2.1. Reagents and antibodies
F-12kaighn’s (F12k) modification medium, RPMI modification medium, pancreatin, penicillin-streptomycin, and phosphate-buffered saline (PBS) were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). Fetal bovine serum was obtained from Gibco (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO) was acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cisplatin was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). TMT®Mass Tagging Kits and Reagents was sourced from Thermo Fisher Scientific (Waltham, MA, USA). The glucose assay kit and lactic acid assay kit were purchased from Jiancheng Bioengineering Institute (Nanjing, China). PI3K (4249S), phospho-PI3K (4228S), AKT (4691S), phospho-AKT (4060S) and phospho-mTOR (5536S) antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Lamin B1(12987-1-AP), beta-actin (20536-1-AP), mTOR (20657-1-AP) and HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (SA00001-2) were obtained from Proteintech Group, Inc. (Chicago, USA). The other chemicals and reagents were analytical grade and commercially available.
2.2. Preparation of Fuzi decoction, FZA and FZP
The preparation process of Fuzi decoction was stated as follows. Fuzi was obtained from Bozhou Yonggang Pharmaceutical Co., Ltd (Sichuan, China) and was authenticated by Professor Shengjin Liu (Nanjing University of Chinese Medicine, Nanjing, China). Fuzi was added 8 times as much water to decoct and boiled for 1 h. Then the filtrate was collected, and the residue was extracted again with repeating the above operation. After that, the twice aqueous solutions were combined by filtration and concentrated to a final extract.
The preparation steps of FZA were summarized as follows. After Fuzi pieces had been beaten into powder, the Fuzi powder was soaked in 4-fold 85% ethanol for 24 h and filtered. The residue was added with 4 times 85% ethanol solution and extracted by heating at reflux 4 times, each time for 2 h. After the residue was washed with small quantity of ethanol, the impregnating solution, extracting solution and washing solution were combined. The mixture was evaporated in vacuum to remove ethanol, and distilled water was added to obtain the loading solution. 50 mL of 0.16 g/mL sample solution was taken and eluted with D101 macroporous resin using distilled water, 80% ethanol respectively. Lastly, the extracts were dried into powder by vacuum freeze-dried technology.
The preparation method of FZP was generalized as follows. After extraction of FZA, the dried drug residue was subjected to ultrasonic extraction for twice with 4-fold ultrapure water, each time for 1 h. The extract was combined and concentrated appropriately, 95% ethanol was added to the crude extract until the final concentration was 80%, followed by precipitating overnight at 4 °C. Next, the crude extract was filtered, and the residue was washed with absolute ethanol and acetone to obtain crude polysaccharide, further deproteinizing by using the Sevag method (trichloromethane: n-butanol = 5:1, v/v), shaking for 20 min and leaving in a separatory funnel for 30 min. After the solution was stratified, the chloroform-water interface was observed, and the supernatant was obtained when a gel-like precipitate of free protein appeared. After dialysis with running water, the supernatant was precipitated with alcohol, then the sediment was washed with anhydrous ethanol and acetone. Finally, FZP was obtained by freeze drying technique.
2.3. Animal experiment
All experiment were performed in accordance with the relevant guidelines and regulations and approved by Nanjing University of Chinese Medicine. The animal procedures described in this study were approved by the Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine (approval number: 202012A008). Nude mice (6-8 weeks) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). A549 cells at a density of 3 × 106 cells /μL in PBS were injected subcutaneously into the right axilla of nude mice. The control group was injected with 0.2 mL PBS solution at the same site. When the volume of implanted tumors reached 50 mm3, the mice were randomized into five treatment groups and administered intragastrically with different treatments daily: PBS (10 mL/kg), FZ (15 g/kg), FZP (7 g/kg), FZA (2 g/kg). An intraperitoneal injection of 4 mg/kg cisplatin was rendered as positive control every two days. The weight of mice was recorded daily during administration and tumor volume was assessed with a vernier caliper every 2 days after inoculation. The tumor volume (V) was calculated by the following equation: V = (Maximal length × Maximal width2)/2. After 2 weeks of treatment, blood was collected from eyeball venous plexus of mice, and mice were killed by cervical dislocation. To evaluate the pathological changes in the NSCLC subcutaneous xenograft mice model, the lung, heart, spleen, liver, kidney tissues, and tumor tissues were collected, fixed in 4% paraformaldehyde, followed by paraffin embedding and cutting, and stained with hematoxylin and eosin (H&E). The rest of tumor tissues was frozen in liquid nitrogen for proteomics study. The blood from the fundus venosus was set aside for 30 min, centrifuged at 1500 rpm/min for 15 min and the upper serum layer was stored in the refrigerator at -80°C for metabolomics analysis.
2.4. Cell viability assay
The human NSCLC cell lines A549, NCI-H1975, NCI-H1650, NCI-H1299 and NCI-H460 were purchased from the cell bank of the Chinese Academic of Science (Shanghai, China). NCI-H1975, NCI-H1650, H1299 and NCI-H460 were maintained in RPMI-1640 while A549 was cultured in F12k media. The culture media were supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum. The cells were cultured in 37 ℃ incubator equipped with 5% CO2. Cell viability was measured by MTT assay following the manufacturer's instructions. The cells were seeded into 96-well plates (5,000 cells/well) and cultured overnight, followed by treatment with different concentrations of FZA for 24 h, 48 h and 72 h. The optical density (OD) values at 490 nm were determined by a microplate reader (EnVision, PerkinElmer). IC50 values were calculated with GraphPrism software.
2.5. UPLC-Q-TOF-MS analysis
The freeze-dried powder was dissolved in pure methanol to prepare a solution with a concentration of 7.5 mg/mL. After vortexed, the mixture was centrifuged at 14000 rpm for 15 minutes. Then the obtained supernatant was filtered for UPLC-Q-TOF-MS analysis. An ACQUITY UPLC system coupled with a Triple TOF™ 5600+ MS system (AB SCIEX, USA), which integrating a switchable electrospray ion source interface, was used to identify the chemical ingredients of FZA. Samples were separated on an ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, 1.7 μm) at the flow rate of 0.3 mL/min along with the column temperature of 35 °C. Mobile phases A and B were 0.1% formic acid aqueous solution and pure acetonitrile, respectively. The linear gradient elution was set as follows: 0-20 min, 5% A-35% A; 20-25 min, 35%-58% A; 25-28 min, 58%-100% A; 28-29 min, 100% A; 29-30 min, 100% A. The sample volume injected was set at 5 μL. The detection wave was 235 nm. The mass spectrometry analysis was mainly performed in TOF-IDA-MS mode under the following parameters: scan range, m/z 50~1500; nebulizer gas GS1 60 psi; heater gas GS2, 60 psi; curation gas, 40 psi; temperature, 600 °C for ESI−; ion spray voltage, 5500 eV; declustering potential, 100 eV; collision energy, 10 eV; collision energy difference, 15 eV.
2.6. TMT-based proteomic analysis
Total protein samples were extracted from frozen tumor tissue in PASP lysis buffer (100 mM NH4HCO3, 8 M urea, pH 8), and then quantified using a Bradford protein assay kit (Cat. P0006, Beyotime). The proteins were separated on a 12% gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis and protein bands were visualized by Coomassie Blue R-250 staining. The protein solution was digested with trypsin and CaCl2 at 37 °C overnight. Peptides desalination was carried out using the C18 desalting column, and the samples were then lyophilized following the manufacturer's instructions. The digested peptides were collected and cleaned with a C18 column, then the digested peptides were labeled with TMT 10-plex reagents according to the manufacturer's instructions (Thermo Fisher Scientific). The TMT-labelled peptides were dissolved in mobile phase A (98% double-distilled water, 2% acetonitrile, pH 10) and fractionated on a Waters reversed-phase BEH C18 column (4.6×250 mm, 5 μm) with a RIGOL L-3000 HPLC System. Fractions were collected one tube per minute and then dried and combined into ten fractions for further LC-MS/MS analysis. LC-MS/MS analysis was performed with an EASY-nLC 1000 System (Nano HPLC, Thermo Fisher Scientific) coupled online to a Q ExactiveTM HF-X Mass Spectrometer with a Nanospray FlexTM (ESI) Ion Source (Thermo Fisher Scientific). The LC-MS/MS raw data were processed using Proteome Discoverer 2.4 (PD 2.4, Thermo) and Uniprot database, with a false-discovery rater (FDR) < 0.01 at the level of proteins and peptides. Proteins with significant P value < 0.05 and fold change >1.2 or <0.8 were considered as differentially expressed proteins (DEPs). Based on the DEPs, Gene Ontology (GO) function analysis as well as the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were carried out. Meanwhile, volcano maps and cluster heat maps were drawn to visualize differential proteins. Furthermore, the String DB (https://STRING.embl.de/) was also employed to screen DEPs and to develop protein-protein interaction (PPI) network.
2.7. Non-targeted metabolomics analysis
The serum sample of each group stored at 80 ℃ were thawed on ice. After vortex mixing, 50 μL was placed in a centrifuge tube and 200 μL ice methanol solution containing 1,2-13C-myristic acid (12.5 μg/mL) was added and vortexed for 3 min. After the serum was centrifuged at 18 000 r/min for 10 min at 4 ℃, the supernatant was taken 100 μL and dried in a concentrator for 2 h at 45 °C. The supernatant was added with 30 μL methoxyamine hydrochloride dissolved in pyridine (w/v, 10 mg/mL), vortexed for 5min, and oscillated in a thermostatic oscillator at 300 r/min for 90 min at 30 °C, then 30 μL BSTFA (1% TMCS) was added and mixed. After 30 min of oscillating at 300 r/min at 37°C, the sample was centrifuged at 18 000 r/min for 10 min at 4 °C and the supernatant was performed with a GC-MS system that consisted of a Trace 1310-TSQ 8000 Evo (Thermo Fisher, San Jose, CA, USA) equipped with a TG-5MS capillary column (0.25 mm × 30 m, 0.25 μm, Thermo Fisher, San Jose, CA, USA) under the following conditions: sampling volume, 1 μL; carrier gas, helium (99.999%); flow rate, 1.2 mL/min; and split ratio, 20:1. The gradient heating program was conducted as follows: 0-1 min, 60 °C; 1-14 min, 60-320 °C; 14-19 min, 320 °C. The TSQ 8000 was equipped with an electron ionization source. The ionization energy was 70 eV, the transfer line was held at 250 °C, and the source temperature was 280 °C. The GC-MS data were acquired after a solvent delay of 3.5 min, and the MS scan range was 50-500 m/z. The GC-MS data were converted into ABF format using the ABF Converter (http://www.reifycs.com/AbfConverter/) and imported into MSDIAL (v.4.10) software. The results were imported into SIMCA 14.1 (MKS Data Analytics Solutions, Sweden) for principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). The MetaboAnalyst website (http://www.metaboanalyst.ca) and one-way ANOVA method were used for evaluating identified metabolites. According to p < 0.05, FC > 1.2 or < 0.83, metabolites were obtained and imported into MetaboAnalyst website for metabolic pathway analysis. All statistical analyses were performed using Graphpad Prism 8.0 and the metabolic analysis website.
2.8. Network pharmacology prediction
The SMILES structures of FZA identified in 2.5 were retrieved from Pubchem database (https://pubchem.nc-bi.nlm.nih.gov/) and were introduced into Swiss Target Prediction wesite to predict the potential targets of FZA. Next, Uniprot database (https://www.uniprot.org/) was used to obtain the standard gene symbols of all the target proteins corresponding to FZA. Then, Genes related to NSCLC diseases were collected from GeneCards database (https://www.genecards.org/). In order to clarify the interaction between FZA target and NSCLC related target, the two targets were intersected and the Venn Diagram were drawn. Cytoscape 3.8.2 software (https://cytoscape.org/) was used to map the active ingredients-target network of FZA in the treatment of NSCLC. The PPI network based on STRING was constructed using the minimum required interaction score of 0.4. All the target proteins by FZA were then subjected to DAVID database for functional enrichment analysis including GO and KEGG. The filtering of retrieval results is with a threshold value of p < 0.05 and the top 20 pathways were screened out according to the number of enriched genes, and visual bar charts and bubble plots were drawn.
2.9. Measurement of intracellular glucose and lactate levels
NCl-H1299 and A549 cells were inoculated in 6-well plates with 5×105 cells/well overnight. After discarding the medium, cells were treated with different concentrations of FZA (0, 50, 100, 200 μg/mL) and cultured for 24 h. Then, the medium was collected, centrifuged and the pH value of the medium was measured with supernatant. Three replicate groups were set up. The cells were collected by ultrasonic crushing, centrifuged at 8000 g, 4 ℃ for 10 min, and the supernatant was taken for BCA protein quantification. According to the method of lactate assay kit and glucose kit, the OD value was determined by microplate reader. The lactic acid and glucose content were calculated and divided by the corresponding protein concentration for normalization.
2.10. Assessment of energy metabolites by GC-MS
A549 cells at logarithmic growth stage were seeded with 1×106 cell/well in 60 mm culture medium and incubated overnight. After that, the medium was discarded. Then, 2 mL of different concentrations of FZA (0, 50, 100, 200 μg/mL) were added and the medium was removed after 24 h of treatment. Three replicate groups were set up. The cells were washed twice with PBS and quenched by adding 1 mL of 60% methanol containing 70 mM HEPES on dry ice (-50 °C). Furthermore, 500 μL of pre-cooled 75% methanol-MTBE (9:1) containing 1,2-13C myristyl (5 μg/mL) was added and frozen in cold nitrogen for 3 min, then thawed at 37 °C for 10 min and repeated three times. The samples were centrifuged at 18,000 rpm for 10 min. The supernatant was transferred into a new tube and evaporated to dryness at 45 °C in centrifugal concentrator (Thermo Fisher, San Jose, CA, USA). After drying, 30 μL of methoxyamine hydrochloride dissolved in pyridine (w/v, 10 mg/mL) was added and vortexed for 5 min. Then, the samples were shaken at a speed of 300 rpm for 1.5 h in a thermostatic oscillator at 30 °C. Subsequently, 30 μL of BSTFA (1% TMCS) was added and shaken for 30 min at 37 °C. After derivatization, the mixture was centrifuged at 18,000 rpm for 10 min and 50 μL supernatant was injected into the GC-MS. The analysis conditions were described in Section 2.7.
2.11. RNA isolation and real-time qPCR
Total RNA was extracted from cells after treatment with FZA using the FastPure® Cell/Tissue Total RNA Isolation Kit (Vazyme, China), and the concentration of total RNA was determined using NanoDrop One Microvolume UV-Vis Spectrophotometer (ThermoFisher, China), then reverse transcribed into cDNA using the Hifair Ⅲ1st Strand cDNA Synthesis SuperMix for qPCR (Yeasen, China), The synthesized cDNA was applied in the Real-time PCR assay utilizing Hieff® qPCR SYBR Green Master Mix (Yeasen, China) on QuantStudio 7Flex Real-Time PCR System(Applied Biosystems, Foster City, CA, United States), according to the manufacturer's instructions. β-actin was used as a reference gene. The primers were synthesized by Sangon Biotech, and the sequences were listed as follows:
LDHA-F:(5′- GGATCTCCAACATGGCAGCCTT-3′).
LDHA-R:(5′- AGACGGCTTTCTCCCTCTTGCT-3′).
GLUT1-F:(5′- TTGCAGGCTTCTCCAACTGGAC-3′).
GLUT1-R:(5′- CAGAACCAGGAGCACAGTGAAG-3′).
2.12. Western blotting analysis
The treated cells were lysed with ice-cold cell lysis buffer containing protease and phosphatase inhibitor cocktail. Dissolved proteins were collected, and the debris was removed by centrifugation at 12,000 rpm for 10 min at 4 °C. The concentration of total protein was determined using the BCA protein assay kit. Equal amounts of lysate protein were electrophoresed by FuturePAGE (ACE, Nanjing, China). The separated proteins were transferred electrophoretically to PVDF membranes. The membranes were blocked with 5% BSA in tris buffer solution-tween (TBST) for 2 h and subsequently incubated overnight at 4 °C with primary antibodies. After washing with TBST, the membranes were incubated with HRP-conjugated Affinipure Goat Anti-Rabbit lgG(H+L) for 2 h at room temperature. Thereafter, reactive bands were visualized by the Chemidoc XRS+ Imaging System (Bio-Rad; Hercules, CA) using super ECL detection reagent (Yeasen, China) and analyzed by Image J software.
2.13. Statistical analysis
All data was present as mean ± SD. Data between two groups was analyzed by Student’s t-test, while one-way ANOVA was used to analyze data for more than two groups. Graphs were generated and statistically analyzed using GraphPad Prism 8 software. Significance is indicated as following: *, p < 0.05; **, p < 0.01; ***, p < 0.001.
