1. Network Pharmacology Analysis
1.1 Collection of PK Parameters
PK parameters of resveratrol were procured from TCMSP data set rendition (http://tcmspw.com/ tcmsp. php)23, which included 499 Chinese spices enrolled in the Chinese Pharmacopeia and 29,384 ingredients24. In addition, data on its absorption, distribution, metabolism and excretion properties can be obtained.
1.2 Resveratrol Targets
Resveratrol targets were acquired from databases and literature. Firstly, its three-dimensional structure was obtained from PubChem as shown in Fig. 2A (https: //pubchem.ncbi.nlm.nih.gov/)25. Then, the targets of resveratrol were collected from the SwissTargetPrediction (http://www. swisstargetprediction. ch)26, DrugBank (http://www.drugbank.ca)27, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, Pharmmapper (http://www.lilabecust.cn/ pharmmapper/)28,29. Acquired targets were then mapped to the UniProt database (http://www. uniprot.org/) 30 for normalization.
1.3 Identification of NPC Targets
The DisGeNET platform (http://www.disgenet.org)31, GeneCards (https://www.genecards. org) 32, TTD (http://bidd.nus.edu.sg/group/cjttd)33, OMIM (http://www.omim.org)34 and the Comparative Toxicogenomics Database (http://ctdbase.org/)35 were selected to collect NPC genes by searching words “nasopharyngeal carcinoma” and reproducible targets were removed.
1.4 Construction of Protein-Protein Interaction (PPI) Data
Intersecting targets for nasopharyngeal carcinoma and resveratrol were obtained using Venny 2.1.0 (http://bioinfogp.cnb.csic.es/tools/venny/index.html) and the overlapping targets were then uploaded to the STRING 11.0 (https://string-db.org/). The pharmacology network that made up the hubs of the networks were resveratrol, NPC, and their connected target genes.
1.5 Analysis of GO Function Enrichment and KEGG Pathway
Useful pathways of resveratrol connected with NPC were dissected with GO enrichment and KEGG pathways analysis. The GO incorporated a biological process (BP), molecular function (MF), and cellular component (CC). The Component-target-disease-pathway interaction network was constructed by Cytoscape version 3.8.2, which was a java based open-source software36. The enrichment degree of P-value < 0.05 was considered statistically significant.
3. Experiments Verification
Resveratrol purchased from Solarbio (Solarbio, Beijing) was dissolved at a stock concentration of 50 mmol/L in dimethyl sulfoxide (DMSO) and the final DMSO concentration was not exceeding 0.1%.
3.2 Cell Culture and Treatment
A poorly differentiated human NPC cell line (American Type Culture Collection, ATCC)38,39 was cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). All cells were incubated at the temperature of 37˚C with 5% CO2.
3.3 Cell Proliferation Assay
The effect of resveratrol on the cell growth of CNE2 cells was examined using the Cell Counting Kit-8 (CCK-8) assay (Abbkine, USA). NPC cells were plated in 96-well plates at 5,000 cells/well. Cells incubated in 96-well plates were treated as indicated and cell proliferation was assessed by CCK-8 assay at 0, 24, 48 and 72-hour resveratrol treatment following the manufacturer’s instruction. Viable counts were determined with absorbance readings at 450 nm by a SpectraMax M5 plate-reader (Molecular Devices, Sunnyvale, CA, USA) and IC50 were calculated on GraphPad Prism 8.
3.4 Hematoxylin-eosin (H&E) Staining
CNE2 cells were treated with 50 and 100 µM resveratrol and washed with PBS. Then, the cells were immobilized over night with 4% paraformaldehyde and stained with H&E. The stained sections were imaged under an inverted phase contrast microscope (Nikon, Japan).
3.5 Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) staining
The Apoptosis of NPC was detected by TUNEL staining. According to the manufacturer’s instructions, the apoptosis cells were detected by TUNEL assay Kit (Alexa Fluor 640, Yeasen, China). Nuclei counterstained with DAPI staining and viewed 400x under inverted microscope (Leica DM400DB microscope, Germany). The rate of apoptosis was measured by the number of normal positive cells (red spot) in the fluorescent picture.
3.6 Wound Scratch Assay
NPC cells were cultured in 24-well plates with 5x104 cells/well and incubated at 37˚C with a 5% CO2 incubator. The cell monolayer was scraped in a straight line to create a scratch with a 1mL pipette tip, and the debris was removed by washing the cells with PBS. Scratches were observed under a microscope (Nikon, Japan) at 24 and 48-hour. The results of the scratches represented the mean of the three experiments.
3.7 Transwell Migration Assay
In the migration assay, the cells were digested and suspended in serum-free medium. A total of 100 µL of cell suspension (5 x 10 4) was added to the upper chamber of the transwell plate and 500 µL of medium containing 10% FBS was added to the bottom chamber. After incubation at 37˚C and 5% CO2 for 48 hours, the cells were gently removed from the upper surface of the polycarbonate film with a damp cotton swab. The polycarbonate film was carefully removed from the upper chamber and the cells were immobilized in precooled methanol for 30 minutes. After staining with 0.1% crystal violet for 20 minutes, the specimens were observed under microscope (Nikon, Japan) and photographed.
3.8 Western Blot Analysis
After treating NPC cells with resveratrol, whole Lysis were harvested, and Western blotting was performed to detect target proteins. MAPK Family Antibody Sampler Kit ERK1/2, JNK, P38 (1:1000; 9926; CST; USA) and Phospho-MAPK Family Antibody Sampler Kit p-ERK1/2, p-JNK, p-P38 (1:1000; 9910; CST; USA) were used. β-actin antibody was purchased from Abclonal (1:50000, a00702, Abclonal, MA, USA). The total protein was obtained by tissue lysis and its concentration was measured. The protein separated by PAGE was moved to PVDF film and added with primary and secondary antibodies. Then, the protein was exposed, developed and fixed on x-ray film with ECL chemiluminescent reagent.
3.9 Immunohistochemical (IHC) staining of NPC Human Tissues
IHC staining was conducted with 36 NPC surgical specimens and, where possible, their noncancerous counterparts. The p-ERK1/2 (1:500), p-JNK (1:50), and p-P38 antibodies (1:200) were selected because of the network target prediction. The sections without first antibody incubation were used as background control. IHC staining was observed and shot with magnifying lens. Spot staining power was examined utilizing Image Pro Plus 6.0 programming (Rockville, USA) and addressed by the mean optical density value.
3.10 Statistical Analysis
Measurement data were expressed by mean ± standard deviation (SD). Statistical significance between different groups was determined by one-way-ANOVA analysis using GraphPad Prism 8.3.0 (GraphPad Software Inc., San Diego, CA, USA), and P < 0.05 (two tails) was considered statistically significant.
3.11 Ethics approval and consent to participate
In accordance with approval from the ethics committee of First Affiliated Hospital of Dalian Medical University (NO. PJ-KS-KY-2022-26), all participants signed an informed consent before enrolled in this research. The stages of this study were performed based on the ethical principles in the Helsinki Declaration.
3.12 Consent for publication
The authors appreciate all the patients in this work for their cooperation and permission for the publication of the article.