5.1 Molecular docking

The docking studies were performed using Molecular Operating Environment software (MOE, 2015.10) and the detailed procedure was described earlier [39]. Briefly, the three-dimensional structure of the target β2AR was downloaded from RCSB PDB (6PS5) and further modified by removing the heteroatoms and water molecules in the MOE software. 642129 molecules to be tested were collected from ZINC 15 database and transferred to MOE to carry out the docking within the β2AR’s active site. In addition, propranolol, a known β adrenergic receptor inhibitor, was selected as a positive control. Its structure was obtained from ChemicalBook (www.chemicalbook.com). The binding effect of the compounds with β2AR was evaluated through their binding scores. Based on the docking results, the promising natural inhibitor of β2AR were further selected to proceed molecular dynamics simulation.

5.2 Molecular dynamics simulation

For understanding the structural-basis of the function mechanism between the selected inhibitors and the protein targets, an initial coordinate for 100 nsec all-atom molecular dynamics simulation was carried out using a GROMACS 2020.6-MODIFIED software and a CHARMM36 force field. The ligand–protein complex was immersed in a cubic box with three-points (TIP3P) water and followed by the addition of counter ions to neutralize the solvated system. Then, the complex system was quickly energy minimized using the steepest descent minimization algorithm. Following this, two-staged equilibration with NVT (Number of particles, Volume and Temperature) and NPT (Number of particles, Temperature and Pressure) was conducted and ensemble for 100 psec. The equilibrated systems were proceeded to molecular dynamic run for 100 nsec under a constant number of particles at 310 K and 1 bar pressure. The results were analyzed and determined for root mean square deviation (RMSD).

5.3 Cell culture

Human A2058 and murine B16F10 melanoma cell lines were both cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; BI) and 1% penicillin–streptomycin at 37°C with 5% CO2.

5.4 Adrenergic receptors’ expression pattern analysis

To make sure whether the melanoma cell lines are suitable for testing the efficiency in blocking β2AR, adrenergic receptors’ expression pattern of these two cells were analyzed using the transcriptome data. Datasets GSE150348 of A2058 cells and datasets PRJNA505989 of B16F10 cells were obtained from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra), respectively [40, 41]. Expression levels of genes were normalized and visualized by pheatmap R package. Besides, the single cell transcriptome datasets of GSE129611 and GSE129218 from GEO were also used to figureout the expression pattern of adrenergic receptors in human and mouse melanocytes [42, 43]. The quality control standard for each dataset was based on the original studies when available. After that, the cell cluster annotations were accomplished depending on the cell makers reported in the previous studies and visualized using the t-distributed Stochastic Neighbor Embedding (t-SNE) method. Average and log-normalized expression levels for adrenergic receptors were plotted using the DotPlot function in the Seurat R package.

5.5 Blocking efficiency assay by western blot

To investigate the inhibitory effect of the selected natural inhibitors on NA signal transduction, A2058 and B16F10 melanoma cells were seeded in 6-well plates and incubated at 37 °C and 5% CO2 for 24 h. Then, the culture medium was exchanged with a fresh FBS-free medium containing different concentrations of propranolol or the selected compound rhynchophylline, and incubated at 37 °C and 5% CO2 for 30 min. After that, 100 µM NA (Sigma, #489350) was added for an additional incubation of 48 h. The inhibition assay was further conducted and detected using western blot.

The treated cells were lysed with a RIPA buffer (Beyotime, P0013) containing the protease (CWBIO, CW2200S) and phosphatase inhibitors (NCM, P003) on ice. Then, the protein was extracted by ultrasonic disintegration for three times and centrifugation at 4°C, 2000 g for 15 min. Followed by the extraction, the protein concentration in the supernatants was measured using a BCA Protein Assay Kit (Beyotime, P0009) according to the manufacturer’s instructions. After that, the prepared sample was separated by polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% skim milk powder (dilution: TBST) at room temperature for 1 h, and incubated with primary antibodies overnight at 4°C (dilution: TBST): anti-p-PKA Substrate (Cell Signaling Technology, 9624S, 1:1000), anti-GAPDH (GeneTex, GTX100118, 1:5000). On the following day, the membranes were incubated with HRP-linked secondary antibodies (Cell Signaling Technology, 7074P2, 1:1000) at room temperature for 1 h and visualized using a WesternBright™ ECL (Advansta, K-12045-D50).

5.6 Transdermal permeation experiment

Transdermal permeation assays were conducted in the vertical Franz-type diffusion cells (DISA, Milan, Italy). In detail, the mice dorsal skin was placed between the donor and acceptor chamber of the Franz cell, and the dermal surface was in contact with the receptor compartment. 4 mL transdermal formulation of propranolol (1 mg/mL; Sigma-Aldrich, PHR1308) and rhynchophylline (1 mg/mL; YuanYe, B20453) dissolved in the solvent (1, 2-propylene glycol: absolute ethanol: peppermint ketone: ddH2O = 50:32:5:13) was set as the donor. 50 mL 1% PBS solution was as the acceptance media and maintained at 37°C by the constant stirring. Then, the experiments were carried out for 24 h. To evaluate the permeation efficiency, 1 mL of the acceptance solution for each test were taken at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h and measured using Ultra-micro-UV spectrophotometer (nanodrop N60).

5.7 Prevention efficiency of hair-graying on resiniferatoxin (RTX)-induced Stress Mice

C57BL/6 female mice of 7 weeks old were housed under specific pathogen-free animal center with 12 h light–dark cycle and allowed for free access to water and diet. For the establishment of stress-induced hair graying models, RTX (AdipoGen Life Sciences, AG-CN2-0534) was prepared in the PBS containing 2% DMSO with 0.15% Tween 80 and was injected in the flank of the depilated mice at 20 µg/kg for 7 days. For the inhibition group, 200 µL 1 mg/mL propranolol or rhynchophylline were applied to the skin of mice twice a day, 1 h before and 11 h after the RTX injection, respectively. After the treatment, the hair graying area of the mice were photographed at day30 and measured using ImageJ software. All the animal experimental procedures were conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the European Community guidelines (EEC Directive of 1986; 86/609/EEC) and Use Committee of Sun Yat-sen University (approval no. SYSU-YXYSZ-20210332).

5.8 RNA sample preparation and sequencing

The final concentrations of NA and rhynchophylline chosen for the treatment of A2058 cells for RNA sequencing were 100 µM, which was determined based on the results of above inhibition assay. Further, a higher concentration of NA with 500 µM was also conducted to determine whether rhynchophylline could counteract the effects of NA at an elevated concentration. A2058 melanoma cells were treated with different concentrations of NA alone or with rhynchophylline as described above for 48 h. To collect the cell samples for RNA extraction, the culture medium was discarded, and the cell was washed with PBS to remove residual medium. Next, 1 mL of Trizol reagent (Life Technologies) was added, and the mixture was vigorously pipetted to ensure complete contact and digestion of Trizol with the cells. The dissolved cell lysate was transferred to a new RNase-free centrifuge tube, where further pipetting was carried out until all cells were dissolved. The samples were sent to Chi Biotech (Guangzhou, China) for RNA extraction. Following this, its purity was assessed using Nanodrop by measuring the OD260/280 and OD260/230 ratios, while its integrity was evaluated using the Agilent 4200 TapeStation.

For mRNA library preparation, 1 µg of total RNA was utilized as the input material per sample. Initially, poly A-tailed eukaryotic mRNA was enriched using the Oligo (dT) magnetic beads. Subsequently, the enriched mRNA was randomly fragmented employing bivalent cations. To synthesize the first strand of cDNA, reverse transcriptase was utilized with segmented mRNA acting as a template, and random oligonucleotides being employed as primers. The second strand of cDNA was then synthesized using 2nd Strand Enzymes and dNTPs. The resulting double-stranded cDNA was subjected to end repair, A-tailing addition and sequencing adapter ligation to generate cDNA fragments of around 200–300 bp in size. PCR amplification was performed, followed by additional purification steps to obtain the final library. To evaluate the quality of the prepared libraries, the Agilent 4200 TapeStation was utilized. After passing the library quality assessment, the prepared libraries were sequenced on a NovaSeq 6000 platform. During the sequencing process, paired-end reads of 150 bp in length were generated, providing valuable sequence information for subsequent analysis.

5.9 RNA data analysis

The raw data in Fastq format obtained from sequencing were subjected to data processing using custom Perl scripts. This preprocessing step involved trimming reads containing adapters, poly-N sequences, or low-quality bases, resulting in clean reads. Subsequently, the paired-end clean reads were aligned to the reference genome database hg38. After the alignment, read counts mapped to each gene were obtained using the featureCounts (version 2.0.3) program [44]. Then, transcripts Per Kilobase of exon model per Million mapped reads (TPM) values were calculated. Before performing differential gene expression analysis, the read counts were normalized using the edgeR (version 3.18.1) program package through scaling factors [45,46,47]. Once the read counts were normalized, the differential expression analysis between two conditions was then carried out. The resulting p-values were adjusted using the Benjamini & Hochberg method for multiple testing. The threshold for significant differential expression was set at a corrected p-value of 0.05 and an absolute fold change of 2. Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, Gene Set Enrichment Analysis (GSEA) of differentially expressed genes were conducted using the clusterProfiler R package (version 4.22) with correction for gene length bias [48, 49].

5.10 Melanogenesis assay

To make sure the effect of NA and rhynchophylline on the melanogenesis, the expression levels of melanocyte stem cells-related genes MITF and DCT were detected by analyzing the RNA-seq data and western blot: anti-MITF (Cell Signaling Technology,97800S, 1:1000), anti-DCT (Invitrogen, PA5-105275, 1:500), anti-β-actin (Cell Signaling Technology, 4970S, 1:1000), anti-GAPDH (GeneTex, GTX100118, 1:5000).

5.11 Calcium signals detection

Aimed to investigate the effects of NA and rhynchophylline on calcium oscillations, A2058 and B16F10 cells were cultured on confocal culture dishes for 24 h. Fluo-4 AM fluorescent calcium probe (Beyotime, S1060, 2 mM) was then added and incubated with the cells for 30 min at 37°C. Following three washes with PBS, the cells were further incubated in Hanks' solution for an additional 30 min. To assess the impact of inhibitors, rhynchophylline was administered 30 min prior to NA addition. Subsequently, NA was added to the dish and the images were captured in a "time series" mode using a confocal laser scanning microscope (Zeiss, LSM880), and continuously for 300 s with 3-s intervals.

5.12 Cell morphology observations

To investigate the impact of NA and rhynchophylline on the cell morphology, the cells were initially seeded onto a culture dish with a diameter of 35 mm at a density of 1 × 105 cells/dish and sub-cultured for 24 h. Subsequently, the culture medium was replaced with fresh medium containing varying concentrations of NA and rhynchophylline. The cellular morphology was then observed and images were captured at different time points.

5.13 Flow cytometry

An Annexin V kit (Yeasen, 40302ES60) was used for the analysis of apoptosis. A2058 and B16F10 cells treated with NA alone or with rhynchophylline were digested with trypsin without EDTA for 1 min. Following that, the cells were centrifuged at 300g, 4 °C for 5 min and washed with cold PBS twice. Subsequently, 1 × binding buffer was added and resuspend the cells. To perform the apoptosis assay, 5 µL of Annexin V-FITC and 10 µL of PI staining solution were further added and incubated with the resuspended cells at room temperature for 10–15 min. The prepared samples were analyzed within 1 h using a flow cytometer (Beckman, CytoFLEX).

5.14 Statistical analysis

All data were analyzed using Graphpad Prism 9 (GraphPad Software). One-way analysis of variance (ANOVA) with a post-hoc Tukey's test was used for statistical comparison of the groups. Statistical significance was set at a p-value (* < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001).