Design
A randomized controlled experiment of animal models.
Time and setting
The experiment was completed in the 309th Hospital of PLA and the Institute of Drug and Instrument Control, General Logistics Department of Chinese People’s Liberation Army from August 2010 to March 2012.
Materials
Animals
A total of 44 male Sprague-Dawley rats aged 6-8 weeks and weighing 180-220 g were provided by the Institute of Drug and Instrument Control, General Logistics Department of Chinese PLA (first class, certificate No. H00117). The rats were housed in the specific-pathogen free laboratory at Experimental Animal Center, Department of Pharmacology, Institute of Drug and Instrument Control, General Logistics Department of Chinese PLA. The rats were allowed to acclimate to their new surroundings for 1 week before experiments.
Drugs and reagents
Resina draconis (dragon’s blood, common bletilla tuber) is a compound Chinese medicine[8]. Stock solution per milliliter contains resina draconis crude drug 2.0 g (adult dose: [1.45 g/(kg·d)], which is provided by the Manufacturing Laboratory, the 309th Hospital of Chinese PLA. Resina draconis dose (5.0 g/kg) was equal to 3.5 times the daily dose of adult in clinic.
Methods
Grouping
The involved 44 rats were randomly assigned to four groups (n = 11): blank control group, TNBS model group, resina draconis group, and 5-aminosalicylic acid treatment group. The rats in the TNBS model group were injected with a mixture of TNBS 30 mg and 50% alcohol 0.25 mL. The resina draconis group was intragastrically treated with draconis [0.75 g(kg·d)] and the 5-aminosalicylic acid treatment group was given 5-aminosalicylic acid [100 mg(kg·d)] in the same way for 10 days. One rat was selected from each group in every experiment.
Establishment of colitis models and drug intervention
With the exception of blank control group, TNBS-induced ulcerative colitis models were established in the TNBS model group, resina draconis group, and 5-aminosalicylic acid treatment group in accordance with the methods of Sobczak et al [9]. The mixture of TNBS 30 mg and 50% alcohol 0.25 mL was injected into the rat intestine. The rats appeared any of the items can be successful model criteria, such as stools, occult blood positive, gross blood. On day 2 following model induction, the rats in the resina draconis and 5-aminosalicylic acid treatment groups were intragastrically treated with resina draconis [0.75 g(kg·d)] and 5-aminosalicylic acid [100 mg(kg·d)] respectively for 10 days. The rats in the blank control and TNBS model groups were administered a normal diet. 10 days later, the rats were intraperitoneally anesthetized with 2% sodium pentobarbital (3 mL/kg). An incision was made on the middle of sagittal line. The specimen of colon 8 cm above the anus was collected and the enteric cavity was incised along mesentery. Intestinal contents were washed with cold saline. Colon tissues were rapidly stored at -70 ℃ nitrogen canister for further use.
Evaluation of indexes in colitis rats following model establishment
In accordance with the method of Cooper et al [10], disease activity index was scored by recording rat weight and stool condition. Myeloperoxidase activity was measured within 1 week[11]. One unit of myeloperoxidase activity was defined as the quantity of enzyme that degraded 1 mol H2O2 at 37 ℃ per g wet tissue. During the assessment of disease activity index, macroscopic colonic damage score, histopathological score and myeloperoxidase activity, the data of dead rats were excluded.
Detection of dendritic cell phenotypes in the mesenteric lymph node
Isolation of dendritic cells with immunomagnetic beads: Mesenteric lymph node was sterilely obtained from the rats to prepare single cell suspension. In accordance with the method of Brenan et al [12], dendritic cells were isolated from the rat mesenteric lymph node using a MiniMACS immunomagnetic separation system (MihenyiBiotec, Germany). Totally seven sets of rats received immunomagnetic bead separation technique.
Dendritic cells surface markers major histocompatibility complex class II and CD86 expression: After isolation, cells were quantified. Cell suspension of each rat was divided into two tubes, which was respectively incubated in PE-0X62 (Serotec, Oxford, UK), FITC-major histocompatibility complex class II (eBioscience, San Diego, CA, USA) and PE-0X62, FITC-CD86 (eBioscience) at 4°C for 30 minutes. The specimens were fixed in 1% paraformaldehyde, and measured using flow cytometry within 24 hours[13].
Determination of nuclear factor-κB expression
In accordance with the method of Tanaka[13], streptavidin-peroxidase immunohistochemical staining was used to detect nuclear factor-κB expression[14]. Nuclear factor-κB p65 positive cells mainly contained mucosal cells, monocytes and macrophages. Brown particles were visible in the cytoplasm and nuclei. Image analysis technique was used to quantify the average number of positive cells within 1 mm × 1 mm so as to evaluate staining intensity.
Total RNA (Code: D9108; TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, Liaoning Province, China) was extracted from about 100 mg tissues by one-step method. Absorbance values were measured with an ultraviolet spectrophotometer at 260 nm/280 nm. RNA purity was calculated. Using a PrimeScriptTMRT reagent Kit (Code: DRR037A; TaKaRa), RNA was reverse transcribed into cDNA. All procedures were performed strictly according to the instructions. A total of 25 μL of products were stored at -20 ℃ for polymerase chain reaction (PCR).
Real-time fluorescent quantitative PCR was conducted[15] by using the high temperature method. Amplified specimens: glyceraldehyde phosphate dehydrogenase was considered as an internal reference (control)[16]. Measurement results: normal specimens served as controls. Primers for amplification were synthesized by Beijing SBS Genetech Co., Ltd., Beijing, China. Rat Toll-like receptor-4: annealing temperature: 55 ℃; upstream primer: 5’-GTG GGT CAA GGA CCA GAA AA-3’, downstream primer: 5’-GAA ACT GCC ATG TCT GAG CA-3’; product size: 526 bp. Rat glyceraldehyde phosphate dehydrogenase (GAPDH): annealing temperature: 52 ℃; upstream primer: 5’-CAC CAT GGA GAA GGC CGG GG-3’, downstream primer: 5’-GAC GGA CAC ATT GGG GGT AG-3’, product size: 408 bp.
Reaction system of PCR was as follows[17]: Taq DNA polymerase, dNTPs, dye, stabilizer, modifier, reaction buffer, reaction product, upstream and downstream primers. After shaking, the specimens were transiently centrifuged. A little mineral oil was added for amplification. Reaction conditions are as follows: 94 ℃ for 3 minutes, 30 cycles of 94 ℃ for 40 seconds, annealing temperature for 50 seconds and 72 ℃ for 90 seconds, followed by
72 ℃ for 10 minutes. A total of 6 μL of amplified products obtained from each specimen were electrophoresed in 1% agarose gel containing goldview nucleic acid stain[18]. DNA Marker (DL2000) served as standard molecular weight marker. The specimens were observed with an ultraviolet transilluminator, photographed with a digital camera. The target electrophoresis strip was analyzed with BIO-PROFIF gel image analytical system (VL Co., France), and corresponding internal reference electrophoresis strip served as a reference. Results were represented by the ratio of their integral absorbance (A260 nm).
Main outcome measures
Expression rates of major histocompatibility complex II and CD86, as well as expression levels of Toll-like receptor-4 and nuclear factor-κB, were observed.
Statistical analysis
Data were analyzed using SPSS 13.0 software, and measurement data were expressed as the mean±SD. Intergroup comparison was conducted using one-way analysis of variance. Intergroup comparison of numeration data was done using Chi-square test. A value of P < 0.05 was considered statistically significant.