Identification of PAC and AAC components by Chromatography
DHI (product approval number Z20026866) was provided by Shandong Buchang Pharmaceutical Co. Ltd. (Heze, China). The chemical analysis department extracted and separated DHI, and extracted it with dichloromethane and chloroform in sequence according to the polarity. The extract is concentrated under reduced pressure to obtain a dichloromethane partial extract, which is the PAC. The extract is concentrated under reduced pressure to obtain a chloroform partial extract, which is the AAC. As we have previously reported (23), chromatographical identification of PAC and AAC components was performed by an ultra-performance liquid chromatography system (Waters Corp., Milford, MA, USA) equipped with a diode array detector, a column oven, an automatic sampler and a binary gradient solvent pump. We performed chromatography on an ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm) at 40°C. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B). The gradient program was as following: 0~7 min, 3~19% B; 7 ~ 13 min, 19% B; 13 ~ 18 min, 19 ~ 25% B; 18 ~ 25 min, 25 ~ 90% B; 25 ~ 30 min, 90% B. We set the flow rate of the mobile phase to 0.4 mL / min and the injection volume was 2 µL at the detection wavelengths of 254 and 286 nm.
Cell culture
EA.hy926 cells (Shanghai Cell Bank, Type Culture Collection Committee, Chinese Academy of Sciences) were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100U/mL penicillin and 100µg/mL streptomycin (Hyclone, Thermo Scientific, USA) and were grown in an incubator containing 5% CO2 at 37oC. When reached the desired confluence, cells were passaged by detaching with 0.25% trypsin-EDTA (Gibco, USA). Precisely weigh several milligrams of the extract from each layer of the extract of DHI, use DMSO as the solvent, uniformly prepare the mother liquor concentration of 100mg/mL, and dilute to the working concentration with cell culture medium.
Cell migration assay
Cell migration was measured in two different assays. EA.hy926 cells were seeded into 96-well plates at a density of 2 × 104 cells per well. Cells were stained with 1µg/mL Hoechst 33342 (Molecular Probes, USA) at 37°C for 30 min after 24 hours of serum deprivation. The cell monolayer was scraped in a straight line to create a ‘‘scratch’’ with a 200µl pipet tip, rinsed twice with PBS to remove the debris and then replaced in 100µl experimental DMEM containing 1% FBS and PAC (50µg/mL), AAC (25µg/mL), or vehicle. The plate was inserted into an incubator at 37°C for 12 h. Images were captured at the beginning, 4h, 8h, and 12h after injury using a High Content Screening (HCS) Microplate Reader (Operetta, PerkinElmer, USA) at x10 magnification. The cell migration was quantified using the line selection tool in ImageJ software by tracing the wound margin of two defined positions in each image. By comparing the images from time 0 to the next time point, the distance of each scratch closure was calculated. The oris™ assay was performed in an optically clear 96-well black skirted fluorescence microplate according to the manufacturer’s instruction (24). Briefly, cells are seeded and allowed to attach and spread in the presence of a silicon stopper. Subsequently, the plug was removed and it was found that the central cell-free region was surrounded by monolayer cells and migration can occur. EA.hy926 cells were cultured in DMEM containing 10% FBS and plated on orisTM cell migration-collagen I coated plates containing cell seeding stoppers. The cells were grown for 16 to 20 hours before the stopper was manually removed. The cells were washed and replaced the medium with DMEM containing 1% FBS, and the indicated concentrations of VEGF, PAC, AAC or vehicle. After incubation with the compound for 12 hours, the media were removed, and the cells were stained with Calcein AM for 30 min at 37℃. After washing twice with PBS, images were captured on the High Content Screening Microplate Imaging Reader. Three technical replicates were done per experiment, and three independent experiments were performed.
In vitro tube formation assay
Tube formation assay was performed following a procedure by Michaud (25). 96-well plates were coated with basement membrane matrix (BD Biosciences, USA). EA.hy926 cells were incubated in a drug-containing medium at a density of 1.5×104 cell/well. After 12 hours of incubation, the cells were stained with 1 µM calcein AM for 30 minutes. Nine fields of view were randomly selected in each well. Imaging was performed using the Operetta HCS System, which analyzes the ability of tube formation by calculating the number of cellular networks. Three technical replicates were done per experiment, and three independent experiments were performed.
Real-time quantitative PCR analysis
The angiogenesis-related genes were examined by real-time quantitative PCR in cultured EA.hy926 cells treated with PAC, AAC, or vehicle and ischemic gastrocnemius muscle. Total RNA samples were extracted from ischemic muscle or ECs using TriQuick Reagent (Solarbio, Beijing, China), followed by reverse transcription of the RNA samples into complementary DNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany) in accordance with the manufacturer’s protocol.
The resulting cDNA was used as a template for real-time polymerase chain reaction amplification. SYBR Green Master Mix reagent was used for quantitative PCR, and GAPDH was used as an internal control to quantify the level of angiogenesis-related genes. C1000TM Thermal Cycler Sequence Detection System (BIO-RAD, USA) was used to perform the amplification and analysis. Samples were compared using a relative CT method. The fold increase or decrease was determined relative to a blank control after normalizing to a housekeeping gene using 2-∆∆CT, GAPDH.
Ingenuity Pathway Analysis
Ingenuity Pathway Analysis (IPA) tool (Ingenuity Systems, Redwood City, CA) was used to analyse the interactions between the 33 genes. Since PAC mostly increased the expression of VEGFA and decreased the expression of TIMP3, the potential up- or down-regulated genes were predicted by the Map function of IPA, and since AAC mostly decreased the expression of FGF2 and TGFB2, the potential up- or down-regulated genes were predicted by the Map function of IPA .
PA's Map function can be used to predict potential up-regulation or down-regulation of genes, and since AAC mostly reduces the expression of FGF2 and TGFB2, IPA's Map function predicts potential up- or down-regulation of genes.
Preparation of standard and sample solutions
Preparing the standard stock solutions of sixteen standards of DHI in 50% methanol aqueous solution with ultrasonic mixing. For sample stock solution, 0.107g PAC and 0.125g AAC were placed in a 25 mL volumetric flask respectively, with 50% methanol aqueous solution and methanol volume to the scale, ultrasonically mixed completely, and cleared by centrifugation.
Animals
Homozygous VEGFR-2-Luc male mice (26) were obtained from three transgenic breeding colonies that were maintained in a specific pathogen-free animal lab of TJAB. Animals were housed in cages in the facility in batches and maintained on a normal diet. All experiments were reviewed and approved by the TJAB Animal Experimental Ethics Committee (TJAB-JY-2011-002) and conducted in accordance with the guidelines for use of animal experiments at Tianjin University of Traditional Chinese Medicine. Before the experiment, the animals were allowed to adapt to the environment for 72 hours in a constant temperature of 22°C in a 12-hour light/dark cycled, food and water were freely available.
Murine hind-limb ischemic model
Mice were anesthetized with isoflurane and unilateral hind-limb ischemia was induced as previously described (He et al., 2016). The entire femoral artery and vein of the right hind limb were exposed, and the exposed vessels were ligated at their proximal and distal ends, both vessels were excised in the middle. The intact perfused contralateral limb of each mouse was used as an internal control. After hind-limb ischemia, saline, simvastatin, DHI, PAC, or AAC were administrated daily for 21days. The dosage of the PAC per mouse was 13.8 mg/kg, and the dosage of the AAC was 69 mg/kg. The preparation method of PAC and AAC in vivo: first dissolve with 10% volume of absolute ethanol, then add 90% volume of normal saline to mix, intraperitoneal injection, once a day. LDPI system was used to perform a continuous, non-invasive assessment of ischemic limb microvascular perfusion. The ratio of perfusion in the ischemic limb to perfusion in the healthy limb was monitored periodically as an indicator of recovery of hind limb perfusion.
In vivo tumor Growth
VEGFR2-Luc mice were used to establish the Lewis lung carcinoma (LLC) animal model. 100µl saline containing 2.5×105 of LLC cells was injected subcutaneously into the axilla of both forelimbs of the VEGFR2-Luc mice. The day after implantation, the animals were randomized into control, DHI, PAC, and AAC-treatment groups. At 11 days after treatment, protruding tumors were found at the injection sites. Using a caliper, the tumor size was measured every three days. At the end of the treatment, mice were sacrificed, and the tumor was removed and weighted.
In vivo bioluminescent imaging
IVIS1 Lumina K Series III system (PerkinElmer) was used to provide a real-time, rapid in vivo imaging enabling acquisition of biologically relevant events within milliseconds. Mice were anesthetized with isoflurane and then 150 mg/kg of D-luciferin (PerkinElmer) was injected intraperitoneally for each mouse. The optical signal intensity of VEGFR-2-luc mice were obtained 5 minutes after D-luciferin administration. The regions of interest (ROI) from the displayed images were identified on the ischemic sites or tumor sites using LivingImage® software and quantified as photons per second (p/s) .
Western blotting
EA.hy926 cells were treated with different drugs in 10 cm dishes for 24 hours. The cells were lysed with lysis buffer containing protease inhibitor, then the lysate was transferred to a 1.5 ml centrifuge tube and incubated on ice for 30 minutes with a vortex. Total protein was obtained by centrifugation at 12,000 x g for 30 minutes at 4°C, and the protein concentration was measured by a BCA kit. After the addition of the loading buffer, the samples were boiled and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% skim milk for 2 hours at room temperature, and then incubated with antibodies against CXCR4 or GAPDH overnight on a 4°C shaker. The membranes were washed three times with PBST and then incubated with the secondary antibodies for 1 hour at room temperature in the dark. The membranes were washed three times with PBST, and then the chemiluminescence signals were detected by ECL Plus detection system and exposure to X-ray film to produce bands within the linear range.
Statistical Analysis
Data analyses were performed with SPSS16.0 statistical software. The results were shown as mean ± SD. Statistical significance was assessed by unpaired Student’s t-test or by analysis of variance test for comparison between multiple groups. A P-value of < 0.05 was taken as statistically significant.