Nigella sativa L. seed extracts promote wound healing progress by activating VEGF and PDGF signaling pathways: An in vitro and in silico study

Chemicals

Bovine serum albumin (BSA) (cat. no. 23209), HUVECs (cat. no. C0035C) and NHDFs (cat. no. C0135C) used in this study were purchased from Thermo Fisher Scientific Inc. Dulbecco’s Modified Eagle Medium (DMEM) (cat. no. D6429), TRIS-buffered saline (TBS) (cat. no. SRE0071) and fetal bovine serum (FBS) (cat. no. F7524) were purchased from Sigma-Aldrich. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from BioVision Inc. (cat. no. 2808). Anti-VEGF mouse monoclonal antibody was procured from Santa Cruz Biotechnology, Inc., (cat. no. sc-53462). Anti-PDGF Receptor β polyclonal antibody (produced in rabbit) was purchased from Sigma-Aldrich (cat. no. SAB4502149). Anti-β-actin mouse monoclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (cat. no. sc-69879). Enhanced Chemiluminescence Detection (ECL) kit was obtained from Amersham BioSciences UK Ltd (cat. no. RPN2209).

Collection of materials

N. sativa seeds were purchased from a local herbal shop in Bangkok, Thailand. After being cleaned with tap water, they were dried under shade conditions, powdered, and air-tight packaged in a container.

Extract preparation

In order to determine the yield on the plant material, 3,000 mL petroleum ether, chloroform, ethyl acetate, and ethanol were continuously shocked with 600 g plant material in a conical flask for 72 h (during the cold percolation process). After the extracts were collected and filtered using Whatman No. 1 filter paper, a rotary evaporator set at 40°C was used to dry them. In order to preserve the dried extracts until further use, they were stored at 4°C until use.

GC-MS analysis and phytocompounds identification

Gas Chromatography-Mass Spectrometer Model Shimadzu GCMS-QP2020 NX (Shimadzu, Japan) equipped with 5 Sil MS 5% diphenyl/95% dimethyl polysiloxane capillary column (measuring 30 mm wide, 0.25 mm diameter, and 0.25 mm thick) was used analyze the extracts of N. sativa seeds. Then, 100 μl of solvent extracts were diluted using 1,400 μl dimethyl sulfoxide (DMSO). Next, 1 μl diluted sample (100/1,400, V/V in DMSO) was injected in the split mode with a split ratio 1:10. Electron impact ionization was used for GC-MS detection with an ionization energy of 70 eV. A low flow rate of 1.0 mL per min of helium at a low pressure was used as the carrier gas in the column. Before the injector temperature was set at 250°C, 60°C was set for 15 min before gradually increasing to 280°C over 3 min. It was conducted at 70 eV with a scanning distance of 0.5 s as well as fragment sizes ranging between 50 Da and 650 Da for the MS analysis, 40 min were spent on the GC operation. Acquisition mode scan ranged from 35 m/z to 500 m/z with scan speed 2,500. Extracts were analyzed for their percentage composition of compounds. NIST20R and Wiley libraries were used to interpret and compare GC-MS data as well as compare retention indices.²⁴

In vitro cell line study

Cell lines and culture

HUVEC and NHDF cell lines were maintained at 37°C during the experiment using a humidified atmosphere containing 5% CO₂. DMEM supplemented with 10% FBS and 1% antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) was used to grow the cells in T-25 flasks. Trypsinization and passage were performed once the cells reached 70% confluency.

Cell viability analysis

In culture media, 10 mg/mL stock solutions of plant extracts were diluted in DMSO. To determine cell viability, cells were seeded into 96 well plates at a density of 5x10³ cells per well and incubated at 37°C and 5% CO₂ for 24 h. Fresh DMEM supplemented with various solvent extracts of N. sativa seeds (petroleum ether, chloroform, ethyl acetate and ethanol) (0, 10, 20, 50, 100 μg/mL) was added, and incubation was carried out for 24 h. After the incubation with extracts, cells were incubated for 2 h in growth media (DMEM) containing 20% MTS solution to assess viability. Microplate readers were used to measure the absorbance of formazan at 490 nm. The crude extracts were dissolved in 0.5% DMSO, which represents the highest concentration of DMSO used in the vehicle culture medium.

Protein expression analysis by western blotting

Laemmli (1970) described sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a method for separating proteins.²⁵ Using equal volumes (50 g) of samples and buffer, sample mixtures were heated at 95°C for 4 min, then cooled on ice. The dye front was reached at the bottom of the running gel after separating proteins with a Bio-Rad mini slab gel apparatus at a constant voltage of 100 V. In this experiment, polyvinylidene difluoride (PVDF) membranes were charged at a constant voltage of 100 V for 1 h in order to transfer protein bands. Incubation with primary antibodies (anti-VEGF (mouse monoclonal antibody 200 μg/mL) and anti-PDGF Receptor β (rabbit polyclonal antibody 100 μg/mL); β-actin (mouse monoclonal antibody 100 μg/mL) at appropriate dilutions followed by blocking with 5% BSA blocking solution at room temperature for 1 h. A secondary antibody (goat anti-mouse monoclonal antibody 400 μg/mL), purchased from Santa Cruz Biotechnology, Inc., (cat. no. sc-2005; 1:10,000) was incubated for 1 h after primary antibody incubation. Incubation with secondary antibody was followed by two washes (5 min each) with Tris-buffered saline, Tween (TBS-T) and placement on Saran Wrap™ (protein-side up). After adding detection reagent mixture to the blot, blots were incubated for 30-60 sec and we drained off excess reagent (ECL). Quantity One 1-D Analysis Software (RRID:SCR_014280) (Bio-Rad) was used to quantify the immunoblot signals. Using a probe consisting of β-actin, similar amounts of proteins were loaded onto the membranes.

In silico analysis

Selection and preparation of ligands

Through GC-MS analysis, a total of 268 phytocompounds were identified in four different extracts of N. sativa. The 3D structures of all the identified compounds were extracted from the PubChem database (RRID:SCR_004284).²⁶ A list of phytocompounds identified are provided in Tables 1-4 as Underlying data.⁴² Using PyRx software (RRID:SCR_018548) with default parameters, energy minimization of each ligand was performed using universal force fields, followed by Gasteiger charges to achieve a good structural conformation for docking.

Selection and preparation of receptors

As part of this study, six different proteins such as TNFα, TGFBR1 kinase, IL-1β, PKC-βII, VEGF and PDGF that participate in wound healing were selected, and their crystal structures were retrieved from Protein Data Bank (PDB). Using Chimera 1.16 (RRID:SCR_002959), any missing residues in the selected target proteins were modelled, nonstandard hetero atoms were removed, polar hydrogens and Gasteiger charges were added, and then energy minimization of each protein performed with 100 steepest descent gradient steps using amber force field (Amber ff14SB). Finally, the energy minimized protein was converted into pdbqt format for molecular docking.

Protein-ligand docking

The Autodock Vina (RRID:SCR_011958) was used for the molecular docking of phytocompounds of N. sativa with selected wound healing target proteins. If the ligand binding site is represented, it will be located at the center of the grid box. A value of eight is set for the exhaustiveness of the model. A configuration file was created based on the dimensions of the XYZ axis determined by Discovery studio’s visualizer. In Autodock Vina 1.1.2, this configuration file was used for docking using the command line. To dock ligands with a degree of flexibility, Autodock Vina uses the Monte Carlo algorithm. Monte Carlo algorithm used in Autodock Vina is relatively faster than other docking programs.²⁷ In addition to the results file, the binding modes were generated as a single file (PDBQT format) in a log format. BIOVIA Discovery Studio (RRID:SCR_015651) visualizer was used to analyze the binding interactions between best docked ligands and receptors. Strong hydrogen bonds (2.2 to 2.5), moderate hydrogen bonds (2.5 to 3.2), and weak hydrogen bonds (up to 3.6) were measured with respect to the hydrogen atom of the heavy atom.

ADME properties prediction

QikProp (RRID:SCR_014906) module was used to predict ADME properties (Schrodinger Suite 2022). To determine a ligand’s pharmacokinetics and pharmacodynamics, the QikProp module analyses its properties, which are resembling those of a drug. Several ADME properties were considered significant, including the molecular weight (MW), H-bond donor, H-bond acceptor, and logarithm of n-octanol/water partition coefficient (log P (O/W)).

Statistical analysis

Data were analyzed using GraphPad Prism (RRID:SCR_002798) version 5 software to assess the significance of individual variations between the control and treatment groups by one-way analysis of variance (ANOVA) and Duncan’s multiple range test. Approximately P<0.05 was considered significant in Duncan’s test.

GC-MS analysis

GC-MS analysis identified a total of 268 phytocompounds in N. sativa seed extracts (Figure 1).⁴² Petroleum ether, chloroform, ethyl acetate and ethanolic extracts showed 65, 70, 67 and 66 peaks, respectively, which are indicating the presence of phytocompounds (Tables 1-4 in Underlying data⁴²). Among these, the highest peak levels were observed such as, 66.81% linoleic acid (PubChem CID: 5280450) at 33.908 min retention time (petroleum ether extract), 42% cis-vaccenic acid (PubChem CID: 5282761) at 33.343 min retention time (chloroform extract), 29.24% ethyl palmitate (PubChem CID: 12366) at 30.050 min retention time (petroleum ether extract), 20.09% oleic acid (PubChem CID: 445639) at 34.550 min retention time (petroleum ether extract), 16.72% palmitic acid (PubChem CID: 985) at 29.904 min retention time (chloroform extract), 16.57% tetradecanoic acid (PubChem CID: 11005) at 25.974 min retention time (petroleum ether extract), 16.37% 3-(3-Chlorophenyl)imidazolidine-2,4-dione (PubChem CID: 285803) at 31.375 min retention time (ethanolic extract), 15.83% methyl linoleate (PubChem CID: 5284421) at 32.22 min retention time (chloroform extract), 15.34% adaphostin (PubChem CID: 387042) at 30.733 min retention time (ethanolic extract), 14.07% glyceryl diacetate 2-oleate (PubChem CID: 5363238) at 31.897 min retention time (ethanolic extract), 12.62% 2-linoleoylglycerol (PubChem CID: 5365676) at 31.502 min retention time (ethanolic extract), 11.34% monopalmitin (PubChem CID: 14900) at 28.131 min retention time (ethyl acetate extract), 11.14% (z)-tetradec-7-enal (PubChem CID: 5364468) at 2.3 min retention time (ethyl acetate extract), 10.54% glycerol, 2-octadecanoate, diacetate (PubChem CID: 539925) at 33.703 min retention time (ethanolic extract), 10.52% glyceryl diacetate 1-linolenate (PubChem CID: 6434505) at 35.075 min retention time (ethanolic extract), 10.45% olealdehyde (PubChem CID: 5364492) at 36.797 min retention time (ethyl acetate extract), 10.25% 2,3-dihydroxypropyl acetate (PubChem CID: 33510) at 10.419 min retention time (ethyl acetate extract), 10.23% 16-trimethylsilyloxy-9-octadecenoic acid, methyl ester (PubChem CID: 6421149) at 34.547 min retention time (chloroform extract), 10.08% propyl ester (PubChem CID: 221069) at 34.71 min retention time (ethyl acetate extract).

Figure 1. Analysis of different solvent extracts of N. sativa seeds using GC-MS.

Effect of N. sativa seeds on cell viability of NHDFs and HUVECs

To check the cytotoxicity of N. sativa seed extracts, two different normal cell lines (NHDF and HUVECs) were used at the different concentrations of crude extracts. Cell viability percentages were plotted against extracts treatment concentrations to obtain treatment-response curves. A concentration-dependent increase in cell viability was observed with N. sativa seed extracts (Figures 2 and 3). In response to 25-50 μg/mL of ethanolic and chloroform extract on both cell lines, the viability of cells were increased by 60-68% following 24 h treatment (Figures 2 and 3).

Figure 2. Effect of N. sativa seed extracts on cell viability in HUVEC cells.

Cells were cultured in DMEM supplemented with 10% FBS were incubated with indicated concentrations of extracts (0–100 μg) for 24 h. Each bar represents the mean ± SEM of six independent observations. Significance was considered as the levels of p < 0.05 level using Duncan’s multiple range test. a - compared to control; b - compared to DMSO control; c - compared with 10 μg treated cells; d - compared with 25 μg treated cells.

Figure 3. Effect of N. sativa seed extracts on cell viability in NHDF cells.

Cells cultured in DMEM supplemented with 10% FBS were incubated with indicated concentrations of extracts (0–100 μg) of 24 h. Each bar represents the mean ± SEM of six independent observations. Significance was considered at p < 0.05 level using Duncan’s multiple range test. a - compared to control; b - compared to DMSO control; c - compared with 10 μg treated cells; d - compared with 25 μg treated cells.

Protein expression analysis

Effect of N. sativa seed extracts on VEGF and PDGF protein expression in NHDF cells

The effect of different solvent extracts of N. sativa seeds on VEGF and PDGF protein expression in NHDF cell lines were investigated. Incubation for 24 h with extracts of indicated concentrations was conducted in DMEM supplemented with 10% FBS using NHDF cells. Densitometry analysis was used to calculate protein expression, which is expressed in relative intensity. Internal control was performed using β-Actin. Based on five independent observations, each bar represents the mean and standard error of the mean. The significance level was determined by using Duncan’s multiple range test at p < 0.05. VEGF and PDGF expression levels were comparatively increased at 25 μg/mL by both ethanolic and chloroform extracts (Figure 4).

Figure 4. Effect of different N. sativa seed extracts on VEGF and PDGF protein expression in NHDF cells.

Protein expression was analyzed by western blotting using specific antibodies. Each bar represents the mean ± SEM of five independent observations. Significance was considered as the levels of p < 0.05 level using Duncan’s multiple range test. a, compared with control; b, compared with ethanolic extract (25 μg) treated cells; c, compared with chloroform extract (25 μg) treated cells; N. sativa, Nigella sativa L.; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; NHDF, normal human dermal fibroblast; P. E, Petroleum Ether extract; CHLO, Chloroform extract; E. A, Ethyl Acetate extract; ETOH, Ethanolic extract.

Effect of N. sativa seed extracts on VEGF and PDGF protein expression in HUVEC cells

Effect of different solvent extracts on VEGF and PDGF protein expression in HUVEC lines. Incubation of HUVECs with indicated concentrations of extracts for 24 h was performed in DMEM supplemented with 10% FBS. Densitometry analysis was used to quantify protein expression, which is expressed as relative intensity. An internal control was performed using β-actin. Five independent observations are represented by a bar with a mean and standard error of measurement. The Duncan’s multiple range test was used to determine significance at p < 0.05. At a concentration of 25 μg/mL, ethanolic and chloroform extracts of N. sativa seed led to comparatively significant increases in the expression levels of VEGF and PDGF proteins (Figure 5).

Figure 5. Effect of different N. sativa seed extracts on VEGF and PDGF protein expression in HUVEC cells.

Protein expression was analyzed by western blotting using specific antibodies. Each bar represents the mean ± SEM of five independent observations. Significance was considered as the levels of p < 0.05 level using Duncan’s multiple range test. a, compared with control; b, compared with ethanolic extract (25 μg) treated cells; c, compared with chloroform extract (25 μg) treated cells; N. sativa, Nigella sativa L.; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; HUVEC, human umbilical vein endothelial cell; P. E, Petroleum Ether extract; CHLO, Chloroform extract; E. A, Ethyl Acetate extract; ETOH, Ethanolic extract.

In silico analysis

In the present study, Autodock Vina was used to predict the binding affinity of a total of 268 selected phytocompounds of N. sativa with the target proteins of wound healing process such as TNFα (PDB ID: 2AZ5), TGFBR1 kinase (PDB ID: 6B8Y), IL-1β (PDB ID: 6Y8M), PKC-βII (PDB: 2I0E), VEGF-A (PDB ID: 3QTK) and platelet-derived growth factor receptor alpha (PDGFRA) (PDB ID: 6JOL). The binding energies of 10 ligands that showed the highest binding affinities are indicated in the heatmap (Figure 6). It is clear from Figure 6, three compounds (1, 2, and 3) namely tricyclo[20.8.0.0(7,16)]triacontane, 1(22),7(16)-diepoxy- (PubChem CID: 543764), adaphostin (PubChem CID: 387042), and obeticholic acid (PubChem CID: 447715), respectively, showed highest binding affinity with all the tested target proteins. The best docked protein ligand interactions are shown in Table 1 and Table 2. The 2D and 3D structures of the top docked complexes are shown in Figures 7-9. ADME properties of those compounds were under the acceptable range (Table 3).

Figure 6. Heat map representation of top protein ligand docking analysis.

Figure 7. Results of molecular docking analysis.

Molecular docking analysis of tricyclo[20.8.0.0(7,16)]triacontane, 1(22),7(16)-diepoxy- complexed with targeted proteins of A) PKC-βII, B) TNFα, C) IL-1β, D) PDGFRA, E) VEGF-A and F) TGFBR1.

Figure 8. Results of molecular docking analysis.

Molecular docking analysis of adaphostin complexed with targeted proteins of A) PKC-βII, B) TNFα, C) IL-1β, D) PDGFRA, E) VEGF-A and F) TGFBR1.

Figure 9. Results of molecular docking analysis.

Molecular docking analysis of obeticholic acid complexed with targeted proteins of A) PKC-βII, B) TNFα, C) IL-1β, D) PDGFRA, E) VEGF-A and F) TGFBR1.