in the FMT-H group.3.The SERT mRNA level in colon tissue of the FMT-C group was significantly higher compared with that in the FMT-H group?P<0.05?,and consistently,Western blot results showed a significant rising trend in SERT protein expression in the FMT-C group.In the Caco-2 cells,the SERT mRNA level stimulated with fecal liquid from the FMT-C group was significantly higher than that from the FMT-H group and the blank control?P<0.001?.Western blot results also showed that fecal liquid from the FMT-C group significantly up-regulated SERT protein in Caco-2cells.4.ELISA analysis showed that the level of 5-HT in colonic tissue in the FMT-C group was lower than that of the FMT-H group?151.59±10.15 vs 198.68±25.94 ng/ml,P<0.01?.The FMT-C group exhibits a decreased level of colonic 5-HT and an extended GITT compared with those in the FMT-H group,while there is a linear correlation between the level of colonic 5-HT and GITT.The level of 5-HT decreased in the FMT-C group localized to colonic chromogranin A-positive?CgA+?was found in immunofluorescence.Less 5-HT cells per field can be found in FMT-C group.Furthermore,the number of CgA+cells per field had a rising trend in FMT-H group,however,there is no significant difference between FMT-H group and FMT-C group.5.Constipation-induced dysbiosis could be transmitted to the mice.Pyrosequencing analysis was used to further identify the different components of gut microbiota in mice after FMT.Further analysis of microbial phylum showed that the proportion of Firmicutes was significantly decreased,whereas Bacteroidetes was increased in FMT-C group.Moreover,analysis at the genus level showed that the relative abundance of Clostridium,Lactobacillus,Desulfovibrio and Methylobacterium were significantly lower,while Bacteroides and Akkermansia have an increased trend in the FMT-C group compared with the FMT-H group.6.Based on the pyrosequencing analysis,the relative abundance of Akkermansia in the FMT-C group was significantly increased.Accordingly,next we detected the number of mucin-producing goblet cells in the colon from the two groups.PAS staining showed that the average number of goblet cells in each crypt of the colon in the FMT-C group was significantly decreased than that in the FMT-H group?14.44±1.68 vs 22.88±0.79,P<0.01?.MUC2 is the prominent component in the gut secreted from goblet cells.Both the mRNA expression of colonic MUC2 and the average number of MUC2-positive cells in the crypts of the mice?7.49±0.33 vs 17.19±0.26,P<0.05?in the FMT-C group was significantly decreased compared with those in the FMT-H group.ConclusionDysbiosis contributes to chronic constipation development via regulation of serotonin transporter,resolution of intestinal 5-HT and inhibition of intestinal smooth muscle contraction in the intestine.