| Objective Chronic constipation is a frequent disorder of the lower gastrointestinal tract with characteristic infrequent bowel movements, passage of hard stools and straining, with onset of symptoms at least 6 months. Chronic constipation is one of the most common Functional Gastrointestinal Disorders, with high population incidence rate, which seriously affectes the quality of life and physical and mental health of patients because of recurrent disease. In recent years, researches have shown that the intestinal microbiota of chronic constipation patients is different from healthy normal peoples in number and diversity. Researches have also shown that administration of antibiotics and microbioecological preparations can improve the symptoms of constipation. 5-hydroxy tryptamine(5-HT)is an important neurotransmitter, more than 90% of the body’s 5-HT is synthesized in the gut. Gut-derived 5-HT regulates diverse functions, including intestinal fluid secretion, gut motility, and gastrointestinal sensation. Serotonin transporter(SERT) is a universally existing cross-membrane transport protein,which is located in the enterochromaffin(EC) cells of the bowel and presynaptic membrane,plays a key role in 5-HT reuptake, thereby terminating the role of 5-HT in effect site. 5-HT and SERT plays an important role in the pathogenesis of chronic constipation. In the present study, we treated mice with intestinal microbiota of chronic constipation patients and healthy normal peoples, then dectected the SERT and 5-HT in mice intestine, dectected the defecation of mice.The aim is to investigate the possible mechanism of intestinal microbiota dysbiosis in the generation of chronic constipation.Methods In the present study, fecal samples were collected from chronic constipation patients(n=5) and healthy normal peoples(n=5) according to the inclusion criteria, then analysed by pyrosequencing. Six-week-old female C57 BL / 6 mice were randomized into two groups(n=20): control group(n=10, fecal microbiota of chronic constipation patients) and experiment group(n=10, fecal microbiota of healthy normal peoples). Streptomycin(25mg) was administered to the mice for 3 days, then fecal microbiota(250ul,1:10 g/ml) was given to the mice by oral gavage once daily for 3 consecutive days, then on alternate days, a total of 7 days. On day 7 the total number within 2h, weight, dry weight, water content of feces and defecating time of first black feces were determined. On day 15, the total number within 2h, weight, dry weight, water content of feces and ink propulsion rate were determined(All of the mice were fasted overnight for 16 h. On the next morning, mice were given ink 0.2 m L/10 g. After a 25 min interval, the mice were killed to collect the small intestinal segments of stomach to ileocecal junction. Ink propulsion rate(%) = migration distance of ink/whole length of small intestine × 100%). All animals were killed after 15 days, and the colons of mice were removed for later use. We detected SERT m RNA and SERT protein levels using Realtime-PCR and Western blot, detected 5-HT levels using ELISA and immunofluorescence stain.Results 1. The results of pyrosequencing showed that, compared with the control group,the abundances and ecological diversities of the chronic constipation patients intestinal microbiomes were decreased, F/B(Firmicutes/Bacteroidetes)was decreased, with higher proportion of Bifidobacterium, Enterococcus, Fusobacterium, and lower percentage of Clostridium, Lactobacillus, Streptococcus, Escherichia-Shigella, Desulfovibrio. 2. On day 7, the fecal weight within 2h of experimental group mice was significantly decreased compared with the control group(271.86±32.72 vs 223.24±47.50,P<0.05). However the differences of other fecal parameters were not significant, including the total number within 2h(15.42±2.63 vs 13.55±3.12,P﹥0.05),dry weight(134.91±25.18 vs 114.82±29.96,P﹥0.05),water content of feces(50.62%±5.42% vs 48.88%±6.08%,P﹥0.05) and the defecating time of first black feces(79.19±12.66 vs 89.08±16.11,P﹥0.05). On day 15, the total number within 2h(12.14±2.90 vs 8.55±1.83,P<0.05), weight(246.72±64.01 vs 151.90±32.42,P<0.01), dry weight(92.93±23.07 vs 65.52±11.76,P<0.05), water content of feces(61.95%±3.70% vs 56.63%±3.01%,P<0.05) were decreased, the defecating time of first black feces(69.06±2.72 vs 83.24±11.31,P<0.05)were increased in the experimental group compared with the control group. The differences of all fecal parameters were significant. 3. In the control group, ink propulsion rate of all the 5 mice were 100%. In the experimental group, ink propulsion rate of 2 mice were 100%. Difference between the two groups was significant according to the chi-square test(5/5 vs 2/5,P<0.05).4. The results of Realtime-PCR and Western-blot showed that SERT m RNA and SERT protein levels in mice intestine were significantly increased in the experimental group compared with the control group(P<0.05). 5. The results of ELISA showed that 5-HT level in mice intestine was significantly decreased in the experimental group compared with the control group(198.77±25.99 vs 151.69±10.18,P<0.05), which is consistent with the results of immunofluorescence stain.Conclusions Intestinal microbiota of chronic constipation patients can regulate 5-HT and SERT levels in the mice intestinal tissues, and affect mice bowel movement. Intestinal microbiota dysbiosis may promote chronic constipation through regulating 5-HT and SERT levels in intestine. |
