A total of 84 male Institute of Cancer Research (ICR) mice (age, 6 weeks, weighting 25-30 g) from the Laboratory Animal Centre of the School of Basic Medical Sciences (Xi'an Jiaotong University and Medical Laboratory Animal Centre of Air Force Medical University, Xi'an, China) were used after acclimatization to housing conditions (22±1˚C; 12/12-h light/dark cycle; relative humidity 45-55%) and free access to normal mice chow and water. The normal mice chow was purchased from Huanyu Biotechnology Co., Ltd. and consisted of corn, soybean meal, flour, fish meal, yeast powder, vegetable oil, amino acids, calcium hydrophosphate, salt, vitamins and trace elements, which corresponded to 18.0% crude protein, 4.0% crude fat, 5.0% crude fibre, 8.0% ash, 1.0% calcium, 0.6% phosphorus and other macro- and micronutrients. The mice were divided into four groups: Control; FC; lactulose and Arc (n=6/group). The mice from the lactulose and the Arc group received oral administration of 500 mg/kg lactulose (Shanghai Aladdin Biochemical Technology Co., Ltd.) or 100 mg/kg Arc (>98% purity; Shanghai Aladdin Biochemical Technology Co., Ltd.) daily for 14 days, respectively. An equal volume of pure water was administered by oral gavage to mice in the control group and the FC group. Induction of FC was performed as described previously (24). The mice from the FC, lactulose and Arc groups were administered loperamide (5 mg/kg; Shanghai Aladdin Biochemical Technology Co., Ltd.) orally by gavage on days 12-14. Alterations in water consumption and food intake were measured using a metabolic cage. Additionally, body weight was measured daily on days 12-15 and faeces and distal colon segments were collected on day 15. Mice were anesthetized with inhalation of isoflurane at 3% induction and 2% maintenance. Blood (1 ml) was collected from the postcava and left to stand for 30 min at room temperature. Mice were sacrificed by exsanguination under anesthesia. Serum was collected following centrifugation at 1,111 x g at room temperature for 10 min. Other mice were euthanized by intraperitoneal injection of 200 mg/kg sodium pentobarbital. In this current study, the criteria for humane endpoint euthanasia included inability to access food and water and obvious anxiety resulting from FC and no mouse was sacrificed according to these. All animal protocols were approved by the Institutional Animal Care and Use Committee of the Shaanxi University of Chinese Medicine (approval no. SUCMDL20190314001).

Determination of intestinal transit ratio (n=6 each group) and transit time (n=6 each group) was performed as described previously (25,26). Briefly, following 14 days of treatment, mice fasted with free access to water for 12 h. For the intestinal transit ratio analysis, the mice were orally administered a meal containing 20% charcoal (Shanghai Aladdin Biochemical Technology Co., Ltd.) and 10% gum arable (Shanghai Aladdin Biochemical Technology Co., Ltd.) at a volume of 0.1 ml/10 g. Mice were euthanized by intraperitoneal injection of 200 mg/kg sodium pentobarbital 30 min after meal ingestion and the intestine from the pylorus to the rectum were collected immediately. The lengths of the total intestinal tract and the distance the charcoal meal had travelled from the pylorus were measured. The intestinal transit ratio was calculated as follows: Intestinal transit ratio (%)=length travelled by the charcoal meal/total length of intestine x100. The transit time was evaluated in mice following gavage of a 0.1 ml volume of 5% Evans blue (MilliporeSigma) in 1.5% methylcellulose (Shanghai Aladdin Biochemical Technology Co., Ltd.). Faeces were monitored at 10 min intervals for the presence of Evans blue staining. The time to the excretion of the first blue faeces was recorded.

Formalin (10%, at room temperature for 24 h)-fixed distal colon segments were embedded in paraffin (at 60˚C for 2 h) and sliced into serial 5-µm thick sections. Hematoxylin and eosin (H&E) staining (2 g/l hematoxylin and 3.5 g/l eosin) at room temperature for 5 min was used to assess the morphometric features. To measure colonic mucosa thickness, colon sections were stained with Alcian Blue Dye Solution (pH2.5) (Leagene Biotechnology) at room temperature for 30 min. Images were acquired with a light microscope (Olympus Corporation) at x200 magnification. Image-Pro Plus software (Version 6.0.0.260; Media Cybernetics, Inc.) was used to measure the thickness of muscle and mucosa.

Following fixation of distal colon samples in 1% osmic acid at room temperature for 2 h, washing in phosphate buffer and dehydrating in an ethanol series, the colon samples were embedded in epoxy resin (SPI Supplies; Structure Probe, Inc.) at 37˚C overnight and polymerized at 60˚C for 48 h. The blocks were cut into ultra-thin 70-nm thick sections and stained with 2% uranyl acetate and 2.6% lead citrate at room temperature for 8 min. A transmission electron microscope (H-7650, Hitachi) was used to detect the morphology and structure of Cajal cells in the colon of mice at x20,000 magnification. The images were acquired with an accelerating voltage of 80 kV.

Commercial ELISA kits for MTL (cat. no. CEA575Mu; Cloud-Clone Corp.) and BDNF [cat. no. EK2127; Multisciences (LIANKE) Biotech., Co., Ltd.] were used to measure their concentration in the serum and colon tissues of mice, according to the manufacturer's protocol. The levels of NO in the colon tissue were assessed using an NO assay kit (cat. no. A013; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions.

The colon tissue was homogenized in RIPA lysis buffer (Beyotime Institute of Biotechnology). Following quantification of protein concentrations using a BCA protein assay kit (Beyotime Institute of Biotechnology), a total of 15 µg/lane (C-Kit, SCF, AQP-3, and AQP-9) or 30 µg/lane (nNOS, eNOS, and iNOS) protein was loaded on an 8% or 12% SDS-gel, resolved using SDS-PAGE and transferred onto PVDF membranes (Thermo Fisher Scientific, Inc.). The membranes were blocked with TBST containing 0.15% Tween-20 and 5% BSA (BioFroxx, Inc.) and incubated with primary antibodies against neuronal NOS (nNOS; 1:1,000; cat. no. A2649; ABclonal Biotech Co., Ltd.), endothelial NOS (eNOS; 1:1,000; cat. no. A15075; ABclonal Biotech Co., Ltd.), iNOS (1:1,000; cat. no. A0312; ABclonal Biotech Co., Ltd.), C-Kit (1:1,000; cat. no. AF6153; Affinity Biosciences), SCF (1:500; cat. no. 26582-1-AP; ProteinTech Group, Inc.), AQP-3 (1:1,000; cat. no. A2838; ABclonal Biotech Co., Ltd.), AQP-9 (1:1,000; cat. no. DF9225, Affinity Biosciences) or β-actin (1:2,000; cat. no. 60008-1-Ig; ProteinTech Group, Inc.) overnight at 4˚C. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; cat. nos. SA00001-1 and SA00001-2; ProteinTech Group, Inc.) at 37˚C for 40 min. Protein bands were visualized using ECL-Reagent (7 Sea Biotech) and imaged. Gel-Pro-Analyzer software (Version 4.0, Media Cybernetics, Inc.) was used for quantitative analysis of blots.

Immunohistochemical analysis of the colon tissue was performed on 5-µm sections. Briefly, antigen retrieval was performed using a citric acid buffer and heating in an 800 W microwave for 10 min, blocked with BSA (1%, Sangon Biotech Co., Ltd.) for 15 min at room temperature and incubated with primary antibodies against C-Kit (1:100; cat. no. AF6153; Affinity Biosciences), SCF (1:100; cat. no. 26582-1-AP; ProteinTech Group, Inc.), AQP-3 (1:100; cat. no. A2838; ABclonal Biotech Co., Ltd.), or AQP-9 (1:100; cat. no. DF9225, Affinity Biosciences) at 4˚C overnight. For immunohistochemistry, following incubation with horseradish peroxidase-conjugated secondary antibody (1:500; cat. no. 31460; Thermo Fisher Scientific, Inc.) at 37˚C for 1 h, sections were stained with DAB) detection kit (MXB Biotechnologies Co., Ltd.) at room temperature for 3 min, examined by light microscopy (Olympus Corporation) at x400 magnification equipped with a digital camera (Olympus Corporation). For immunofluorescence, the sections were incubated with Cy3-conjugated secondary antibody (1:200; cat. no. A27039; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. DAPI (100 ng/ml, Shanghai Aladdin Biochemical Technology Co., Ltd.) was used for counterstaining at room temperature for 5 min. Images were captured with a fluorescence microscope (Olympus Corporation) at 400x magnification. The evaluation of immunofluorescent and immunohistochemical staining was performed using Image-Pro Plus software (Version 6.0.0.260, Media Cybernetics, Inc.).

Total RNA in the distal colon tissues of mice was extracted using TRIpure™ (BioTeke Corporation) and quantified using a spectrophotometer (NanoDrop™ 2000; Thermo Fisher Scientific, Inc.). Following cDNA synthesis with Moloney murine leukaemia virus reverse transcriptase (BeyoRT™ II M-MLV, Beyotime Institute of Biotechnology) according to the manufacturer's instructions, RT-qPCR was performed on an Exicycler 96 PCR system (Bioneer Corporation) with initial denaturation at 95˚C for 5 min followed by 40 cycles of denaturation at 95˚C for 10 sec, annealing at 60˚C for 10 s, and extension at 72˚C for 15 s. mRNA levels were quantified using the 2^-ΔΔCq method and normalized to the internal reference gene β-actin (27). The primer sequences were as follows: Tumor necrosis factor-α (TNF-α) forward, 5'-TCTCATTCCTGCTTGTGG-3' and reverse, 5'-CTTGGTGGTTTGCTACG-3'; interleukin (IL)-1β forward, 5'-CTCAACTGTGAAATGCCACC-3' and reverse, 5'-GAGTGATACTGCCTGCCTGA-3'; IL-6 forward, 5'-TAACAGATAAGCTGGAGTC-3' and reverse, 5'-TAGGTTTGCCGAGTAGA-3' and β-actin forward, 5'-CTGTGCCCATCTACGAGGGCTAT-3' and reverse, 5'-TTTGATGTCACGCACGATTTCC-3'.

The data are presented as the mean ± standard deviation (n=6) and analyzed using one-way ANOVA followed by post hoc Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

FC was evaluated based on the changes in faecal matter, including defecation frequency and faecal moisture analysis. Loperamide was used to establish the FC model. Loperamide resulted in a significant decrease in faecal numbers (4.00±0.89), faecal weight (0.11±0.02 g) and water content (49.59±5.79%) in FC mice compared with that in the control mice (20.50±3.45, 0.47±0.09 g and 67.18±6.38%, respectively; Table I). Body weight and feeding behaviour did not vary between the control and FC groups. Additionally, the length travelled by the charcoal meal in the intestinal tract was shorter in loperamide-treated mice compared with that in the control (red arrows delineate the travelled location of the charcoal meal; Fig. 1A). Furthermore, no difference was observed in the total length of the intestine in each group, but the length traveled by the charcoal was shorter in FC mice than that of control (Fig. 1B and C). Measurement of intestinal transit showed a decrease in intestinal transit ratio and an increase in the transit time in the FC mice (Fig. 1D and E). The aforementioned results indicated that the FC model was established successfully. Arc treatment not only improved the faecal characteristics but also accelerated intestinal transit in FC mice.

Subsequently, changes in the colonic pathology in FC mice were assessed. H&E staining of the colon sections showed that the muscular layer was significantly thinner in the colon of FC mice compared with that in the control mice (Fig. 2A and B; double-headed arrows delineate the muscular layer). Furthermore, results of H&E staining could be confirmed by alcian blue staining, which provided more evidence on the protective effects of Arc on FC. The results of alcian blue staining revealed that the thickness of the colonic mucosa was significantly decreased in the FC group compared with the control group (Fig. 2C and D; double-headed arrows delineate the colonic mucosa). These changes were reversed in Arc-treated mice. Thus, Arc mitigated colonic damage in the FC mice.

To investigate the underlying mechanism by which Arc attenuated FC, levels of excitatory (MTL) and inhibitory neurotransmitters (NO), as well as BDNF, were determined in the serum and colon tissue of mice. Arc ameliorated the decrease in MTL and BDNF levels, as well as the increase in NO observed in loperamide-treated mice compared with those in the FC mice (Fig. 3A-E). Western blot analysis showed higher expression of nNOS and eNOS and lower expression of iNOS in the Arc group compared with the FC group (Fig. 3F and G). Moreover, the mRNA levels of TNF-α, IL-1β and IL-6 were increased in FC mice compared with those in the control. Additionally, the inflammatory induction was significantly inhibited by Arc (Fig. S1A-C). These findings showed that Arc regulated levels of neurotransmitters and inflammation in FC mice.

The levels of C-Kit and SCF and the condition of mitochondria/ER were used to evaluate ICC injury. Immunohistochemical analysis showed that C-Kit and SCF were primarily present in the submucosa and muscle layers (Fig. 4A-C). Arc reversed the decrease in C-Kit and SCF levels in the colon of FC mice, which was confirmed by western blot analysis (Fig. 4D and E). Electron microscopy results showed that the ICC in the colon of control mice was rich in cell organelles, including endoplasmic reticulum and mitochondria. Injured ICC in the FC group were characterized by swollen mitochondria with disrupted cristae and partial cytoplasmic depletion. By contrast, the damage to the ICC in the Arc group was notably decreased (Fig. 4F). These results showed that Arc reduced ICC damage in the colon of FC mice.

Based on the involvement of AQPs in FC progression (28), the expression levels of AQPs in the colon of mice were detected. Increased expression of AQP-3 and AQP-9 was observed in the colon of FC mice and this was significantly reduced by Arc treatment (Fig. 5A-E). Therefore, Arc downregulated the levels of AQPs in the colon of mice with FC.