Drugs and regents
Scutellarin was obtain from Must Biotechnology Corporation (Chengdu,China) and was dissolved in dimethyl sulfoxide (DMSO). AOM and DSS were purchased from Sigma-Aldrich (American); fetal bovine serum(FBS)was obtain from QuaCell Biotechnology Corporation (Guangdong, China). MTT kit, BeyoFast TM SYBR Green qRT-PCR Mix༈2×༉and Apoptosis and Necrosis Assay Kit were acquired from Beyotime Biotechnology Corporation (Shanhai, China). And PrimeScript TMRT Reagent Kit was purchased from TaKaRa Biotechnology Corporation (Beijing, China), while BCA Protein Assay Kit from Multi Science Biotechnology Corporation (Shanghai, China).
Cell culture and passage
The SW480 cells were purchased from Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). When they were cultured to about 90%, trypsinized and centrifuged with moderate culture medium (Sigma-Aldrich, American) which contained 10%FBS and 1% penicillin-streptomycin, as well as discarded the supernatant. Next, the cells were resuspended in culture medium and added to three new culture bottles with new culture medium, and the morphology of the cells was observed under a microscope (leica, Germany). Finally, the cells were cultured in an incubator (Thermo, American).
Cell viability assay
SW480 cells in logarithmic growth stage were spread on 96-well plates with a density of 5 × 103 cells in each well, 6 multiple wells in each group. Four groups were designed: 160 ug/ml, 80 ug/ml, 40 ug/ml and normal control group (0.2%DMSO) were established according to the preliminary experimental results. After the cells were attached to the well, the drugs were administered for 24 h and 48 h respectively. And the culture medium containing MTT was added to measure the absorbance value of each well at 570 nm on a preheated microplate reader (Thermo Scientific Varioskan Flash, Type 3001). Calculating the inhibition rate of cells in each well based on the measured absorbance data, cell inhibition rate (%) = (control group OD value-drug group OD value) / control group OD value × 100%.
Effect of Scutellarin on the morphology of SW480 cells
SW480 cells at logarithmic growth stage were collected, digested and inoculated into a 96-well plate with 1 × 104 cells in each well. The cells were treated with Scutellarin 160 ug/ mL, 80 ug/ml, 40 ug/ml and 0.2%DMSO for 48 h, and the cell morphology was observed under an inverted microscope.
Cell migration experiment
SW480 cells were seeded in a 24-well plate, each well was added 500 ul cells with a density of 1 × 106 cells/ml, and each group had 3 duplicates. Then observing cells until they adhered to the well and grew. When the cells grew to about 90%, using a 10 ul pipette tip to make a straight scratch, then washing the dead cells in the 24-well plate with PBS, finally giving 500 ul different concentrations of Scutellarin. After 0 h, 12 h and 24 h, observing the scratch migration of each group.
Clone formation assay
SW480 cells were seeded in a 6-well plate, after the cells were stick to the well, culture medium of Scutellarin of different concentrations and the normal control group with 0.2%DMSO were added. Next, cells were cultured in the incubator for 7 days. Taking out the 6-well plates from the incubator, then washing cells twice with PBS, staining with crystal violet for 10 min, and the experimental results were photographed and recorded after washing with PBS again.
Hoechst 33342 dye staining
1 × 104 cells were inoculated into each well of the 96-well plate, cultured with Scutellarin containing medium for 24 h, next fixed with fixative solution for 10 min, and then added with Hoechst33342 working solution for staining at 37℃ incubators for 5 min. Fluorescence microscope was used to detect and record the cells.
Apoptosis and necrosis assay
SW480 cells with a density of 1 × 106 were seeded in a 6-well plate and cultured for 24 h with different concentrations of Scutellarin. Adding 5 ul Hoechst and PI dye into the 6-well plate and mixing immediately. The samples were incubated at 4℃ for 30 min, washed with PBS and photographed under a fluorescence microscope.
Activity detection of Caspase-3 and Caspase-9
SW480 cells were collected after 24 h of different concentrations of Scutellarin treatment, then added lysis buffer, lysed in ice bath for 15 min. After centrifugation at 4℃ for 15 min, protein concentration was determined by Bradford method to achieve 1–3 mg/ml protein concentration. 40 ul test buffer was added into the reaction system, 50 ul test sample and 10 ul AC-devd-PNA (2 mM) was mixed and incubated at 37℃ for 2 h, finally, detected on a microplate reader.
C57BL/6 male mice (n = 50) were purchased from Sichuan Provincial People's Hospital (Chengdu, China), and randomly divided into 5 groups according to body weight, 10 mice in each group. 5 groups were AOM + DSS model control group, 100 mg/kg Scutellarin group, 50 mg/kg Scutellarin group, 25 mg/kg Scutellarin group and blank control group, respectively. The first four groups were intraperitoneally injected with AOM 12.5 mg/kg, while the blank control group was intraperitoneally injected with 0.9% NaCl solution, with the dose volume of 0.01 ml/kg. 1 week later, the first 4 groups were given 2.5% DSS solution free drinking for 1 week, followed by 2 weeks of pure water which repeated for 3 cycles. Starting from week 2, the drug groups were intraperitoneally injected with different concentrations of Scutellarin every day until the end of three cycles. Weight, fecal status, and blood in the stool were recorded weekly.
The colorectal tissues of the mice in each group were paraffin-embedded and cut into 5 um thin slices. Immunohistochemical staining: Bax, Bcl-2 monoclonal antibody (1:100, Proteintech Group, Inc.), incubated at 4℃ overnight, then incubated with the secondary antibody at room temperature for 50 min. After the tissue slices were colored with DAB, they were re-stained with cymbalin, sealed with neutral gum, and finally observed and photographed under a microscope.
Western blot analysis
SW480 cells and colon tissues treated with Scutellarin of different concentrations were collected, centrifuged at 4℃ for 10 min, then added with pyrolysis liquid. Finally, the total protein content of samples was determined by BCA method. Equal amount of protein was taken from each sample, and the same volume of sample loading buffer was added. After boiling for 5 min, each group of protein was separated by using 8% SDS-PAGE, subsequently transferred to a PVDF membrane. The PVDF membranes were blocked with 3% BSA for 1.5 h and probed with primary antibodies against GAPDH (1:5000, in 1% BSA, Multi Science Biotechnology Corporation), Bax and Bcl-2 (1:1000, in 1% BSA). After placing the membranes in a refrigerator overnight at 4℃, adding the secondary antibody (1:5000, in antibody diluent). Place them on a shaker at room temperature for 90 min, and proceeding with the enhanced chemiluminescence reagent (ECL) luminescence kit. Quantity one software was used to analyze, and the ratio of the optical density value of the target protein to the internal reference protein (GAPDH) was regarded as the experimental result for statistics.
Quantitative RT-PCR analysis
Total RNA was extracted from different groups of cells and tissues, then using NanoDrop 2000 micronucleic acid analyzer (Thermo, American) to detect the purity and concentration of total RNA. The PrimeScript RT Reagent Kit was used for reverse transcription of cDNA, next detecting its purity and concentration with NanoDrop 2000 micronucleic acid analyzer again. At last, BeyFastTM SYBR Green qRT-PCR Mix was used for quantitative fluorescence PCR reaction. qRT-PCR reaction condition is: predenaturation at 95℃ for 2 min; 95℃ 15 sec, 60℃ 30 sec, 72 ℃ 30 sec, × 40 cycles. PCR primers were designed based on nucleic acid sequences provided by GenBank database (Table 1).
The data was analyzed by one-way analysis of variance (ANOVA) with SPSS19.0 software, expressed as mean ± standard deviation .