Preparation and phytochemical analysis of FZJD
FZJD comprises the following eight herbal plants: Hedysarum multijugum Maxim. (Huang-Qi), Fallopia multiflora (Thunberg) Harald (He-Shou-Wu), Bistortae Rhizoma (Cao-He-Che), Actinidia arguta (Siebold & Zucc.) Planch. ex Miq. B (Teng-Li-Gen), Smilacis Glabrae Rhizoma (Tu-Fu-Ling), Codonopsis Radix (Dang-Shen), Fructus Lycii (Gou-Qi), and Atractylodes macrocephala Koidz (Bai-Zhu) in the ratio 2:1:1:1:1:1:1:1. FZJD ointment was provided by the Pharmaceutical Preparation Center of Guang’anmen Hospital, China Academy of Chinese Medical Sciences. Bai-Zhu was soaked in water for 24 h. Next, the mixture was subjected to steam distillation in another container and obtained 450 mL distillate. The residue was boiled with water for 1 h and the decoction was mixed with 450 mL of distillate. The other herbs were decocted twice with water for 1 h each. The decoction from the two steps was mixed and filtered. The filtrates obtained from Bai-Zhu and other herbs were mixed and the mixture was concentrated through evaporation under reduced pressure to obtain the final ointment (relative density of 1.20-1.25 g/cm3 at 50 °C). The chemical constituents of FZJD ointment were analyzed using high-performance liquid chromatography (HPLC). The test solution was prepared by dissolving FZJD ointment in methanol. HPLC analysis was performed in an Agilent 1200 HPLC (DAD) system equipped with a C18 analytical column (250 × 4.6 mm, 5 μm). The mobile phase for gradient elution comprised acetonitrile and water. The following standards were used for quality control: calycosin-7-glucoside and astragaloside IV (H. multijugum Maxim); rhein, emodin, physcion, and 2,3,5,4′ tetrahydroxystilbene 2-O-β-D glucoside (F. multiflora); gallic acid (Bistortae Rhizoma); epicatechin (A. arguta); astilbin (Smilacis Glabrae Rhizoma); lobetyolin (Codonopsis Radix); betaine (Fructus Lycii); atractylenolide I, II, and III (Atractylodes macrocephala); fructose, glucose, and sucrose (saccharides). All standards were purchased from the National Institutes for Food and Drug Control (Beijing, China).
Animals
Male 615 mice (aged 4–6 weeks; weighing 18–20 g) and male Sprague-Dawley (SD) rats (aged 4–6 weeks; weighing 190–200 g) were purchased from the Vital River Company (Beijing, China). All animals were housed in a temperature and humidity-controlled facility. The animals had free access to food and water. All animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals, Ministry of Science and Technology, China. This study was approved by the Ethical Committee of Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing (IACUC-GAMH-2020-006).
Cell culture
The murine GC cell lines, mouse forestomach carcinoma cell (MFC) and mouse aorta endothelial cell (MAEC) were purchased from the Institute of Basic Medical Science Chinese Academy of Medical Science (Beijing, China). MFCs and MAECs were cultured in Dulbecco’s modified Eagle medium (DMEM) medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin (all from Thermo Fisher Scientific, Waltham, MA, USA). All cells were maintained at 37 °C and 5% CO2 in a humidified chamber.
Establishment of a tumor-bearing mouse model and drug administration
The mice were allowed to acclimatize for one week. The MFCs (2×105 in 0.2 mL) were injected into the right axilla of 615 mice. The animals randomly divided into the following four groups (n=10/group) were treated with the test agents the next day: Control group, administered with phosphate-buffered saline (PBS) by oral gavage (OG; 0.2 mL/day) and intraperitoneally injected with PBS (0.1 mL for thrice a week); 5-FU group, administered with PBS by OG (0.2 mL/day) and intraperitoneally injected with 5-FU (20 mg/kg bodyweight, thrice a week); FZJD group, administered with FZJD (0.625 g/mL) by OG (0.2 mL/day) and intraperitoneally injected with PBS (0.1 mL; thrice a week); FZJD+5-FU, administered with both FZJD and 5-FU following the treatment protocols of FZJD and 5-FU groups. The treatment period for all groups was two weeks. The mice were sacrificed by cervical dislocation on the day after the final drug administration. The tumor tissues were excised and weighed.
Preparation of FZJD-containing serum
The SD rats were randomly divided into the following two groups: blank serum group, administered with saline; FZJD serum group, administered with FZJD (0.4375 g/mL) by OG (2 mL; twice a day for five consecutive days). The animals had free access to food and water until 12 h before blood collection. All rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium. The blood samples collected from the abdominal aorta at 1 h post-final OG administration under sterile conditions were incubated at room temperature for 4 h and centrifuged at 3000 rpm for 15 min. The serum samples of the same group were mixed well and heat-inactivated in a water bath at 56 °C for 30 min. Next, the serum sample was filtered through a 0.22-µm membrane filter and stored at −80 °C.
Identification and sorting of TEMs
The bone marrow-derived macrophages (BMDMs) were isolated from the tumor-bearing mice (tumor-bearing group, TB) and wild-type mice (normal group, NR). The MFC-bearing mouse model was established as described previously. After 2 weeks, the mice were sacrificed. The mouse skin was disinfected for 5 min using 75% ethanol. BMDM isolation was performed as previously described with minor modifications [16]. Femurs and tibias were excised from the mice under sterile conditions and the muscle tissues were removed carefully. The bone ends were cut and the marrow was flushed out using a syringe filled with DMEM into a small Petri dish. The supernatant was collected from the Petri dish and filtered through a 75-µm membrane filter into 15-mL tubes. The samples were centrifuged at 1000 rpm and 4 ℃ for 10 min. The supernatant was discarded and the pellet was incubated with 10 mL of red blood cell lysis buffer for 30 s. Next, the pellet was resuspended in 10 mL of complete medium. The mixture was centrifuged at 1000 rpm for 5 min. The supernatant was removed and the pellet was resuspended in culture medium supplemented with 50 ng/mL macrophage colony-stimulating factor (M-CSF) (RD systems, Minneapolis, MN, USA) and cultured for 72 h. For flow cytometric analysis and cell sorting, the BMDMs were blocked with the whole IgG for 15 min at 4 °C. The samples were immunostained with anti-CD45 (fluorescein isothiocyanate (FITC)-conjugated clone 30-F11) (Thermo Fisher Scientific, Waltham, MA, USA), anti-F4/80 (allophycocyanin (APC)-conjugated clone BM8), anti-CD11b (APC/Cyanine7-conjugated clone M1/70), and anti-CD202b (TIE2) (phycoerythyrin (PE)-conjugated clone TEK4) (all from BioLegend, San Diego, CA, USA). Flow cytometry analysis was performed using the Aria III flow cytometer.
Cell migration assay
Cell migration assay was performed using the Transwell plates with a pore size of 8 µm (Corning, Cambridge, NY, USA). The TEMs (2×105) were seeded into the upper chamber. The bottom chamber was filled with the following three culture media supplemented with 300 ng/mL ANG-2 (R&D systems, Minneapolis, MN, USA) which divided into the following three groups: the control medium group (CM); the blank serum-supplemented medium group (BSM), supplemented 10% serum derived from SD rats belonging to the blank serum group; the FZJD serum-supplemented medium group (FSM), supplemented 10% serum derived from SD rats belonging to the FZJD group. After 24 h, the cells in the membrane were fixed, stained, and counted using light microscopy.
Tube formation assay
Briefly, Matrigel (BD Bioscience, San Diego, CA, USA) was added to the 24-well plates after thawing overnight. All plates were incubated at 4 °C for 30 min and 37 °C for 30 min to allow gel polymerization. The TEMs (2×105/well) and MAECs (5×104/well) were seeded on the gel. The cells were then incubated in CM, BSM and FSM (containing 200 ng/mL ANG-2) for 6 h. Then stained with Calcein-AM (Solarbio, Beijing, China), and the number of capillary-like structures was counted using a fluorescence microscope.
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
The TEMs (2×105/well) were seeded in CM, BSM and FSM (containing 200 ng/mL ANG-2) for 30 min. Total RNA was extracted from the cells using TRIzol reagent (TIANGEN, Beijing, China), following the manufacturer’s instructions. The concentration and purity of the RNA were determined using a ultraviolet (UV) spectrophotometer. The extracted RNA was reverse-transcribed into cDNA using the PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa, Tokyo, Japan). The primers used for qRT-PCR analysis are listed in Table 1. The expression levels of target genes were normalized to those of Gapdh. The fold changes in gene expression were calculated using the 2-ΔΔCt method.
Table 1 Primers used for qRT-PCR
Primer name
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Primer sequence
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Vegfa
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Forward
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5′-ACACATTGTTGGAAGAAGCAGCCC-3′
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Reverse
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5′-AGGAAGGTCAACCACTCACACACA-3′
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Mmp9
|
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Forward
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5′-TGGGGGTTAGGGACAGAAAT-3′
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Reverse
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5′-GAACAATAACGCACAGACCC-3′
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Cox2
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Forward
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5′-CCAGAGCAGAGAGATGAAA-3′
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Reverse
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5′-GGTACAGTTCCATGACATC-3′
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Gapdh
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Forward
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5′-TTCCTACCCCCAATGTATCCG-3′
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Reverse
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5′-CCACCCTGTTGCTGTAGCCATA-3′
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Immunohistofluorescence (IHF) staining
IHF staining was performed using the 10-µm thick paraformaldehyde-fixed tumor tissue sections. The sections were incubated with anti-TIE2 (rabbit anti-mouse, ab95722, 1:50; Abcam, Cambridge, UK) and anti-CD31 (goat anti-mouse, AF3628-SP, 1:100) (R&D Systems, Minneapolis, MN, USA) primary antibodies. Next, the sections were incubated with Alexa Fluor 488-conjugated goat anti-rabbit (A-11008) and Alexa Fluor 555-conjugated donkey anti-goat antibodies (A-21432) (Thermo Fisher Scientific, Waltham, MA, USA). The nuclei were stained with 4′,6-diamidino-2-phenylindole. Images were captured using a NIKON TE-2000U inverted fluorescence microscope. All images were processed identically using Adobe Photoshop CC2019 software.
Statistical analysis
All statistical analyses were performed using SPSS 18.0. The data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA) was used for multiple comparisons. Student’s t test was for analyzing unpaired data. The differences were considered significant at P < 0.05.