Participants and Questionnaires
IBS-D patients and healthy controls (HCs) were recruited from Gastroenterology Outpatient Department at Qilu Hospital, Shandong University. IBS-D patients were diagnosed by a gastroenterologist according to ROME IV criteria. The HCs were free of gastrointestinal symptoms and to attend routine health check-up. Exclusion criteria included history of any organic syndromes (coeliac disease, tumors, inflammatory bowel disease, gastrointestinal infections and psychiatric disorders), history of major abdominal surgery or using probiotics, non-steroidal anti-inflammatory drugs, anti-inflammatory drugs, Proton-Pump Inhibitors, prokinetics, antianxiety drugs, antidepressants in the past 4 weeks, accompanying with pregnancy, lactation, bladder irritation, chronic pelvic pain syndrome, dysmenorrhea, endometriosis, other painful gynecological diseases, clotting disorder and obvious mental disorders. In addition, the individuals with long-term alcohol consumption were also ruled out. All participants provided written informed consent and the study was approved by the ethics committee of Qilu Hospital of Shandong University (ethic’s number: 2019036).
The information of subjects was collected by completing questionnaires (Additional file 1). The common information of HCs and IBS-D patients included sex, age, body mass index (BMI), Hamilton anxiety scale (HAM-A) , Hamilton depression scale (HAM-D) . In addition, the questionnaires of IBS-D patients included clinical symptoms and IBS Severity Scores (IBS-SSS) .
Subjects underwent colonoscopy and 3 biopsies from cecum and 3 biopsies from descending colon respectively were obtained using endoscopic biopsy forceps (MIN-BF-23; MICRO-TECH, Nanjing, China) during colonoscopy withdrawal. One of biopsy samples from cecum and descending colon respectively were ﬁxed in formalin and the others were stored in liquid nitrogen immediately.
The mucosa was fixed in formalin for 72h and then was embedded in paraffin. The paraffin block was cut into 4 μm sections using a cryostat and was mounted onto glass slides. The sections were incubated in rabbit anti-CgA antibody (1:100; Abcam, Cambridge, UK) in a humidified box at 4°C overnight and then in Alexa Fluor 488-conjugated anti-rabbit IgG (1:400; Abcam) in a black humidified box at room temperature for 1 hour and in fluoroshield mounting medium with DAPI (Abcam) for 5 minutes. Finally, the slides were observed using fluorescence microscope (BX53, OLYMPUS, Tokyo, Japan). EC cells were identified in the crypt epithelium and showed strong nuclear and cytoplasmic staining. The number of EC cells was quantiﬁed with methods described by the previous studies [26-28]. Total of 5 nonoverlapping high-power ﬁelds (ﬁnal magniﬁcation 200×) were chosen and 2 persons who were blinded to clinical data analyzed the number of EC cells.
Quantitative Real Time Polymerase Chain Reaction
The total RNA was extracted using AllPrep DNA/RNA/Protein Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Quantitative Real Time Polymerase Chain Reaction (QRT-PCR) reactions were made by the SYBR® Green Realtime PCR Master Mix (QPK-201, TOYOBO) in a 20μl reaction volume containing the following reagents: 1μl of cDNA preparation; SYBR® Green Realtime PCR Master Mix (TOYOBO); 7.4μl PCR grade water, and 0.8μM of forward and reverse primers. QRT-PCR reactions were run in triplicate on an Applied Biosystems Stepone real-time PCR System (Thermo, Waltham, MA, USA) using the following conditions for pre-denaturation, denaturation, annealing and extension (40 cycles): 95˚C for 1 min, 95˚C for 15 s and 58˚C for 15 s, followed by 72˚C for 45s. The specific primers sequences were as follows (5＇ - 3＇): TPH1: forward CTGGTTATGCTCTTGGTGTCTTTC, reverse TGCAAAGGAGAAGATGAGAGAATTTAC； β-actin: forward ATGATGATATCGCCGCGCTC, reverse CCACCATCACGCCCTGG.
Total of 20μg extracted protein as experimental samples were separated by 10% SDS–PAGE and transferred to polyvinylidenedifluoride membranes (Bio-Rad, Hercules, CA, USA). Then, the membranes were incubated into 5% skim milk for 2 h at room temperature. After that, the membranes were exposed to primary antibody overnight at 4˚C and were incubated for 1 h at room temperature using secondary antibody. Finally, the enhanced chemiluminescence kit (Merck Millipore, Darmstadt, Germany) was used to reveal the reaction products. The antibodies used in this study were as follows: rabbit anti-TPH1 (1:500; Abcam), anti-β-actin antibody (1:1000; Zhongshan Gold Bridge, Beijing, China), horseradish peroxidase-conjugated goat anti- rabbit IgG (1:1000; Zhongshan Gold Bridge), and goat anti-mouse IgG (1:1000; Zhongshan Gold Bridge). The results were quantified by using Image J software (National Institutes of Health, Bethesda, MD, USA).
Mucosal microbiota assessment
The V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers (5＇ - 3＇): forward ACTCCTACGGGAGGCAGCAG and reverse GGACTACHVGGGTWTCTAAT by thermocycler PCR system (GeneAmp 9700, ABI, USA). Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Raw data files were demultiplexed, quality-filtered by Trimmomatic and merged by FLASH with the following criteria: (i) The reads were truncated at any site receiving an average quality score < 20 over a 50 bp sliding window. (ii) Primers were exactly matched allowing 2 nucleotide mismatching, and reads containing ambiguous bases were removed. (iii) Sequences whose overlap longer than 10 bp were merged according to their overlap sequence. Operational taxonomic units (OTUs) were clustered with 97% similarity cutoff using UPARSE (version 7.1) and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier algorithm against the Silva (SSU132) 16S rRNA database using confidence threshold of 70%. Venn diagrams were made to express the difference of OTUs and genera between the groups. Correlation analysis was conducted to show the relation between variables (EC cells number, HAM-A, HAM-D, IBS-SSS, Degree of Abdominal Pain, Frequency of Abdominal Pain, Frequency of Defecation) and microbial abundance.
Data were analyzed by SPSS 22.0 and R 3.1.1. The data were expressed as means ± SD for continuous variables and percentages for categorical variables. Differences between two groups were evaluated by independent - samples t-test or non-parametric test according to the data in normal distribution or not. Categorical variable data was analyzed by the chi-square test. The correlation between variables and bacterial relative abundance were assessed by using Spearman correlation analysis. P value less than 0.05 was regarded as significant.