Adult male and female Kunming mice (n=8 per group, weighing 20-22g) were obtained from the Experimental Animal Center of the Air Force Military Medical University, China. Mice were housed in the experimental animal center in controlled, pathogen-free conditions and kept at a constant temperature (22°C) with12:12-hour light-dark cycle and free access to standard laboratory tap water. All animal study protocols were developed according to the International Guidelines for Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of the Shannxi University of Chinese Medicine.
POI was induced using a small bowel manipulation method as previously reported 10, 11. In brief, mice were anesthetized via intraperitoneal injection of 2% chloral hydrate at a dose of 0.15ml/10g, and the abdomen was shaved and sterilized with 75% ethanol. A 1 cm midline abdominal incision was made so that the small intestine from the ligament of Treitz to the terminal ileum could be carefully externalized onto wet gauze using two saline-moistened cotton swabs. Then, this section of the small intestine was systematically touched with two saline-moistened cotton swabs for 5 minutes, and this was repeated 3 times. The sham treatment group only underwent the laparotomy procedure without any bowel manipulation.
After suturing the abdomen, mice were randomly assigned to receive either T-DCQT, M-DCQT, or normal saline solution (N.S) treatment.
2.2 Evaluation of intestinal motility
Intestinal motility was evaluated 24h and 48h after surgery10. Briefly, mice were gavaged with a charcoal marking mixture (10% charcoal suspension in 10% gum arabic, 0.1 ml per 10 g of body weight) (Sigma-Aldrich Co., St. Louis, MO).
Mice were sacrificed 20 min later via enflurane inhalation and intestinal motility was expressed as follows: small intestinal transit (%) = the distance the charcoal mixture traveled through/the whole length of the small intestine × 100.
2.3 Sample collection and detection
At 24h and 48h post-operation, mice were re-anesthetized and the ileum was removed. Blood was exsanguinated from the inferior vena cava. Blood samples were centrifuged at 4000g for 10 min at 4°C. The ileum tissue and plasma samples were then stored at -80°C for subsequent experiments.
2.3.1Evaluation of pathological alterations
Intestinal sections were fixed in 10% neutral-buffered formalin, embedded in paraffin, and cut into 5-μm sections on a microtome. The sections were stained with hematoxylin and eosin (H&E). Three specimens from each treatment group and five representative areas from each slide were selected for blinded evaluation of damage of the ileum post-surgery/treatment.
Some formalin-fixed, paraffin-embedded sections were used to perform immunohistochemical staining for NF-κB，p38, and TLR4. In brief, tissue sections were incubated with either a mouse NF-κB primary antibody (SC-8008), a rabbit p38 primary antibody (SC-535), or a mouse TLR4 primary antibody (SC-293072) (1:100; Santa Cruz, USA) at 4°C overnight. The sections were then washed in 10 mM phosphate-buffered saline (PBS; pH 7.4) (Thermo Fisher Scientific) at room temperature three times and then incubated with a goat anti-rabbit horseradish peroxidase (HRP)-conjugated IgG secondary antibody (1:10000; Cell Signaling Technology®) at room temperature for 30 min. The sections were then washed 3 times in 10 mM Tris-buffered saline (pH 8.4) for 2min at room temperature and then washed with 10 mM PBS (pH 7.4) three times at room temperature. The sections were then incubated with 200 µl of 3, 3'-diaminobenzidine substrate (Thermo Fisher Scientific) for 5 min at room temperature, followed by washing with distilled water at room temperature for 5 min.
Following protein staining, counter staining and scoring was performed by a pathologist. The level of immunostaining in the tissue samples was evaluated using a standard scoring system based on the proportion of positively stained cells and the level of staining intensity.
2.3.2RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
The primers used for qPCR studies are listed in Table 1.
Total RNA was isolated from fresh ileum and colon tissues using TRIzol (Invitrogen, New York, NY, USA; cat.no. 15596-026). The concentration of RNA and absorbance ratio for 260 nm/280 nm (OD260/280) was verified using a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). After DNase I treatment, the RNA was transcribed into cDNA using a Prime Script RT Reagent Kit (TaKaRa, Tokyo, Japan; cat. no. RR037A) according to the manufacturer’s instructions in an Eppendorf Mastercycler Personal Thermo cycler with the following method: 37°C for 15 min, followed by 85°C for 5 s, and then cooling at 4°C. qRT-PCR was conducted in a total reaction volume of 20 μL with SYBR Premix EX Taq II (TaKaRa, Tokyo, Japan; cat. no. RR820A) with the following method: 30 seconds at 95°C, followed by 40 cycles of 5 seconds each at 95°C and 31 seconds at 60°C. Melt curve analysis was performed to confirm the specificity of the qRT-PCR. qRT-PCR studies were performed with all samples in triplicate, and the results were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and then analyzed with the 2^−ΔΔCt method.
2.3.3 Protein preparation and western blotting
According to previously described methods, ileal tissue segments were homogenized and lysed with ice-cold 1 × RIPA buffer containing inhibitors. The protein concentration was determined using the BCA method. The supernatant was isolated and mixed with Laemmli buffer and then boiled for 10 minutes at 95°C. Equal amounts of protein were loaded into 10% gels to be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After separation by SDS-PAGE, the protein in the gel was transferred to a polyvinylidenedi fluoride (PVDF) membrane. Following transfer, the membrane was blocked for 2 h with 5% nonfat milk in TBST to minimize binding of nonspecific antigens. The membranes were then washed with TBST and incubated with primary antibody overnight at 4°C.
Primary antibodies used included anti-NF-κB, TLR4, p-p38, and p38 MAPK. After incubation in primary antibody, the membranes were washed in TBS and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein bands were detected using an ECL chemiluminescent detection system. The densities of target protein bands were then normalized to that of β-actin. Western blot analysis was conducted with Image-studio version 4.0.
2.3.4 Liquid chip method to detect the expression of inflammatory cytokines。
The expression levels of the plasma cytokinesIL-1α, IL-6, MCP-1, MIP-1α, MIP-1β, IL-17, INF-γ, IL-4, IL-10, IL-13, and TNF-α were determined using Luminex technology (Milliplex MAP Human Cytokine/Chemokine Panel; Cat no: HCYTOMAG-60 K, Millipore, St. Charles, MO). This bead-based assay utilizes fluorescent color-coded beads pre-coated with capture antibodies that target specific cytokines. Plasma samples were filtered through 0.22 μm spin filters and run in duplicates for each assay. Duplicates did not vary by more than 5%. Samples were assayed using Luminex200 (USA)12.
T- and M-DCQT are composed of spray-dried medicinal herbs (crude drug) were listed in Table 2, and they purchased from Shaanxi Traditional Chinese Medicine Pieces Co., Ltd. (Shaanxi, China). The crude formula components were extracted, concentrated, and used as follows.
According to the manufacturer, the T-DCQT decoction was prepared in the standard ratio of 12:15:12:9. One dose of the mixture was steeped in cold water for 2h. After decocting the mixture twice (first decoction was 90 min long, second decoction was 60 min long; Rheumpalmatum L. was added 70 min late into the first decoction period), the T-DCQT decoction was mixed and filtered. 48g of crude drug in sterile distilled water was concentrated into 96ml to generate a 0.5g/ml solution. Taking into account the effective dose of T-DCQT in patients and the difference in body surface area between human and animals, T-DCQT was administered to mice at a dose of 0.1 ml/10 g body weight. The procedure for preparing the M-DCQT decoction was similar to the procedure used to prepare T-DCQT.
The mice in the T-DCQT and M-DCQT groups received their respective drug decoction via transanal enema at a dose of 0.1ml/10g body weight, twice per day (at 8 hours interval). The drug enema is administered by insertion 2 cm into the rectum and is kept in place for 15 min. In other experimental groups, normal saline was delivered by transanal enema.
2.5 Experimental design
In pilot experiments, mice were euthanized at 4, 12, 24, and 48 h post-operation (n=8-10 per group). Based on the previous experiments, we selected 24h and 48 h as the designated time points for all subsequent experiments and analyses.
Mice were randomly divided into the experimental groups, which were the control group, sham group, POI group, T-DCQT group, and M-DCQT group. Mice in each experimental group were also randomly divided into 24 or 48h experimental subgroups (n=8 per subgroup).
2.6 Data analysis
Results are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using IBM SPSS statistics 19. Data quality was verified with Levene’s test for equality of variances. The results were then analyzed with one-way ANOVA, followed by the least-significance difference (LSD) test. Some results were also analyzed using the Kruskal-Wallis and Mann-Whitney U tests. Differences with P < .05 were considered statistically significant.