Chemicals and drugs
Azoxymethane (AOM) and dextran sulfate sodium (DSS) salt (Mr ~40,000) were purchased from the Sigma-Aldrich (St. Louis, MO, USA). Limonin (95% purity) were obtained from Xi'an Xin Sheng Bio-chem Co.,Ltd. (Xi'an city,Shaanxi province, China).
Five weeks old male Balb/c mice (25-30 g) were purchased from the Animal Resource Unit, Faculty of Veterinary Medicine, Universiti Putra Malaysia (Serdang, Selangor, Malaysia), given commercial rat chow (Gold Coin, Malaysia) and tap water ad libitum, in plastic cages (3 mice/cage) with a 12-h light-dark cycle at room temperature. Male mice were used because males are more prone to CRC than females, and females may be influenced by fluctuating hormones or estrus cycle. The animal study protocol was approved by the Institutional Animal Care and Use Committee (IACUC), Universiti Putra Malaysia (UPM/IACUC/AUP-R069/2017).
Mice were divided into three groups (n=6) G1: healthy control, G2: non-treated CRC-induced (Azoxymethane/Dextran Sulfate Sodium AOM/DSS) control, G3: CRC-induced + 50 mg Limonin /kg body-weight. The dose for limonin was chosen based on previous studies (Shimizu et al., 2015).
(The CRC was induced by a single intraperitoneal injection of azoxymethane AOM (10 mg/kg body weight) to the mice in groups 2 and 3. After a week of AOM injection, mice received 2% dextran sulfate-sodium (DSS) in the drinking water for 7 days, followed by regular drinking water for recovery thereafter (Tanaka et al. 2000). Limonin were completely homogenized in distilled water (0.25 mg/ml) and given as drinking water to G3. The average daily Balb/c mice drinking water intake was 0.16 ml/g body weight or 5±1 ml/mouse/day. The similar (insignificantly different) progressive body weight gains indicated that the mice consumed about the same amount of food and water throughout the 20 weeks experimental duration after the CRC-induction (AOM/DSS). Oral gavage was not used to avoid any additional stress to the mice, which may increase their mortality. Limonin did not seem to affect the water or food intake of the mice in the treatment group.
Mice were weighed monthly and the disease activity index (DAI) was scored based on the calculated sum of the individual scores of stool consistency and blood in stool as follows: stool consistency score = 0: normal, 2: loose, 4: diarrhea; blood in stool score = 0: normal, 2: reddish, 4: bloody (Li, Shen and Luo, 2016). Following an intraperitoneal injection of ketamine: xylazine (100 mg/kg:10 mg/kg), the mice were sacrificed by exsanguination (intracardiac puncture) 17 weeks post-treatment. The intracardiac puncture allowed for sufficient blood to be collected for the subsequent biochemical analysis. The blood was allowed to clot for 30 min, centrifuged at 3000 g for 10 min at 4°C, and the serum stored at -20°C until analyses. Colon and spleen were collected and either stored at -80ºC or fixed in 10% formalin for further use.
Macroscopy and histopathology
The colon of each mouse was cut, cleaned, colon length measured and tumor incidence/numbers was counted. The colon length was measured using a ruler, measured and recorded to the nearest cm. The tumor incidence (%) was determined as the percentage of mice having at least one tumor when examined under the macroscope. Colon samples were fixed in 10% buffered formalin for 24 h and handled through automated programmed tissue processing machine. The tissue was embedded in paraffin and tissue blocks sectioned at thickness of 5 µm and stained with hematoxylin and eosin (H&E) for light microscopic examination. Due to the short CRC developmental period, only a few tumors were present in each AOM/DSS mouse. The inflammation score was graded as previously described (Saadatdoust et al. 2015). The inflammation thickness ranged from 0 to 3 (0 = no inflammation, 1 = mucosa, 2 = mucosa plus submucosa and 3 = transmural). The thickness of an inflammation is measured throughout the layers of the colon wall, such as the mucosa, sub-mucosa or transmural tissues. The inflammation score was calculated based on the average scores from 0 to 3 (0 - no inflammation; 1 – mild (infiltration of inflammatory cells into the mucosa); 2 – moderate (infiltration of inflammatory cells into the mucosa and submucosa); 3 – severe (infiltration of inflammatory cells into the transmural layer).
Enzyme-linked immunosorbent (ELISA) assay
The serum malondialdehyde (MDA), reduced glutathione (GSH) and prostaglandinE2 (PGE2) levels were determined using commercial ELISA kits (Elabscience Biotechnology, Wuhan, China) following the manufacturer’s protocol.
RNA extraction and RT-qPCR analysis
Total RNA from large intestine of the mice was extracted with RNeasy Mini Kit (Qiagen, Hilden, Germany). Briefly, ~20 mg of large intestine was disrupted in liquid nitrogen utilizing mortar and pestle and homogenized in 600 μL of Buffer RLT. The homogenate was transferred to a new tube and centrifuged for 3 min at full speed. The supernatant was transferred to a new tube, added 70% ethanol, mixed with pipetting, and passed through RNeasy mini column (Qiagen, Germany). The column was washed with wash buffer and contaminating DNA was digested on the column with DNase 1 (Qiagen, Hilden, Germany). RNA was eluted off the column using 30 μL of RNase free water. The RNA yield was determined by measuring absorbance at 260 nm and purity was assessed according to the ratio of absorbance readings at 260 nm to 280 nm using the Nano Drop TM ND-1000 (Thermo Fisher, USA).
Total RNA of each sample (772 ng) was reverse transcribed with RT2 First Strand kit (Qiagen, Hilden, Germany). Quantitative PCR for selected genes (Table S2) was performed according to Custom RT2 Profiler PCR array using RT2 SYBR Green qPCR Mastermix (Qiagen, Hilden, Germany). Thermal cycling and fluorescence detection were performed using a CFX96 Touch qPCR System (Bio-Rad, California, USA). The average relative mRNA expressed for each experimental group (n=3) was calculated using 2^ (-Avg. (Delta (Ct)) normalized to β-actin, since β-actin was the most stable gene compared to the other house-keeping genes analyzed in this study. Fold change (2^(-∆∆Ct)) were calculated by dividing the average normalized gene expression (2^(-∆Ct)) in the test group with the average normalized gene expression (2^(-∆Ct)) in the control healthy group. The target genes analyzed, and primer design are as in Supplementary Materials (Table S2).
Flow cytometry (immunophenotyping) analysis
The spleen was prepared by passage through a 70 μm cell strainer to obtain single-cell suspensions. The cell suspension was washed with 5 mL of phosphate buffered saline (PBS) and 5 mL of complete DMEM medium (10% FBS and 1% penicillin/streptomycin). The cell suspension was centrifuged at 500 g, 4ºC for 5 minutes. Then, the pellet was washed with 3 mL of PBS, followed by incubation with 3 mL of ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM EDTA) for 3 minutes and centrifuged at 500g for 5 minutes. The single-cell suspension was washed with BSA solution twice and centrifuged at 300 g, 4ºC for 5 minutes. The BSA (fetal bovine serum albumin) is a nutrient, stabilizer and surfactant in cell cultures, to protect enzymes/proteins and to prevent their adhesion to the surfaces of reaction vessel walls.
The cell pellets were stained with 5 μl of monoclonal antibodies specific for mouse (CD4+, CD8+ and CD19+) on ice for 20 minutes. The antibodies were from BD Pharmingen™ and they were (i) PerCP-Cy™5.5 Rat Anti-Mouse CD4, Cat No: 550954, Concentration: 0.2 mg/ml, Isotype: Rat DA, also known as DA/HA IgG2a, κ; (ii) APC Rat Anti-Mouse CD8a, Concentration: 0.2 mg/ml, Isotype: Rat LOU, also known as Louvain, LOU/C, LOU/M IgG2a, κ, Cat No: 553035, (iii) PerCP-Cy™5.5 Rat Anti-Mouse CD335 (NKp46), Concentration: 0.2 mg/ml, Isotype: Rat IgG2a, κ, Cat No: 560800; (iv) PE Rat Anti-Mouse CD19, Concentration: 0.2 mg/ml, Isotype: Rat LEW, also known as Lewis IgG2a, κ, Cat No 557399.
The spleen cells were washed with 1 mL of BSA solution and centrifuged at 300 g for 5 minutes. The cell pellets were suspended in BSA solution and analysed by BD LSRFortessaTM Cell Analyzer (BD Biosciences, San Jose, USA) and BD FACS DivaTM software (BD Biosciences, San Jose, USA).
All animals in all treatment groups were included in the data collection and analysis are presented as the mean ± S.D, unless otherwise stated (Please refer to the raw data provided as Supplementary materials). Comparisons between CRC and CRC + limonin were analyzed by t-test. Multiple group comparisons were performed by one-way ANOVA followed by Duncan post hoc test. Data was analyzed by SPSS 22.0 software and p<0.05 was considered significant. Correlations were evaluated using the Spearman test and using best curve fit online software at https://mycurvefit.com/.