Aqueous extract preparation and high performance liquid chromatography (HPLC) analysis
Ruyong formula (RYF) is composed of Radix Codonopsis (15g), Astragalus membranaceus (20g), Angelica Sinensis (12g), Radix Bupleuri (12g), Rhizoma Chuanxiong (6g), raw Radix Paeoniae Alba (9g), Fructus Forsythiae (12g), Radix Glycyrrhizae (6g), Trichosanthes kirilowii Maxim (15g) and Pericarpium Citri Reticulatae Viride (6g). All the dried herbs were purchased from Binjiang outpatient department of Zhejiang Chinese Medical University. Prescription dosage of RYF was milled into powder and soaked in 8 times prescription dosage distilled water, and then extracted with boiling water under reflux for two hours. The extract was filtered, and the extraction was repeated once. Subsequently, the filtrates were combined and evaporated under vacuum (RV8V, IKA, Werke GmbH & CO. KG, Staufen, Germany) and then lyophilized with a freeze dryer (VirTis, AD 2.0 EL, SP Scientific, USA) into powder. The percentage yield of RYF aqueous extract was 9.61% (w/w).
Chromatographic analysis was performed using C18 column (250mm × 4.6μm, 5μm; Waters) maintained 25℃. A binary mobile phase consisting of acetonitrile (A) and water-acetic acid (B; 100:0.2, v/v) was used at a flow rate of 1.0 ml/min. Gradient elution was performed according to the following elution program: 0-6 min, 10-20% A; 6-8 min, 20% A; 8-15 min, 20-23% A; 15-17 min, 23-25% A; 17-27 min, 25-50% A; 27-37 min, 50-80% A; 37-45 min, 85-10% A. The injection volumn was 10 μl and the detection wavelength was set at 284 nm. Each of the reference standards (Calycosin-7-glucoside, ferulic acid, lobetyolin, ligustilide) was dissolved in methanol to prepare the stock solution. The lyophilized powder was accurately weighted (0.109 g), then dissolved in water (1.13 g dried herbs/ml) and the solution was centrifuged at 12,000 rpm for 10 min, and the 10 μl supernatant was injected into the HPLC system for analysis.
Animals and cells
BALB/c mice (4-6 w, Female) were purchased from BK Corporation (China). All animal experiment was performed under semi-sterile conditions. The animals were stored in special-pathogen-free (SPF) environment during entire experiment. Water and food were provided normally. Animals were maintained under the standard 12 light/dark cycle at 22-23℃ with relative humidity of 50-60%. All animals’ protocols were approved by the Institutional Animal Care and Use Committee at Zhejiang Chinese Medical University Laboratory Animal Research Center.
The mice breast cancer cell line (4T1), Plasmid SSR#69 (designed by Professor Westerman) and PAMPHO plasmid, were favored from Professor Tong-chuan He from the University of Chicago. LipofectAMINE 2000 was purchased from Invitrogen (California, USA). The human embryonic kidney cell 293 (HEK-293) was purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Thymic epithelial cells (TECs) was obtained from BALB/c mice . All of the cell lines were cultured in DMEM (Gibco, USA) containing 10 % fetal bovine serum (Gibco, USA) at 37℃ with 5% CO2.
Mice mammary tumor model establishment
After two days of acclimatization, mice were with 100 μl of 4T1 cell suspension subcutaneously into the 2rd pair mammary fat pad on the right at a cell density of 1.0×107 cells/ml (the normal group were injected with a vehicle of PBS). Two days after tumor inoculation, animals (n=4) were randomly divided into five groups (four mice per group): (i) cyclophosphamide (CTX, Baxter, Shanghai, China) group (30 mg/kg, i.p. once every two days); (ii) model group (0.2 ml, intragastrically, normal saline, once a day); (iii) RYF-L (low concentration) group (0.145g crude herbs/kg, intragastrically, once a day); (iv) RYF-M (medium concentration) group (0.29g crude herbs/kg, intragastrically, once a day); (v) RYF-H (high concentration) group (0.58g crude herbs/kg, intragastrically, once a day). Four health mice were prepared in normal group (0.2 ml, intragastrically, normal saline, once a day). All treatments lasted for 20 days. Throughout the study, body weights were monitored and the longest (L, mm) and shortest (W, mm) diameter of tumor were measured with a vernier caliper weekly. Tumor volumes (V, mm3) were calculated with Eq. 1. Mice were sacrificed 2 hours after the last administration. The visceral weight and index of thymus, lung, spleen and lymph node were measured with Eq. 2. The anti-tumor rate were calculated with Eq. 3 after collected and weighed the tumor. Calculated the pulmonary metastasis rate according to the number and volume of nodes on the lung.
Thymus tissue sections were inflated with 4% paraformaldehyde, embedded in paraffin and cut at a thickness of 5μm. Tissue sections of each specimen were stained using hematoxylin and eosin (H&E, Sangon Biotech, Shanghai) according to a standard protocol for histopathological examination using fluorescence microscope (Axio Scope.A1, Zeiss, Germany). The pathological changes in the structure of the tissue sections in each group were evaluated. The numbers of cells in thymus cortex and medulla regions were measured using Image J. 3% hydrogen peroxide was used to block endogenous peroxidase activity at room temperature for 15 min. Subsequently, the unspecific binding sites were blocked with 5% BSA/PBA for 40 min. The sections were then incubated for overnight with primary antibody at 4℃ in a humid chamber for E-cadherin (Cell Signaling Technology) and Vimentin (Cell Signaling Technology). PBS was used as negative control. The dilutions used for the primary antibodies were 1:200. The sections were subsequently incubated with rabbit anti-mouse enhanced polymer antibodies (Zsbio Co., Ltd.) at 37℃ for 2 h. Then the reaction products were developed with 3, 30-diaminobenzidine (DAB), counterstained with haematoxylin, differentiated with 1% hydrochloric acid ethanol, washed with distilled water, dehydrated with graded ethanol, vitrified by xylene and sealed with neutral gum. The stained sections were observed with fluorescence microscope after the sections dried. Immunoreactivity was analyzed with Image J to evaluate the expression of proteins.
Cell proliferation analysis
The establishment and evaluation of iTECs were described in our previous literature . Logarithmically grown iTECs were seeded in 96-well plates (1×104 /well). Cells were divided into different concentration treatment groups of RYF extraction (100 μl of 25, 50, 100, 200, 400 μg/ml). There was no cells and only 100 μl cell cultural medium in control group. Negative control group used 100 μl of cell cultural medium with 0.1% DMSO (Sangon Biotech) replaced RYF extraction. There were three wells of cells used as repeated in each group. The cells were incubated at 37 ℃ with 5% CO2 for 48 h. 20 μl of MTT solution (5 mg/ml, Biofrox) was then added into each well. After 4 h incubation, cell culture medium was discarded, and 150 μl of DMSO was added into each well. Plates were shaken at room temperature for 10 min. Absorbance value was measured at 570 nm using Synergy H1 Multi-Mode Microplate Reader (Bio-Tek, USA). Then calculated the inhibition rate of cell proliferation (%) with MTT assay.
Logarithmically grown iTECs cells were seeded in 24-well plates (3×104 /well) for 24 h. There was 100 μl of cell cultural medium with 0.1% DMSO (Sangon Biotech) added in to iTECs negative control group and different concentration treatment groups of RYF extraction (300 μl of 50, 100, 200 μg/ml). The cells were incubated at 37 ℃ with 5% CO2 for 48 h, and then cell culture medium was discarded. 300 μl of crystal violet staining solution was added into each well and stained at room temperature for 30 min. Subsequently repeated washed with water and dried naturally and then observed the proliferation. Results were expressed as percentage of cell proliferation with respect to negative control group cells (as 100%).
TGF-β1 induced iTECs EMT
TGF-β1 was used to induce EMT of iTECs. Briefly, iTECs in logarithmic growth phase were seeded in 24-well plates (3×104 /well) for 24 h, and then starved with serum-free medium for 24 h. Cells were divided into five groups: (i) normal group (DMED high sugar medium); (ii) TGF-β1 group (10 ng/ml TGF-β1); (iii) RYF-L group (50 μg/ml RYF extraction and 10 ng/ml TGF-β1); (iv) RYF-M group (100 μg/ml RYF extraction and 10 ng/ml TGF-β1); (v) RYF-H group (200 μg/ml RYF extraction and 10 ng/ml TGF-β1). There were three wells of cells used as repeated in each group. The TGF-β1 was administered first, then after 48 h the RYF extraction were added and cultured for 24 h. The morphology of iTECs cells induced by TGF-β1 was observed with microscope. The phenotype marker of E-cadherin, Vimentin and a-Tubulin was observed by immunofluorescence staining method described in 2.7. Quantitative real-time PCR analysis detected the expression level of Zeb-1 and Snail 1 and other mRNA for phenotypic markers and phenotype-related transcription factors.
The cells were seeded on 24-well plates (3×104 /well) for 24 h and were repeated washed with PBS respectively. The sterile coverslips with cells were fixed in 4% paraformaldehyde (PFA) for 15 min. Then performed with Triton-100 (Beyotime), and blocked with 0.5% bovine serum albumin (BSA, Biofrox) for 40 min at room temperature. The cells were incubated with primary antibody, rabbit anti-mouse CK5 (1:100, Affinity), rabbit anti-mouse CK8 (1:100, Abcam) or FITC-rabbit anti-mouse E-cadherin (1:200, BD Pharmingen), rabbit anti-mouse a-Tubulin (1:100, Abcam), rabbit anti-mouse Vimentin (1:100, Cell Signaling) for overnight at 4℃. The cells were followed by incubation with secondary antibodies, FITC-mouse anti-rabbit IgG (1:100, Santa Cruz) and PE-Goat Anti-Mouse IgG (1:100, Thermo) at room temperature for 2 h. Cell nuclei were stained with Hoechst 33258 (1 μg/ml, sigma) for 5 min. The labeled section were viewed with fluorescence confocal microscopy (Zeiss, Germany).
Quantitative Real-time Pcr Analysis
The cells were seeded on 6-well plates (1×105 /well) for 24 h respectively. The steps of total RNA extraction, reverse transcription, and fluorescence quantitative detection of SV40 mRNA was as described . Trizol Reagent (Thermo); First Strand cDNA Synthesis Kit (Sangon Biotech). PCR amplification was performed with GAPDH as a reference to detect the expression of target mRNA by the 2−ΔΔCt method in different group cells. There were three wells of cells used as repeated in each group. The primer sequences were listed in Table 1. Primer synthesis was performed by Shanghai Sangon.
Sequences of primer pairs for quantitative real-time PCR.
Forward primers (5' − 3')
Reverse primers (5' − 3')
TCTTCGTCT CCG GCT CTG
Logarithmically grown iTECs cells were seeded in 24-well plates (3×104 /well) until the cell density reached 80%-90%. Then discarded the culture medium, washed by serum-free DMEM, added 1.2-2.0 ml serum-free DMEM and put in the culture incubator for 20 min. The pBGLuc-Smad plasmid was infected and transfected by LipofectAMINE 2000. Subsequently, the successful infection and transfection iTECs were divided and treated by the method described in 2.8. The luciferase level of all groups cell supernatant liquid (50 µl) were analyz ed with single-channel bioluminescent detector according to the Luciferase gene kit’s instruction and calculated the relative activation degree of Smad pathway (%). The expression of Smad mRNA of each group was detected by PCR.
The statistical analysis was performed with SPSS 20.0 software and the results were expressed as mean ± SD. One way ANOVA were used to compare means of multiple groups and least significant difference test was used for comparison between two groups. Differences were considered to be statistically significant when P < 0.05.