ShaoYao decoction ameliorates colitis-associated colorectal cancer by downregulating proinflammatory cytokines and promoting epithelial-mesenchymal transition

SYD was composed of nine commonly used herbs: Radix Paeoniae Alba, Radix Angelicae Sinensis, Rhizoma Coptidis, Semen Arecae, Radix Aucklandiae, Radix Et Rhizoma Glycyrrhizae, Radix Et Rhizoma Rhei, Radix Scutellariae, Cortex Cinnamomi. The raw herbs for SYD were purchased from the affiliated Nan Fang Hospital of Southern Medical University. These were mixed in the ratio of 30:15:15:6:6:6:9:15:7.5 (dry weight). Aqueous extracts of SYD were extracted at 80°C by stirring for 1 h using 10 volumes of distilled water (v/m). The extracts were centrifuged at 1,500 g at room temperature. To obtain the semisolid SYD solution, the supernatant was collected and subjected to condensation under reduced pressure at 70°C [17]. The quality of SYD was confirmed by HPLC analysis (Additional file 1: Figures S1 and S2, Table S1). SYD was suspended again in 0.9% saline at a final concentration of 2 g/mL. The solution was stored in aliquots at -20°C.

All procedures involving laboratory animal use were in accordance with the guidelines of the Instituted Animal Care and Use Committee of Southern Medical University. All protocols were submitted and validated by Animal Care Ethics Committee of Southern Medical University (No. 2012–055). 6 and 8 weeks male C57BL/6 J mice weighting 15–20 g (specific pathogen-free) were obtained from the Laboratory Animal Center of Southern Medical University. The animals were maintained under controlled conditions (22°C, 12-h/12-h dark/light cycle) in a conventional animal colony. There were 65 mice used in the study, 15 mice in control group, 30 in model group and 20 in SYD group. At the end of the study, there were 15 surviving mice in control group, 7 in model group and 12 in SYD group, respectively.

Procedures of induction of caCRC model by AOM and DSS was shown in Figure 1A [16]. At week 2, mice were injected with AOM (10 mg/kg, i.p.). After 1 week, 3% DSS (International Lab, Chicago, IL, USA) was added to the drinking water for 7 days followed by 14 days of tap water for recovery. This cycle was repeated twice.

SYD (7.12 g/kg) or equivalent normal saline was administered by gavage using a tube twice a day. The mice were killed by cervical dislocation at week 16 and the colons (from the ileocecal junction to the anal verge) were removed. After measuring the length and weight, the colons were cut longitudinally along the main axis, washed with phosphate-buffered saline (p H 7.4, 4°C) and macroscopically inspected. The number, size and location of pre-neoplastic and neoplastic lesions (dysplasia and carcinoma) in the colons were recorded based on gross examination. The size of dysplasia was measured by an ocular micrometer. After gross examination, the colons were cut into pieces at about 1 cm intervals. Samples were then fixed in 10% buffered formalin (p H 7.4) or kept in 10 volumes of RNAlater Solution (Ambion, Life technologies, Carlsbad, CA, USA). Remaining samples were flash-frozen in liquid nitrogen and stored at -80°C for further immunohistochemistry, PCR and protein analysis.

Paeonolsilatie sodium was purchased from Jinling Pharmaceutical Co., Ltd. Dimethyl sulfoxide (DMSO) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640, FBS and antibiotics were purchased from Invitrogen (Gibco,Grand Island,NY, USA). Snail homolog 1 (Drosophila) was purchased from FulenGen (Genecopoeia,USA).

MTT assay was used to analyze the effects of paeonol and SYD on cell viability in two kinds of colorectal adenocarcinoma cell lines SW480,HCT116. Cells were routinely maintained in RPMI 1640, supplemented with 10% FBS and antibiotics (50 U/ml of penicillin and 50 μg/ml streptomycin) at 37°C in a humidified atmosphere containing 5% CO2. Cell number was measured on a standard microscope by manual counting of cells using a grid with an area of 0.25 mm². 1 × 10⁴ Cells were seeded on 96-well plates in a 100 μl volume. The MTT assay was performed by addition of MTT to a final concentration of 0.5 mg/ml of medium. After 4 h, the medium was removed, and 150 μl of DMSO was added to each well. After 10 min of shaking at room temperature, 150 μl of soluble material was placed into a new 96-well plate, and absorbance at 562 nm was read with a background subtraction at 660 nm.

HCT116 cell line was cultured in RPMI 1640, supplemented with 10% FBS and antibiotics (50 U/ml of penicillin and 50 μg/ml streptomycin) at 37°C in a humidified atmosphere containing 5% CO2. For transfection,cells were seeded in a 6-well plate 24 h before the experiment. The cells were transfected with Snail 1 plasmid using the Attractene reagent (Qiagen, Germany) according to the manufacture’s protocol.

For histopathological examination, formalin fixed, paraffin-embedded colon tissues were cut into serial sections (5 μm) and stained with haematoxylin and eosin (H&E). Histological alternations such as dysplasia and carcinoma were verified. Assessment of dysplasia and cancer was based on the criteria set forth in the Mouse Models of Intestinal Cancers consensus report: location, presence/absence of prolapse/herniation, size, dysplasia grade, invasion level, presence of desmoplasia and presence of irregular architecture [18]. Carcinoma was defined as high-grade dysplasia of colonic mucosa which invaded beyond the muscularis mucosa and into the submucosa [16].

Paraffin-embedded colon sections were deparaffinized, rehydrated, and pre-treated with hydrogen peroxidase in PBS buffer. Heat-induced antigen retrieval was performed. After blocking with the appropriate antisera sections were incubated with anti-Proliferating cell nuclear antigen (PCNA) (Thermo scientific, clone PC10, 1:300), anti-β-catenin (Cell Signaling Technology, CST, clone 6B3; 1:100 dilution), anti-p53 (Leica-microsystems, clone CM5, 1:100 dilution), anti-COX-2 (Thermo scientific, 1:100 dilution), anti-E-cadherin ( CST, clone24E10, 1:200 dilution), anti-N-cadherin (Millipore, clone EPR1792Y, 1:50 dilution), anti-fibronectin (Epitomics, clone F14, 1:200 dilution), anti-vimentin (CST, clone D21H3, 1:50 dilution), NF-κB (Abcom, clone E379, 1:100 dilution), F4/80 (Santa Cruz Biotechnology, clone BM8, 1:50 dilution). After incubation with HRP-conjugated secondary antibody and tyramide amplification followed by streptavidin-HRP, positive signals were visualized by DAB kit and counter-stained with hematoxylin. Five randomly selected fields from each section were examined at a magnification of 400× and analyzed using NIS-Elements. The positive content was calculated using the following formula: positive content (PC) = mean optical density × positive area.

Colonic tissues were homogenized in liquid nitrogen, dissolved in lysis buffer [7 mol/L urea, 2 mol/L thiourea, 4% (w/v) CHAPS, 20 mmol/L Tris, 65 mmol/L DTT, 0.2% pharmalyte 3/10 ampholyte] supplemented with 8 μl of protease inhibitor cocktail (The protease inhibitor cocktail include AEBSF hydrochloride 500 μM, Aprotinin 150 nM, E-64 protease inhibitor 1 μM, EDTA disodium 0.5 mM and Leupetin hemisulfate 1 μM. Calbiochem, San Diego, CA, USA). Samples were then centrifuged at 15,000 g for 30 min at 4°C. Protein concentrations were determined by modified Bradford assay and resolved by SDS-PAGE, transferred to PVDF membranes and blocked in 5% non-fat milk in Tris-buffered saline (TBST, 100 mM NaCl, 50 mM Tris, 0.1% Tween-20, pH 7.5). Membranes were incubated overnight with primary antibodies [anti-PCNA (CST, clone PC10, 1:1000 dilution), anti-β-catenin (CST, clone 6B3, 1:1000 dilution), anti-p53 (CST, clone 1C12, 1:1000 dilution), or anti-COX-2 (CST, clone D5H5, 1:1000 dilution), anti-E-cadherin (CST, clone 24E10, 1:1000 dilution), anti-N-cadherin (Millipore, clone EPR1792Y, 1:50000 dilution), anti-Vimentin (CST, clone D21H3, 1:1000 dilution), anti-Snail (CST, clone C15D3, 1:1000 dilution)] at 4°C. This was followed by incubation with horseradish peroxidase (HRP) conjugated secondary antibodies. Protein bandings were visualized with enhanced chemiluminescence reagents (ECL, GE Healthcare Bio-science, Uppsata, Sweden). The images were captured with a CCD system (imagestation 2000 MM, Kodak, Rochester, NY, USA). Quantitative analysis of signals was performed using Molecular Imaging Software Version 4.0, provided by Kodak 2000 MM System. The optical density was normalized by β-actin.

The levels of interleukin-1β (IL-1β), IL-6 and TNF-α in the serum were measured using mice cytokine multiplex kit (Millipore, Billerica, MA, USA). Quantification of cytokines was performed using the Luminex system (Austin, TX, USA) [19].

Body weight loss and bloody stools were observed in mice acutely exposed to DSS. These symptoms were relieved during the recovery period (Figure 1B). The colon weight was increased and the length was shortened significantly in the mice that received both AOM and DSS compared with the control mice (Table 1). The significant increases in colon weight and colon length ratio were caused by apparent mucosal thickening. SYD substantially ameliorated the body weight fluctuation and mucosal thickening. The Kaplan–Meier survival curves showed that SYD significantly increased the survival rate of the mice (Figure 1C).

The colonic neoplasm formation rate was 100% in the mice treated with AOM/DSS. Neoplasms were principally distributed in the middle and distal colon (Figure 2A). The total multiplicity of colorectal neoplasms decreased by 42.8% after SYD treatment (P < 0.01, Table 2). Moreover, SYD significantly retarded the development of large neoplasms (diameter > 3 mm) by 59.2% (Figure 2B, Table 2). The tumour weight and tumour volume were decreased by SYD (Figure 2C). The neoplasms in the colon were mostly tubular adenoma or adenocarcinoma according to the criteria of Mouse Models of Intestinal Cancers (Figure 2D). The neoplasms was mostly high-differentiation with invasion in model group.SYD treatment could ameliorate level of differentiation and invasion of neoplasms (P = 0.041, Table 3).

The expression levels of proliferating cell nuclear antigen (PCNA) and β-catenin increased in dysplastic and neoplastic lesions (Figure 3A and Figure 3B). Intense nuclear staining for p53 (CM5 clone: detects both mutant and wild-type forms) was detected in the neoplastic epithelium and in nondysplastic crypts, implicating the involvement of p53 mutation in our AOM/DSS-induced caCRC model [18]. The expression levels of these neoplastic markers suggested that AOM/DSS-induced caCRC phenotypically resembled human caCRC even though the expression of COX-2 was not high in adenocarcinoma (Figures 3A and 3B, Table 4).

SYD significantly decreased cell viability in a time- and dose-dependent manner; the dose ranged from 0 mg/mL to 8 mg/mL. The viabilities of SW480 and HCT116 cells decreased to 50.2266% and 53.6704% after 24 h of treatment with 2 mg/mL SYD (Figure 4A). Paeonol, an ingredient of Radix Paeoniae Alba, significantly decreased cell viability in a time-and dose-dependent manner; the dose ranged from 0 mM/L to 0.8 mM/L. The viabilities of SW480 and HCT116 cells decreased to 48.0659% and 51.3992% after 48 h of treatment with 0.4 mM /L Paeonol (Figure 4B).

SYD and Paeonol decreased the expression levels of PCNA, β-catenin, p53, and COX-2 in CRC cells in a dose-dependent manner. (Figure 5A and Figure 5B, Table 5 and Table 6).

E-cad was moderately expressed in the epithelial cells of the control group but was downregulated in the epithelial cells of the model group. N-cadherin (N-cad) and fibronectin were strongly expressed and vimentin was moderately expressed in the model group, suggesting the occurrence of EMT (Figures 6A). SYD significantly increased the expression of E-cad and decreased the expression of N-cad, fibronectin, and vimentin (Figures 6A, Table 4). Western blot analysis further confirmed the effects of SYD on the expression of E-cad, N-cad, and vimentin. Snail was upregulated in the model group, and it was downregulated by SYD (Figure 6B, Table 4).

Overexpression of Snail, a transcription factor crucial in EMT, induced the upregulation of N-cad and vimentin. SYD and Paeonol decreased the expression of N-cad induced by Snail overexpression. (Figure 5C, Table 5 and Table 6)

The number of macrophages and the expression of NF-κB were increased significantly in the model group compared with those in the control group; SYD counteracted these effects (Figure 7A). Serum IL-1β, IL-6, and TNF-α were upregulated in the model group and were significantly inhibited by SYD (Figure 7B).