Preparation of AJ extract

Dry AJ powder (Shanghai Yuanye Bio-Technology Co., Ltd., China) was extracted with 70% ethanol using heat reflux extraction three times (for 3 h, 2 h, and 1 h, respectively), followed by decompressing concentration to obtain the concentrated extract. The extract was then filtered, freeze-dried, and stored at −4 °C. The freeze-dried powder was dissolved in 10% dimethyl sulfoxide (DMSO, Beyotime, Shanghai, China) and then filtered through a 0.22-μm syringe filter to form a reserve solution.

Cell culture and treatment

Human NSCLC cell lines HCC827 and H1299 were purchased from Procell (Wuhan, China) and cultured in Roswell Park Memorial Institute (RPMI)-1640 containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, MA, USA) and 1% penicillin-streptomycin solution (Procell) under 5% CO2 at 37 °C. NSCLC cells were treated with AJ at different doses (0–400 μg/mL) for 24 h to detect the toxicity of AJ in NSCLC cells. Cells were then divided into four groups: control: untreated NSCLC cells; AJ: NSCLC cells treated with AJ at 25 μg/mL for 24 h; RT: NSCLC cells treated with RT at 8 Gy for 24 h; RT + AJ: NSCLC cells treated with AJ at 25 μg/mL and RT at 8 Gy for 24 h.

Cell Counting Kit-8 (CCK-8) assay

After treatment with different concentrations of AJ or RT, NSCLC cells were seeded into 96-well plates at a density of 5 × 103 cells/well and incubated with 5% CO2 at 37 °C. After incubation for 24 h or 48 h, 10 μL CCK‑8 reagent (C0037, Beyotime, China) was added to each well and incubated for 1 h at 37 °C. The optical density (OD) value at 450 nm was detected with a microplate reader (BioTek, VT, USA). The cell survival rate was calculated as \(\text\,\text\,\left(\mathrm}\right)=\frac\,\text}\,\text}\times 100\).

Colon formation assay

After treatment with AJ or RT, NSCLC cells in the logarithmic growth phase were digested with 0.25% trypsin, resuspended in complete medium, and counted. Subsequently, cells were seeded into 6‑well plates at a density of 1000 cells/well and cultured for 14 days. The medium was changed every 3 days during culture. After cloning, the cells were washed once with phosphate-buffered saline (PBS, Beyotime) and fixed with 1 mL 4% paraformaldehyde for 30 min. After washing once with PBS, the cells were stained with 1 mL crystal violet for 15 min. After washing with PBS several times, the cells were dried and then photographed with a camera. The colony numbers were counted by Image J software (National Institutes of Health, MD, USA).

Apoptosis detection

Apoptosis of NSCLC cells was detected using the Annexin V‑FITC Apoptosis Detection Kit (C1062S, Beyotime). NSCLC cells were digested with 0.25% trypsin and then transferred into centrifuge tubes. After centrifugation at 1000 g for 5 min, cells were resuspended and counted. A total of 5 × 104 cells were centrifugated at 1000 g for 5 min and then resuspended with Annexin V‑Fluorescein Isothiocyanate (FITC) binding buffer. Subsequently, cells were incubated with Annexin V‑FITC and propidium iodide (PI) for 15 min in the dark at 25 °C. All cell events were gated by P1 to exclude dead cells and cells in aggregation. The apoptosis of cells was analyzed using flow cytometry (Becton, Dickinson and Company, NJ, USA).

Western blot assay

Total protein of NSCLC cells was extracted using radio immunoprecipitation assay (RIPA) lysis buffer (P0013, Beyotime) and the protein concentration was detected using the Bicinchoninic acid (BCA) Protein Assay Kit (P0011, Beyotime). The protein sample was split on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels and then transferred onto Polyvinylidene fluoride (PVDF) membranes. After blocking with 5% bull serum albumin (BSA), membranes were incubated with primary antibodies overnight at 4 °C and secondary antibody (Goat Anti-Rabbit IgG H&L [HRP], 1:2000, ab6721, Abcam, Cambridge, UK) for 2 h at room temperature. The membranes were visualized using the ECL kit (36208ES60, Yeasen, Shanghai, China) and analyzed by Image J software. The primary antibodies were c‑caspase‑3 (1:5000, ET1602-39, HuaBio, Wuhan, China), Bax (1:20000, ET1603-34, HuaBio), Bcl‑2 (1:5000, ET1603-11, HuaBio), p‑PI3K (1:1000, #13857, Cell Signaling Technology, MA, USA), PI3K (1:1000, #4249, Cell Signaling Technology), p‑AKT (1:1000, #9272, Cell Signaling Technology), AKT (1:1000, #9271, Cell Signaling Technology), p‑NOS (1:1000, #2977, Cell Signaling Technology), NOS (1:1000, #9575, Cell Signaling Technology), and GAPDH (1:1000, ab9485, Abcam).

Xenograft tumor model

BALB/c nude mice (20 ± 2 g) were purchased from Chengdu Dossy Experimental Animals co., Ltd. All mice were housed in Specific Pathogen Free (SPF) conditions with free access to food and water. The protocols were approved by the ethics committee of our hospital (GPTAP001). The mice were injected subcutaneously into the right hindlimb with 1 × 106 HCC827 cells. Thereafter, the mice were divided into five groups when the longest tumor diameter reached 6–8 mm (day 10). Mice received RT at 8 Gy [21, 22] on days 10, 12, and 14 using an RS 2000pro Biological X‑ray Irradiator (Rad Source). Thus, the total radiation dose was 24 Gy. For radiotherapy, the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate solution at 80 mL/kg (bodyweight) and then secured to a cardboard plate with medical tape. A 15 mm thick protective lead plate with holes was placed on the mice. The size of the lead plate hole is slightly larger than the size of the tumor hole, which ensures exposure of the tumor and protection of the animal organs. At the same time, different doses of AJ (low dose: 5 mg/kg; middle dose: 10 mg/kg; high dose: 20 mg/kg) were administered to the mice by oral gavage once a day for 15 days (days 10–24). Tumor size was measured every 5 days using a vernier caliper and calculated as volume (mm3) = length × width2/2. The mice were anesthetized with chloral hydrate and sacrificed to collect tumors at the 25th day for weighing.

Bioinformatic analysis

The active components of Ampelopsis japonica (AJ) were analyzed in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database [23] (https://old.tcmsp-e.com/tcmsp.php). The screening criteria were oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. DrugBank database (https://www.drugbank.ca/unearth/advanced/bio_entities) was used to analyze the targets of the active components of AJ. For the search of lung cancer-related genes, enter “lung cancer” on the GeneCards website (https://www.genecards.org/) to search for lung cancer-related genes and download the gene list. A Venn diagram was used to obtain the overlapping genes between lung cancer-related genes and the targets of AJ, and a total of 71 genes were obtained. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of these 71 genes was performed using R software (R Foundation, Vienna, Austria) to explore the mechanism of AJ in NSCLC.

Statistical analysis

The results were shown as mean ± standard deviation (SD) of three independent experiments, each preformed in triplicate. The data were analyzed using GraphPad Prism 8.0 (GraphPad Software, CA, USA). The differences among multiple groups of data conforming to a normal distribution were analyzed using one-way ANOVA followed by Tukey’s test; the differences among multiple groups of data that did not conform to a normal distribution were analyzed using the Kruskal–Wallis test followed by Dunn’s test. P < 0.05 was considered statistically significant.