Subject recruitment and sample collection

Patients with chronic constipation and healthy controls were conducted according to Rome III criteria to recruit. Briefly, with onset of symptoms at least 6 months before the diagnosis, and symptoms were consistent with the following diagnostic criteria in the recent 3 months: (1) Two or more of the following conditions must be included: straining; passage of lumpy or hard stools; sensation of incomplete evacuation; sensation of anorectal obstruction; manual maneuvers needed to facilitate defecations; less than three times of defecation every week. (2) Rarely loose stools without laxatives. (3) Inconformity of the diagnostic criteria of irritable bowel syndrome52. Slow transit constipation (STC) is diagnosed according to colon transmission test. All the patients took 20 medical barium sulfate diluted in porridge and erect position abdominal X-ray photographs were then taken at 4, 8, 12, 24, 48 hours after meal53. Patients with STC are found to retain two or more markers after ingestion 48 h, so taking an abdominal X-ray after 48 h is sufficient to make a confirmation in patients with 48 h. Detailed information including age, sex, defecation frequency, and Bristol score of the subjects were collected in Table 1. Healthy controls are the health physical examination personnel without any diseases. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23) which revised in 1996. Meanwhile, the protocols were reviewed and approved by Ethical Committee of General Hospital, Tianjin Medical University, China. Written informed consent was obtained from all participants and their families.

Table 1 Detailed information of the donors. Full size table

Fresh fecal samples were collected from the constipation patients (n = 5) and healthy controls (n = 5) for FMT. The fecal samples collected from each group were mixed to form fecal liquid at room temperature, and the procedures of preparing the fecal samples for FMT were performed as described in the previous studies54. Briefly, fecal samples were dealt with relatively aseptic conditions. Each fecal sample (1 g) was suspended with 10 ml sterile phosphate-buffered saline. The suspensions were filtered through filters with pore diameters of 2.0, 1.0, 0.5 and 0.25 mm. After homogenization and centrifugation, fecal suspensions were obtained and stored in a refrigerator at −80 °C.

Mice and treatment

C57BL/6 mice were purchased from Beijing Animal Research Center, China, weighting 16–18 g, 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 a mixture of 500 mg of ampicillin, 250 mg of vancomycin, 500 mg neomycin and 250 mg of metronidazole (Sigma-Aldrich, St. Louis, MO, USA) daily for 3 days by gavage, considering as the antibiotic depletion model55. 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 experiments56,57,58 (Fig. 1A ). Signs of illness were monitored and body weight was recorded daily. The mice were sacrificed on the 15th day, and colon tissues were collected and stored in 10% formalin solution or −80 °C refrigerator. The animal experiments followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23), revised in 1996. Animal protocols were approved by the Institutional Animal Care and Use Committee at Tianjin Medical University, Tianjin, P. R. China.

Cell culture and treatment

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% CO 2 . Cells were grown as standard monolayers on six-well plate until they reached approximately 70–80% confluency59. Cells were serum starved (0.5%) at 37 °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 according to the previous study55, and the blank serum was used as the blank control.

Defecation function of the mice

Mice in each group were fed in the single cages, we detected the frequency of pellet expulsion, fecal weight, fecal dry weight and fecal water content after giving antibiotics, we used the data of parameters as blank controls. Then the mice were fasted for 16 hours on the 6th and 14th day of the experiment. Freely feeding mice were observed for 2 hours, and the frequency of pellet expulsion and fecal weight were determined. Fecal water content (%) was measured by comparing the weight of the pellets at the end of the experiment and after drying (24 hours at 37 °C). Water content (%) = (wet weight − dry weight)/wet weight × 100% according to the previous studies60, 61.

Gastrointestinal transit time

GITT is the time it takes for food to leave the stomach and travel through the intestines. In our study, five mice in each group were randomly selected for testing GITT and the other five mice were sacrificed for the small intestine advancement test. On the 15th day of experiment, five mice in each group were randomly selected in the single cages. The mice were fasted for 16 hours before experiment, and fed with Indian ink (0.2 mL/10 g body weight-average molar mass) the next day. After that, the time for expulsion of the first blue pellet was determined62. Five other mice were subjected to the small intestine advancement test. The mice were also fasted overnight for 16 h and fed with ink. After a 25 min interval, the mice were killed and the segments from stomach to ileocecal junction were collected. Ink propulsion rate (%) = migration distance of ink/whole length of small intestine × 100%63.

Periodic acid schiff (PAS)

The distal colon was removed from all the mice. The tissue was flushed with PBS, fixed in 4% formaldehyde overnight at room temperature and paraffin-embedded. The specimens were then cut into 5 μm sections and stained with Alcian blue periodic acid–Schiff (PAS) (the goblet cells were stained red). The results were expressed as the number of goblet cells per intestinal villus.

Immunohistochemistry and Immunofluorescence

Formalin-fixed tissues were dehydrated and embedded in paraffin. After being embedded, tissues were sectioned into 4 μm slices which were stained with primary antibodies, rabbit monoclonal anti-MUC2 (1:250, Santa Cruz, USA) overnight at 4 °C. The biotinylated anti-rabbit secondary antibody was stained with horseradish peroxidase (HRP)-streptavidin solution. Finally the sections were counterstained with hematoxylin. Five random areas from a single section were checked for the percentage of positive cells by an independent blinded pathologist. Data were quantified by calculating the average percentages of positive cells in each mouse as the positive rate of cells.

Formalin-fixed tissues were processed for immunofluorescence to evaluate the expression of 5-HT and transgelin protein. Tissues were incubated with primary antibodies (overnight, 4 °C): Part of transgelin, anti-transgelin protein antibody (SM22-alpha-antibody-ab14106) (green, Southern Biotech) was added to the tissue sections for 18 hours at 4 °C. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody(1:5000, Abcam, USA). DAPI (4,6-diamidino-2-phenylindole, blue, Southern Biotech) was lastly applied on the sections. Part of 5-HT, goat anti-mouse 5-HT polyclonal primary antibody(1:500, Abcam, USA) were added to the tissue sections, and then rabbit anti-mouse Chromogranin A polyclonal antibody(1:5000, Abcam, USA) were used. Each of the sections were washed three times with 1 × PBS for 5 minutes and incubated 60 minutes with fluorochrome-conjugated secondary antibodies (5-HT, green; CgA, red) diluted to 2 μg/mL in PBS in the dark. DAPI (4,6-diamidino-2-phenylindole, blue, Southern Biotech) was lastly applied on the sections. We observed and photographed with a fluorescence microscope (Lycra, Germany) for 5-HT and confocal microscopy for transgelin (Nikon, Japan). For 5-HT and CgA staining, numbers of positively-stained puncta were scored blindly, normalized to a field of intestinal mucosa using Image-Pro Plus software, and then averaged across biological replicates.

ELISA analysis

The 5-HT levels were detected in supernatant of colon tissue homogenates by ELISA according to the manufacturer’s instructions. Appropriate amount of mouse colon tissue was through homogenate, centrifugation to get the supernatant. Samples wells and blank wells were set up to be measured. Meanwhile the three repeat wells were set up. The absorbance at 450 nm (OD value) was measured. The linear regression equation was calculated according to the concentration of the standard and the corresponding OD value of the standard curve, and then the sample OD value of the corresponding 5-HT concentration (ng/ml) was calculated.

Real-time PCR analysis

Total RNA of the intestinal tissues or Caco-2 cells was extracted using the RNeasy mini kit (TIANGEN, Carlsbad, CA, USA), and cDNA reverse transcription was applied for using the TIANScript RT Kit (TIANGEN, Inc. Beijing, China) according to the manufacturer’s instructions. The Oligonucleotide primers for target genes were shown as follows: GAPDH, (glyceraldehyde-3-phosphate dehydrogenase, 5′-AGGTCGGTGTGAACGGATTTG-3′ and 5′-TGTAGACCATGTAGTTGAGGTCA-3′), SERT (5′-TGG GCG CTC TAC TAC CTC AT -3′ and 5′-ATGTTGTCCTGGGCGAAGTA-3′) and MUC2 (5′-TCGCCCAAGTCGACA CTCA-3′ and 5′-GCAAATAGCCATAGTACAGTTACACAGC-3′). The 2−ΔΔCt was used to calculate relative mRNA expression.

Western blot analysis

The lysates from the colon tissues or Caco-2 cells were solubilized using RIPA buffer supplemented with protease inhibitors (Solarbio, Beijing, China) and homogenized. And then protein was electrophoresed on a 10% Tris gel with running buffer; then it was blotted to PVDF membrane. Membranes were blocked with nonfat milk and incubated overnight with primary antibodies, anti-SERT antibody (1:1000, Abcam, USA) with anti-β-actin antibody (1:5000, Abcam, USA). Immunoreactive bands were detected after incubation with secondary antibodies (1:5000, EARTHOX, USA). Proteins were quantified densitometrically using Image J software.

16 sRNA pyrosequencing analysis

Fecal DNA was extracted from stool samples of the two groups according to the E.Z.N.A method. Stool DNA Kit QIAamp DNA Stool Mini Kit (Omega Bio-Tek, Norcross, GA, USA) following to the manufacturer’s guidelines. Specific primers with barcode targeting V3-V4 hypervariable region of the 16 S rRNA gene were used for PCR amplification in triplicate64, 65. The Miseq library was constructed and sequenced on the Illumina 2 × 300 bp MiSeq platform after amplification. The optimizing sequences were usually mapped into operational taxonomic units (OTUs) and picked at 97% similarity in Mothur (version v.1.30.1)66, 67. Based on the results of OTUs, community diversity was estimated by Shannon and Simpson index. Similarities were shown by dendrogram among the samples. Principal component analysis (PCA) was carried on the resulting matrix of distances between the two groups.

Statistical analysis

Statistical analysis was performed on SPSS 22.0 (SPSS, Chicago, IL, USA). Measurement data were expressed as mean ± SD. Differences were tested by one- way ANOVA for paired samples. The results of small intestine advancement test were compared by Chi-square test and corrected by Fisher’s. P value < 0.05 was defined as statistically significant.