Reagents, kit, cell culture and animals
Reagents
Dulbecco's modified eagle medium (DMEM) medium, fetal bovine serum (FBS) and penicillin-streptomycin were obtained from Gibco invitrogen cell culture (Carlsbad, USA), cis-Platinum was obtained from Sigma (Saint Louis, USA).
4',6-Diamidino-2-Phenylindole (DAPI) staining kit
First rinsed off the medium with PBS (Beijing Dingguo Changsheng Biotechnology Co.Ltd.,Beijing, China), the membranes were fixed with 4% PFA for 30 min. Rinse three times with PBS each for 5 minutes. After stain with DAPI (Beyotime Biotechnology, Shanghai, China) for 5 minutes, rinsed again three times with PBS each for 5 minutes. Finally, cells were photographed by microscope at 200-fold magnification.
Cell Culture
Human umbilical vein endothelial cells (HUVECs) were purchased from Allcells (Shanghai, China) and cultured in special medium for HUVEC cells at 37°C with 5% CO2. CCE was dissolved in cell cultivated medium to prepare different concentrations at 10, 3, 1, 0.3, 0.1, 0.03, and 0.01mg/mL.
Animals
Male C57BL/6 mice at 7–8 weeks of age were obtained from Slaking Experimental Animal Co. LTD. (Changsha, China). Mice were kept in a specific pathogen free (SPF) animal lab and were fed with sterile food and water freely. Animal care and experiments were performed according to the Institutional Animal Care and Use Guidelines.
Preparation of Chinese cordyceps extracts
Chinese cordyceps extraction (CCE) was prepared from the cordyceps produced by Sunshine Lake Pharma Co., Ltd (Dongguan, China). Briefly, 500 g artificial cultivated Chinese cordyceps (including sclerotia that the caterpillar of Thitarodes xiaojinensis infected by Ophiocordyceps sinensis and the stroma) were cut into small pieces, and soaked in 5000 mL distilled water. The mixture was homogenized in ice bath with a homogenizer (IKA, Staufen ,Germany) at 16,400 rpm for 3 min, and then sonicated for 30 min. The matrix was cooled at − 20°C for 40 min, and sonicated again for another 30 min to obtain a uniform matrix. After extraction, the residues were removed by using suction filtration, and the filtrate was lyophilized to obtain Chinese cordyceps extract (CCE). The nucleoside content of CCE was analyzed by high performance liquid chromatography (HPLC) as previous reported (Huo et al. 2017). Briefly, the HPLC analysis was performed using a Waters HPLC system (Waters Corporation, Milford, USA). The extracted solution was separated in a C18 column (Agilent ZORBAX SB-AQ, Palo Alto, USA) by maintaining the isocratic low rate (0.8 mL/min) of the mobile phase (0.4 M KH2PO4, pH 3.7: methanol 90: 10) and detected at 260 nm absorbance. Peaks of various compounds in the extraction were identified by their retention times and co-injection tests with corresponding standard compounds.
Bioassay
Matrigel Tube Formation Assay
Matrigel (Corning, Inc., Corning City, USA) was thawed at 4°C overnight. Each well of a pre-cooling 96-well plate was coated with 50 µL matrigel and then allowed to gel at 37°C for 30 min. The HUVECs were suspended in 50 µL culture medium (5 × 103 cells per well) with designated concentrations of CCE and seeded into the coated plates in triple. After incubating at 37°C and 5% CO2 for 6 h, the capillary tubes formed were photographed under a phase contrast microscope at magnification of 100× (Olympus IX51, Tokyo, Japan). The total branching length and the total segments length were quantified by using Image J software. The inhibition rate was calculated using untreated wells as control.
The ex vivo angiogenesis assay
The Rat aortic ring assay was slightly modified method as described previously (Ernens et al. 2015). In short, a 6-weeks old male SD rat was sacrificed under anesthesia with pentobarbital sodium (3%). The thoracic aorta was stripped out and washed twice with cold DMEM medium. Then the artery was cut into segment rings of about 1.0 mm. The aortic rings were then sandwiched into a 48-well plate with 300 µL matrigel per well. After the gel solidifies, 200 µL DMEM medium with penicillin-streptomycin (100 IU/mL-100 µg/mL), and fetal bovine serum (1%) were added into each well. After 24 h, the aortic rings were treated with various concentrations of CCE. Incubation was carried out for another 5 days in a humidified atmosphere containing 5% CO2 at 37°C, then aortic rings were photographed using a phase contrast microscope (Nikon TS2R-FL, Tokyo, Japan) with a magnification of 40×. The total branching length and the total segments length were quantified and analyzed by Image J software.
Bioassay with Lewis xenografts mice model
The subcutaneous tumor mouse model was established by inoculating 2 × 106 of Lewis lung cancer cells in the right armpit region of C57BL/6J mice. When the tumor volume increased to approximately 200 mm3, the mice were randomly divided into 4 groups (n = 10) and treated with intraperitoneally injected with physiological saline, 3 mg/kg cis-platinum (DDP), 25 mg/kg CCE and 100 mg/kg CCE, respectively. CCE were administrated once daily for 12 consecutive days, while DDP was administrated every 2nd day. Tumor size was measured three times per week during the treatment. Tumor volumes were calculated using the formula V = a * b2 * 0.5.19 Mice were sacrificed by cervical dislocation at the end of the experiment, and the tumor tissues were collected for immunohistochemical analysis.
Immunohistochemical analysis
Tumor tissues harvested from the sacrificed mice were fixed in 10% formalin, paraffin-embedded and sectioned. After deparaffinization and rehydration, 3-µm thick tumor tissue sections were pre-treated in 0.01 mM sodium citrate (pH 6.0) solution at 100°C for 3 min, followed by staining with rabbit anti-human CD31 antibody (Cell Signalling, Boston, USA) at final dilution of 1:1000. Positive staining was carried out by using Detection System HRP Polymer Kit (Lab Vision, Fremont, USA). The slides were then rinsed in distilled water, counterstained with Gill's haematoxylin (Fisher Scientific, Fair Lawn, USA), and mounted. For angiogenesis analysis, all morphological structures with a lumen interspersed by CD31-positive endothelial cells were identified as blood vessels. Images were photographed at high magnification (200×) using Leica DM6 microscopy system with DFC7000T digital camera (Leica, Munich, Germany). Quantification of microvessels was carried out as previously described. Images were quantified by means of computer assisted image analyzer for mean vascular density (MVD) (Gao et al. 2011).
Statistical analysis
Statistical analysis was carried out using SPSS 16.0 software (SPSS Inc., Chicago, USA), with Student’s two tailed t-test for two groups or one-way ANOVA followed by Dunnett’s post hoc test for multiple groups. The results were presented as mean ± SEM and p < 0.05 was considered statistically significant.