2.1 Materials
SNS was composed of Bupleurum, Citrus aurantii, Paeonia, Roasted licorice and these four drugs were in same ratio as described in Pharmacopoeia. Dried herbs were provided by Zhangjiakou Zhenxin Pharmaceutical Group Co., Ltd (Zhangjiakou, Hebei, China), and were identified as eligible medicinal material. Balsalazide (H20041706) was purchased from Shanxi Zhendong Ante Biopharmaceutical Co., Ltd. (Shanxi, China). Cell culture reagents and supplies were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). The RT-PCR primer was synthesized by Beijing Dingguo Biology Co., Ltd. TNBS, acacia, dimethyl sulfoxide (DMSO), dexamethasone, and LPS were purchased from Sigma Chemical Co., (St. Louis, MO, USA). NO and MPO detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
2.2 Animals
Healthy male Sprague/Dawley (SD) rats (6–8 weeks old, 180–200 g) were obtained from Sibeifu (Beijing) Biotechnology Co.,Ltd. (Beijing, China; Certificate of Conformity: No. 110324210106095731). All animals were housed in cages in a room with controlled temperature (22 ± 2℃), relative humidity (40–60%), and a 12-h light/dark cycle throughout the study. All rats were fed on a standard diet (crude protein 16%, crude fat 4%, crude fiber 12%, and ash 8%), had free access to water. All experiments were approved by the Animal Welfare and Ethics Branch of the Biomedical Ethics Committee of Hebei North University.
2.3 Preparation of SiNi San (SNS)
The preparation of SNS was as follows: The herbs (composed of Bupleurum, Citrus aurantii, Paeonia, Roasted licorice, the ratio of four herbs was 1: 1: 1: 1 and the total quantity was 500g) were placed in a container, and soaked in 70% ethanol (EtOH) of about 7 times the amount of herbs for 2 h; the herbs were boiled for 1 h and then filtered. Next, the herbal drugs were added with 70% EtOH of about 5 times the amount of the drugs, then decocted and boiled for 1 h and filtered again. At last, the two filtrates were mixed together, concentrated and dried to get 117.5g of SNS, and then stored at 4°C. The major components of SNS were saikosaponins A, naringin, peoniflorin and glycyrrhizic acid, the content respectively were 2.16%, 3.05%, 2.33%, 2.41%, as determined by HPLC/UV [15].
2.4 TNBS-induced IBD
After one week of adaption, 48 rats were randomly divided into six groups, each consisting of eight rats. The groups were as follows: normal group (N), control group (C), Balsalazide group (BST), and SNS groups (H, M and L). Oral administration volumes were 10ml/kg body weight. Balsalazide, SNS were suspended in 5% acacia, and the final dose respectively were 1g/kg (BST), 500mg/kg(H), 250mg/kg(M) and 125mg/kg(L). The normal and control groups rats were given 5% acacia with the same volume. All the groups were treated with the same gavage once a day for three days.
IBD was induced by the TNBS, as described previously with slight modifications [16–17]. On the third day, rats were fasted for 24 h and had free access to water. On the fourth day, they were slightly anesthetized with 10% chloral hydrate and a catheter was inserted through the anus approximately 8cm proximal to the anal verge, so that the TNBS (5% TNBS solution was mixed with equal volume of 50% ethanol solution) at a dose of 100mg/kg were injected into the colon, and the normal group was injected the same volume saline. Then the rats were sustained a head-up position for 10 min. All the groups were administrated with corresponding compounds starting from 24 hours after TNBS administration, for another 5 days. During the experiment, diet and water status, body movement, body weight, diarrhea incidence, and bloody stool were recorded daily.
2.5 Disease activity index and analysis of colon injury
Body weight, stool character and incidence of bloody stools were recorded daily after modeling. The disease activity index (DAI) was determined by previously established scoring system [18] (Table 1). On the nineth day, the blood of rats was collected from orbit, and then, rats were sacrificed under 10% chloral hydrate by cervical dislocation. The colon segments were removed for the assessment of colon damage, and the colon macroscopically damage was scored based on the literature-reported method [19, 20] (Table 2). Routine hematoxylin and eosin (H&E)-stained colon sections according to previously described morphological criteria and the damages were both assessed blindly by two investigators according to a modified histological grading scale, which takes both inflammatory cell infiltration and tissue damage into consideration [21] (Table 3).
2.6 Detection the expression of inflammatory factors and mediators in serum and colon tissues of rats
Blood samples of the rats were centrifuged at 3500 × g for 10 min, and the serum was transferred into new Eppendorf tubes for use. Colon samples (40–50 mg) of the rats were homogenized with ice saline (1:9 w/v) in a digital homogenizer. After centrifugation at 3500 × g for 10 min, the supernatant was collected for further detection. All the samples were stored at low temperature.
Commercial kits were used to detect the levels of NO and MPO in both serum and colon tissue. Inflammatory-related cytokines and mediators included tumor necrosis factor-α(TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were analyzed using the methods of RT-PCR as described below.
2.7 Immunofluorescence detection
We used the following method to detect the expression of ZO-1 and claudin-2 in the colon tissue of rats, so as to determine the effect of SNS on the colon permeability of TNBS-induced IBD rats. Firstly, the colon segments were partly post-fixed with 4% paraformaldehyde for 12h. Then the colon was sliced into 10 µm thick, and was incubated in rabbit anti-claudin-2 (1:100, ab53032, Abcam, USA) and rabbit anti-ZO-1 (1:100, ab96587, Abcam, USA) overnight at 4°C. After washing with PBS for 3 times (5 min each time), fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Santa Cruz, CA) was applied for 1 h in the dark, and then washed three times with PBS. DAPI was used to counterstain Nuclei. Images of ZO-1 and claudin-2stain were acquired by Axio Imager 2 (Zeiss, Oberkochen, Germany) at a 200× magnification.
2.8 Detection the inflammatory damage of LPS induced on RAW264.7 cells
RAW264.7 cells obtained from the cell center at the Chinese Academy of Medical Science and Peking Union Medical College (Beijing, China) were cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 mg/ml), L-glutamine (4.5 mg/ml), and glucose (4.5 mg/ml), and incubated at 37℃ in a humidified atmosphere containing 5% CO2 and 95% air. The media were refreshed every other day.
2×106 cells/ml RAW264.7 cells were seeded on a 24-well plate and incubated overnight. The second day, the media were changed, which contained LPS (0.5µg/ml) with or without SNS (200 µg/ml, 100 µg/ml, 50 µg/ml), and the positive drug dexamethasone (1 µg/ml), and then, the cells were incubated for 24 h. The cell supernatant was collected to detect NO levels, and cells were harvested for RT-PCR analysis.
2.9 Evaluation of trans epithelial electric resistance (TEER) and cell permeability to FD-4
Caco-2 and RAW264.7 cells were obtained from cell center at the Chinese Academy of Medical Science and Peking Union Medical College (Beijing, China). The cells were cultured in Dulbecco's modified Eaglemedium (DMEM) medium containing 10% fetal bovine serum (FBS) and were incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The media were refreshed every two days.
As the previously published protocols with slight modification [22], 1.5×105 cells/ml Caco-2 cells were seeded in transwell cell cultured chambers (6.5 mm diameter inserts, 3.0 µm pore size) (Corning Costar, Cambridge, MA), and the growth medium were changed every two days. Caco-2 cell monolayer was formed after cultured for ten days. Then, 2×106 cells/ml RAW264.7 cells were seeded in the bottom of the transwell. Two days later, the RAW264.7 cells were administrated with SNS (200 µg/ml、100 µg/ml、50 µg/ml), and with or without LPS (0.5µg/ml) for 24h. The cell culture was replaced with serum free medium and incubated for another 30min, then the TEER (Ω/cm2) was evaluated.
After the detection of TEER, 100 µl of 1 mg/ml FD-4 was added into each well of the transwell upper chambers, then the cells were incubated in 37°C for 30min. After that, 100µl of medium from the bottom of the transwell was added into a black well to detect the fluorescence content at an excitation wavelength of 480 nm and emission wavelength of 520 nm using SpectraMax M5as previous literature reported [23].
2.10 RT-PCR analysis
Total RNA was extracted from colon tissues and Caco-2 cells using TRIzol reagent (Sigma, USA) in accordance with the manufacturer's instructions. After RNA concentration was determined by spectrophotometer, complementary DNA (cDNA) was generated using RT-PCR reagent (Thermo Fisher Sci. Inc., Vilnius, Lithuania). Then RT-PCR was conducted using the SYBR Green Quanti Tect RT-PCR kit (Thermo Fisher Sci. Inc., St. Austin, TX, USA) through Light Cycler 96 qPCR system (Roche, Basel, Switzerland). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) acted as a control for total mRNA amount. The results were detected using the 2− △△CT method. The PCR primer sequences were shown in Table 4.
2.11 Statistic Analysis
Values were expressed as means ± SEM. All the grouped data were statistically analyzed with the SPSS 11.0 software. Significant differences between the normal group or control group were evaluated by one-way analysis of variance (ANOVA), and Tukey’s studentized range test was used for post hoc evaluations. A p-value < 0.05 was considered to indicate statistical significance.