Cultivated Lactobacillus acidophilus (1.1854) and Streptococcus thermophilus (1.1855; both China General Microbiological Culture Collection Center, Beijing, China) were centrifuged at 3,000 × g for 10 min, after which the supernatant was discarded, washed with a moderate amount of saline, and suspended in saline in order to adjust the concentration of the bacteria liquid to 7.5×10⁹ CFU/ml (L. acidophilus: S. thermophilus, 1:1). L. acidophilus and S. thermophilus were subsequently mixed to achieve C. quadrangularis shoot fermented bacterial liquid in the proportion of 1:1. Fresh C. quadrangularis shoot (5 kg) were washed and crumbled, and 500 ml mixed fermented bacteria liquid was subsequently added at 42°C (fix format) for 16 h for the main fermentation step. Following this, prophase fermentation was performed at 4°C for 12 h to attain the fermented C. quadrangularis shoot.

Fresh C. quadrangularis shoot (CQS) and fermented C. quadrangularis shoot (FCQS) were smashed using a grinder and washed for 10 min with saline three times. The liquid was discarded and 95% alcohol (10X) was added to clean the residue and the mixture was subsequently centrifuged at 750 × g for 5 min. The supernatant was discarded, the small piece of clean solid was squashed and 3 drops of 1% ethanol solution of methylene dye were prepare the slide containing C. quadrangularis shoot fiber. The slide was observed at ×20 magnification under a microscope (Y-2A; Nikon Corp., Tokyo, Japan).

Following pasteurization, CQS and FCQS were dried, smashed and subsequently mixed with the feed at a ratio of 1:9 according to the weight prior to being pressed into a mold. These CQS and FCQS feeds were then added to the experimental mouse feed to a total of 10% CQS and FCQS.

Seven-week-old female imprinting Kun Ming mice (n=50; weight, 25–30 g) Experimental Animal Center of Chongqing Medical University, Chongqing, China) were randomly divided into normal, control, bisacodyl treatment, SCQ and FSCQ groups (n=10/group). The mice were maintained in a temperature-controlled facility (temperature 23±1°C, relative humidity 50±5%) with a 12-h light/dark cycle. During the experiment, mice in the normal group were not treated. Mice in the control group were not treated during the first 6 days; however, after this they were administered activated carbon water by lavage once daily for 3 days. Mice in the bisacodyl treatment group were administered bisacodyl (100 mg/kg body weight) once daily, and after 6 days were administered activated carbon water by lavage once daily without bisacodyl for 3 days. Mice in these three groups were administered water and were permitted ad libitum access to feed. Mice in the SCQ and FSCQ groups were administered feed containing 10% SCQ and FSCQ, respectively, and ad libitum access to water during the whole experiment, and activated carbon water was administered by lavage during the final 3 days. All groups were restricted from consuming food after 9 days for 24 h, and were administered 10% activated carbon ice water at a concentration of 0.1 ml/10 g by lavage. Each group was divided into two subgroups. Five mice from each group were observed to determine the time point at which they discharged their first black stool defecation, whereas the remaining five mice were euthanized by cervical vertebra dislocation 30 min after activated carbon water lavage to observe the GI transit of activated carbon in the small intestine. GI transit was calculated as follows: GI (%) = distance traveled by activated carbon in the small intestine/the total length of small intestine × 100% (13). These experiments followed a protocol approved by the Animal Ethics Committee of Chongqing Medical University (Chongqing, China).

Mouse blood (0.1 ml) was centrifuged at 3,000 × g for 10 min. The supernatant, which contained the mouse serum, was used to determine the indices of motilin (MTL), endothelin-1 (ET-1), vasoactive intestinal peptide (VIP) and acetylcholine enzyme (AchE), which were measured according to the manufacturer protocols outlined in the MTL, ET-1, VIP (Cusabio Biotech Co., Ltd., Wuhan, China) ELISA kits and AchE kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).

The rats were sacrificed using the cervical vertebra dislocation method. Following dissection, the small intestine was placed in 10% formalin solution for 24 h and 95% ethanol to dehydrate prior to xylene treatment. Transparent blocks were embedded in melted paraffin, sliced by microtome and stained with hematoxylin eosin, and observed at ×10 magnification under a Nikon Y-2A microscope (14).

Total RNA (2 µg) was extracted from the small intestine using RNAzol (Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) reagent from the small intestine of each group. RNA concentration was adjusted to 1 µg/µl. and 2 µl RNA extracts were respectively added to 1 µl oligodT18, RNase, dNTP and MLV enzymes, 5X buffer (GE Healthcare Life Sciences, Chalfont, UK) and 10 µl was used to synthesize cDNA under the conditions of 37°C for 120 min, 99°C for 4 min, and 4°C for 3 min. Expression of c-Kit, stem cell factor (SCF), glial cell-derived neurotrophic factor (GDNF), Transient receptor potential cation channel subfamily V member 1 (TRPV1) and nitric oxide synthase (NOS; Tiangen Biotech Co., Ltd., Beijing, China) was amplified through RT-PCR and compared to GAPDH gene expression (Tiangen Biotech Co., Ltd.). cDNA (2 µl) was mixed with 1 µl of each primer (10 µM) and 16 µl DNase-free water in a PCR premix tube (AccuPower PCR PreMix; Bioneer Corporation, Daejeon, Korea), and PCR was performed in an automatic thermocycler (T100, Bio-Rad, Hercules, CA, USA) for 40 cycles of 94°C for 5 min, 58°C for 30 sec, and 72°C for 90 sec, followed by a 10 min cycle at 95°C. The primers were as follows: Forward: 5′-AGA CCG AAC GCA ACT T-3′ and reverse, 5′-GGT GCC ATC CAC TTC A-3′ for c-Kit; forward, 5′-AAA CTG GTG GCG AAT C-3′; reverse, 5′-CAC GGG TAG CAA GAA C-3′) for SCF; forward, 5′-TTT TAT TCA AGC CAC CAT C-3′; reverse, 5′-AGC CCA AAC CCA AGT CA-3′ for GDNF; forward, 5′-AGC GAG TTC AAA GAC CCA GA-3′ and reverse, 5′-TTC TCC ACC AAG AGG GTC AC-3′ for TRPV1; and forward, 5′-CCA CAT CTG GCA GGA TGA GAA-3′ and reverse, 5′-AGG CAC AGA ACT GAG GGT ACA-3′ for NOS (Tiangen Biotech Co., Ltd., Beijing, China); and forward, 5′-CGG AGT CAA CGG ATT TGG TC-3′ and reverse, 5′-AGC CTT CTC CAT GGT CGT GA-3′ for GAPDH gene expression (Tiangen Biotech Co., Ltd.). Subsequently, 2% agarose gels in 1X Tris-acetate-EDTA were used to assess the amplification of genes by PCR (15).

Following the extraction of tissue protein with RIPA lysate, the total protein concentration of each group was homogenized with ice-cold radioimmunoprecipitation assay (RIPA; Easybio, Beijing, China) buffer in ice-cold PBS, then the extraction was centrifuged at 13,000 × g for 30 min at 4°C. The protein (30 µg) was determined using an ultraviolet spectrophotometer, and the concentrations were adjusted to the same level. Protein samples were separated by 10–12% SDS-PAGE (Schleicher and Schuell, Keene, NH, USA) for 4 h and transferred to nitrocellulose membranes. Membranes were sealed for 3 h in sealing fluid, washed 3 times with Tris-buffered saine (TBS; Easybio, Beijing, China) and incubated with primary antibodies against c-Kit (cat no. ab62154, Abcam, Cambridge, MA, USA), SCF (cat no. ab83866; Abcam), TRPV1 (cat no. ACC-030, Alomone; Beijing, China), GDNF (cat no. ab18956; Abcam), NOS (cat no. sc-49058; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (cat no. ab8226; Abcam) for 2 h. After washing with PBS containing 0.05% Tween 20 (PBS-T), blots were and then incubated with horseradish peroxidase-conjugated goat-anti-rabbit IgG secondary antibody (Cell Signaling Technology, Inc.) for 1 h at room temperature. Following washing 3 times with TBS, treatment with an ultra-sensitive light liquid on a light sensitive X-plate (cat no. BE6031, Easybio, Beijing, China), the nitrocellulose filters were developed and fixed (16). Protein expression levels of c-Kit, SCF, TRPV1, GDNF and NOS were determined, with β-actin as the reference gene.

Experimental data are presented as the mean ± standard deviation. Differences between the mean values for individual groups were assessed with a one-way analysis of variance with Duncan's multiple range tests. P<0.05 was considered to indicate a statistically significant difference. SAS 9.2 (SAS Institute Inc., Cary, NC, USA, 2009) was used for statistical analyses.

As demonstrated in Fig. 1, the fiber gaps in FCQS increased and the structure became more relaxed, whereas the fiber gap of CQS was dense and tighter.

Following treatment with activated carbon, the duration until the first black stool defecation was highest in the control mice (192 min), as compared with 85 min in the normal mice (Fig. 2). Treatment with bisacodyl, which is an anti-constipation drug, decreased the duration between treatment and first black stool defecation to 99 min. Notably, the FCQS-fed mice (117 min) exhibited a significantly shorter time than CQS-fed mice (148 min; P<0.05) and control mice (P<0.01); however the duration exhibited by FCQS-fed mice remained longer than bisacodyl-treated and normal mice.

After inducing constipation, the defecation status of the mice in the different groups altered. The defecation count was highest in the normal mice, whereas the control group mice exhibited the lowest defecation count (Fig. 3). The defecation count of the FCQS group was higher than the CQS group, but remained lower than the bisacodyl group. The stool of the control mice was very dry, and CQS-treated mice also exhibited dry stool. Mice in the normal, bisacodyl and FCQS groups exhibited normal moist stool.

On the final day of experimentation, all mice were treated with activated carbon by lavage. All the activated carbon was able to pass through the small intestine in normal mice 30 min post-treatment and this was set as 100% (Fig. 4; Table I). Activated carbon passed through the small intestine with difficulty in the control mice. The control mice exhibited a GI transit value of 37.9%. Bisacodyl-treated mice exhibited a high GI transit value at 88.3%. FCQS and CQS-treated mice also exhibited increased GI transit compared to the control mice, and FCQS-treated mice (73.8%) exhibited increased GI transit, as compared with CQS (61.7%; P<0.01).

In normal mice, the small intestinal villi were orderly, and the thicknesses of the small intestinal wall was homogeneous (Fig. 5). The villi of control mice were injured and broken, and the thickness of the small intestinal wall was heterogeneous. The small intestinal villi of bisacodyl-treated mice exhibited minimal damage and the villi of CQS and FCQS-treated mice exhibited more severe injury than bisacodyl-treated mice. CQS-treated mice exhibited more severe intestinal villi damage than FCQS-treated mice.

MTL, ET-1, VIP and AchE levels in the serum were highest in the normal mice (Table II), whereas the levels were lowest in control mice (P<0.05). Serum levels from bisacodyl-treated mice were similar to those in normal mice and were higher than CQS and FCQS-fed mice. Notably, respective FCQS and CQS treatment increased these levels compared with the control mice (P<0.05), and fermentation further increased the levels of MTL, ET-1, VIP and AchE compared with the CQS-fed mice.

c-Kit and SCF expression levels in the small intestine of mice were determined by RT-PCR and western blotting (Fig. 6). c-Kit and SCF mRNA and protein expression levels were highest in the normal mice, whereas the control mice exhibited the lowest levels of c-Kit and SCF. Expression levels of bisacodyl-treated mice were higher than all other groups, with the exception of the normal group. FCQS-treated mice exhibited increased expression levels, as compared with the control (P<0.01) and CQS-treated mice (P<0.05).

TRPV1, GDNF and NOS are enteric nerve-related genes (16). FCQS was able to increase GDNF mRNA and protein levels, and decrease TRPV1 and NOS levels, as compared with the control mice (P<0.01) and CQS-treated mice (P<0.05; Fig. 7). TRPV1, GDNF and NOS mRNA and protein expression levels in FCQS-treated mice were similar to those exhibited by normal and bisacodyl-treated mice.