Chemicals and reagents
TNBS (Cat #: P2297) and lipopolysaccharide (LPS) were supplied by Sigma-Aldrich, Germany (L6529-1MG). Mesalazine (89-57-6) was purchased from Ethypharm Pharmaceutical Co Ltd (Shanghai, China). Human recombinant S100A9 protein was purchased from Abcam (AB95909). Rat IL-1β, IL-6, and TNF-α ELISA kits were supplied by MultiSciences (Hangzhou, China). PCR primers for quantitative real-time PCR analysis were synthesized by GeneWiz (Suzhou, China). The primary antibodies for p38 (8960, 1:2000 for Western blotting), p-p38 (4511, 1:1000), JNK (9252, 1:1000), p-JNK (9255, 1:2000), ERK1/2 (4695, 1:2000), p-ERK1/2 (4370, 1:1000), NF-κB/p65 (8242, 1:2000), p-NF-κB/p65 (3033, 1:1000), Bcl-2 (3498, 1:1000), cleaved-caspase 3 (9664, 1:1000), and S100A9 (73425, 1:1000) were purchased from Cell Signaling Technology (Danvers, USA), and Bax (sc-20067, 1:2000), p53 (sc-126, 1:1000), IL-6 (sc-57315, 1:1000), TLR4 (sc-293072, 1:1000), NOD2 (sc-56168, 1:1000), and GAPDH (sc-365062, 1:2000) were purchased from Santa Cruz Biotechnology Co. Ltd (Santa Cruz, USA). Anemoside B4 (20 g, purity more than 99 %) was isolated from the roots of P. chinensis and its structure was determined as previous described .
Sprague-Dawley (SD) rats (male, 220-250 g) were obtained from the Experimental Animal Center of Soochow University. All animals were given free access to water and food with air filtration (22 ± 2 °C, 12-h light/12-h dark). All animal experiments were conducted in accordance with the procedure approved by the Ethical Review Committee for Laboratory Animal Welfare of the Soochow University.
Induction of colitis and experimental design
All animals were divided into five groups (n = 7 per group): vehicle group, TNBS + saline group, TNBS + anemoside B4 group (5 mg/kg), TNBS + anemoside B4 group (10 mg/kg), and TNBS + mesalazine group (200 mg/kg). All rats were fasted for 24 h and weighed before injection, and then were anesthetized by chloral hydrate intraperitoneally (300 mg/kg, i.p.). In the vehicle group, 40 % ethanol was slowly instilled into the colon of anesthetized rats for 1 min, while for the other four groups, TNBS solution (80 mg/kg in 40 % ethanol v/v) was instilled for 1 min. Rats in the vehicle group and TNBS + saline group were intraperitoneally injected with saline, and those in the TNBS + anemoside B4 groups were intraperitoneally injected with anemoside B4, while those in the TNBS + mesalazine group were treated with mesalazine by gavage once per day for 7 days. Anemoside B4 (5 mg/kg, 10 mg/kg) or saline was injected twice per day for 7 days after treatment with TNBS for 24 h.
Assessment of colonic damage
During the experiment, rats were weighed every day and disease activity index (DAI) was recorded for seven days after the induction of colitis according to previous reference . The DAI is the combined score of weight loss compared to initial weight, stool consistency, and bleeding. In brief, no weight loss was registered as 0, weight loss of 1 % to 5 % from baseline was assigned 1 point, weight loss of 6 % to 10 % from baseline was assigned 2 points, weight loss of 11 % to 20 % from baseline was assigned 3 points and weight loss of more than 20 % from baseline was assigned 4 points. Stool consistency scores were 0 (normal, well-formed pellets), 2 (loose stool, pasty and semi-formed stools that did not adhere to the anus), and 4 (diarrhea, liquid stools that adhered to the anus). Bleeding scores were 0 (no blood), 2 (Hemoccult positive and visual pellet bleeding), and 4 (gross bleeding, blood around anus).On the 8th day, rats were sacrificed by anesthesia. The colons were harvested and rinsed with saline. Colon length was recorded as an indirect marker of inflammation. Myeloperoxidase (MPO) activity was measured to assess inflammation. Colon samples were fixed in 4 % paraformaldehyde for histological examination and immunofluorescence staining. The remaining colon samples were kept at -80°C for further assessment by quantitative proteomics, enzyme-linked immunosorbent assay (ELISA), qPCR, and Western blotting.
Histopathological analysis of rectums
For histological assessments, sections of rectums with a thickness of 5 μm were stained with hematoxylin and eosin (H&E) to visualize cell morphology using an optical microscope. Light microscopy (CX31 research microscope, Olympus Optical Co. Ltd., Tokyo, Japan) and panoramic viewer camera system were used to examine, scan, and analyze the sections. Histological score was measured by inflammation severity: 0 (normal colonic mucosal), 1 (crypt damage less than 1/3), 2 (crypt damage less than 1/3-2/3), 3 (mucosal erosion), 4 (muscosal erosion or ulcer with significant infiltration of inflammatory cells) according to a previous reference .
Label-free quantitative proteomics
On the 8th day after rats were treatment with TNBS (80 mg/kg) and/or anemoside B4, colon tissues from the rats in TNBS + saline group (n = 3) and TNBS + anemoside B4 group (10 mg/kg) (n = 3) were extracted, lysed in a HEPES buffer (pH 8.0) containing 8 M urea to obtain total proteins. Disulfide bonds in these proteins were reduced with 2 mM dithiothreitol at 37 °C for 45 min. The free thiols were further alkylated with 8 mM chloroacetamide for 1 h at room temperature. The excess chloroacetamide was then quenched by adding 2 mM dithiothreitol for 45 min. After acetone precipitation, proteins were resuspended in a HEPES buffer containing 8 M urea and incubated with the MS sequencing grade Lys-C (TaKaRa Bio, Japan) for 4 h at 37 °C. Then the samples were diluted with 10 mM HEPES (pH 8.0) four times and continued for digestion with MS sequencing grade trypsin (TaKaRa Bio, Japan) at 37 °C for 20 h. Next, the peptides were desalted with C18 Zip-tips (Merck Millipore, MA, USA), concentrated, separated by capillary high performance liquid chromatography and analyzed in an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA). Data were analyzed using MaxQuant according to a previous procedure . Proteins with FDR £ 0.01 were considered as positive identification. The independent sample t-test in IBM SPSS software (Ver 19) was used to calculate the P-values for the identified proteins. The proteins with fold change > 1.50 or < 0.67 and P < 0.05 were considered as significantly differentially expressed proteins.
Western blotting analysis
Western blotting analysis was performed as previously described . In brief, proteins from tissues or cells were extracted in ice-cold RIPA buffer supplemented with protease inhibitor mixture (Thermo Fisher Scientific, Germany), separated by SDS-PAGE, and transferred into PVDF membranes. After blocking with 5% bovine serum albumin for 1 h at room temperature, membranes were incubated overnight at 4°C with the following primary antibodies: p38, p-p38, JNK, p-JNK, ERK1/2, p-ERK1/2, p65, p-p65, Bcl-2, cleaved-caspase 3, S100A9, Bax, p53, IL-6, TLR4, NOD2, and GAPDH. The membranes were washed three times and incubated again with the horseradish peroxidase (HRP) conjugated goat secondary antibodies (Beyotime, Haimen, Jiangsu, China). Then, the membranes were immersed with HRP substrate (Millipore Corporation, Billerica, USA) and signals were detected with the ChemiDoc XRS Imager (Bio-Rad, USA).
Quantitative real-time RT-PCR
According to the manufacturer’s instructions, total RNA was extracted with TRIzol reagent (Ambion, USA) and quantified by a NanoDrop ND-2000 spectrophotometer (Thermo Fisher). After quality check, mRNA was reverse-transcribed to cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). SsoAdvanced™ Universal SYBR® Green (Bio-Rad) was used for qPCR measurement. Standard curves were constructed with the CFX96™ real-time PCR detection system (Bio-Rad). The cycling conditions for the qPCR were as follows: 95 °C for 3 min, followed by 37 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The relative expression levels of genes were calculated using the 2−ΔΔCt method based on the threshold cycle (Ct) value.
Colon tissue was homogenized in tissue protein extraction reagent. The homogenized tissue was centrifuged at 12,000 g for 10 min. The supernatant was collected and stored at -80°C until further analysis. TNF-α, interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the supernatant were analyzed using ELISA kits according to the manufacturer’s instructions.
Ethynyl-2′-Deoxyuridine (EdU) cell proliferation assay
The proliferation of colon cells was detected by the BeyoClick™ EdU-488 Kit (Beyotime, Haimen, Jiangsu, China). Briefly, 4 h before rats were sacrificed, they were injected intraperitoneally with 5 mg/kg EdU solution and then the colon was collected and rinsed with saline. Colon samples were then processed using the BeyoClick™ EdU-488 kit according to the manufacturer’s instructions. The EdU positive cells were detected with a confocal laser scanning microscope (LSM 710, ZEISS, Germany).
Apoptosis detection assay
To further define the apoptotic cells in rat’s colon, the In Situ TUNEL Apoptosis Detection Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to detect the apoptotic cells according to the manufacturer’s instructions. Briefly, rats were sacrificed after the colitis experiment. Then the colon samples were collected and stained with the detection kit. Terminal deoxynucleotidyl transferase (TdT) was used to label DNA strand breaks. Fluorescein labels incorporated in nucleotide polymers were detected by fluorescence microscopy and the percentage of TUNEL-positive cells was counted.
The protein levels of CD11b and S100A9 in colon tissues were detected by immunofluorescence staining. The colon sections were infiltrated with 0.5 % Triton X-100 in PBS, blocked with 3 % BSA, incubated overnight at 4 °C with FITC-anti-CD11b (1:100, Thermo Fisher Scientific, USA) or FITC-anti-S100A9 (1:100, Cell Signaling Technology) antibodies. The sections were counterstained with a secondary antibody for 1 h. The immunofluorescent cells were visualized and digital images were captured using a confocal laser scanning microscope (LSM 710, ZEISS, Germany).
For measurements of MPO activity, colon samples were homogenized in 1:10 (w/v) of 50 mM PBS (pH 6.0) using a Polytron homogenizer. The homogenate was centrifuged at 12,000 rpm for 10 min. Then, MPO activity of homogenate was detected using a commercially available ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. MPO activity was normalized by the weight of total sample and expressed as U/g tissue.
Cell culture and treatment
Caco-2 cell line was purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China) and routinely maintained in DMEM (Gibco, USA) supplemented with 10 % fetal bovine serum (Gibco, USA), 100 µg/ml streptomycin and 100 U/ml penicillin (Gibco, USA).
Cells were cultured at 37 °C with 5 % CO2. For all experiments, Caco-2 cells were treated with vehicle or 1 μg/ml LPS or recombinant human S100A9 protein for 1 h, and then treated with different concentration of anemoside B4. At 6 h or 24 h after treatment of anemoside B4, total protein samples were extracted from cells and prepared for qPCR or Western blotting experiments.
Data and statistical analysis
Statistical analysis was performed using SPSS 16.0 statistical analysis software. Data were expressed as the mean ± SD. All statistical analyses were performed with a threshold for significance (alpha) set at 0.05. P < 0.05 was considered as statistically significant except the quantitative proteomics analysis. Two-way ANOVA followed by Dunnett’s multiple comparisons test was used to analyze the body weight change and DAI scores in the animal experiments. One-way ANOVA followed by Dunnett’s multiple comparison test was used for other data except the body weight change and DAI scores.