Platycodin D inhibits the proliferation and migration of hypertrophic scar-derived fibroblasts and promotes apoptosis through a caspase-dependent pathway

Hypertrophic scar (HS) samples were collected from patients of the Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, and written consent was provided. None of the patients had received treatments before surgery. The subcutaneous fat tissue was removed, and the HSs samples were immersed in 0.25% dispase II (Roche, Germany) at 4 °C overnight. The dermis was then separated from the epidermis and digested with 0.25% collagenase I (Sigma, USA) at 37 °C for 4–6 h. The isolated HSFs were cultured in complete high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, USA). Cells from the third passages (P3) were used in all experiments [18].

HSFs were seeded in 6-well plates at a concentration of 1 × 10⁵/mL in 2 mL of complete medium (CM) per well. A total of 15 wells were divided into five groups (n = 3), and each group was treated with 0, 2, 4, 6 or 8 μM PD. A total of 18 wells were divided into six groups (n = 3), and each group was treated with PD (0 μM), TGF-β1 (5 ng/mL), TGF-β1 (5 ng/mL) + PD (2 μM), TGF-β1 (5 ng/mL) + PD (4 μΜ), TGF-β1 (5 ng/mL) + PD (6 μM) or TGF-β1 (5 ng/mL) + PD (8 μΜ). PD was purchased from Yuanye Bio-Technology (Shanghai, China) and dissolved in ddH₂O to prepare a 10 mM stock solution. Human TGF-β1 (Peprotech, USA) was diluted to prepare a 20 ng/mL stock solution.

HSFs were treated with different concentrations of PD for 24 h and lysed with TRIzol reagent (Invitrogen, CA, USA), and total RNA was extracted and purified. cDNA was obtained by reverse transcription of 1000 ng of RNA. The sequences of the primers used in the present study were as follows: ColA1 forward: 5'-GAGCGGTAACAAGGGTGAGC-3', reverse: 5'-CGGTGGTTTCTTGGTCGGT-3'; Col3A1 forward: 5'-TTGAAGGAGGATGTTCCCATCT-3', reverse: 5'-ACAGACACATATTTGGCATGGTT-3'; α-SMA forward: 5'-GTGTTGCCCCTGAAGAGCAT-3', reverse: 5'-GCTGGGACATTGAAAGTCTCA-3'; TGF-β1 forward: 5'-GGCCAGATCCTGTCCAAGC-3', reverse: 5'-GTGGGTTTCCACCATTAGCAC-3'; GAPDH forward: 5'-ACAACTTTGGTATCGTGGAAGG-3', reverse: 5'-GCCATCACGCCACAGTTTC-3'. The gene expression levels of Col I, Col III and α-smooth muscle actin (α-SMA) were determined using the TB GREEN Premix Ex Taq^™ kit (Takara, Japan) on an ABI QuantStudio6 Pro (Applied Biosystems USA), and gene expression levels were normalized to GAPDH. Three samples were extraction and independently performed three replicates of the qRT-PCR experiment.

After 48 h of treatment with different concentrations of PD, HSFs were lysed in radioimmunoprecipitation assay (RIPA) buffer for 30 min on ice. After lysis was complete, the liquid was collected and centrifuged for 10 min. The supernatant was used to measure the protein concentration (BCA protein assay kit, Thermo Fisher Scientific, USA). Extracted protein (20 mg) was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After being blocked with 5% bovine serum albumin (BSA), the membrane was incubated overnight at 4 °C with primary antibodies. The primary antibodies were anti-type I collagen (Col I), anti-α-SMA, anti-matrix metalloproteinase 2 (MMP2), anti-type III collagen (Col III), anti-TGF-β1, anti-TGF-β receptor I (TGFβ RI), anti-phospho-Smad2/3 (p-Smad2/3) and anti-Smad2/3 (all from Cell Signaling Technology, USA). The next day, the PVDF membrane was incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA) on a slow shaker at room temperature for 1 h. Then, the membrane was washed 3 times and analyzed with Odyssey V3.0 image scanning (Li-COR. Inc., Lincoln, NE, USA). β-actin (Cell Signaling Technology, USA) was used as a loading control.

Briefly, HSFs were seeded in 96-well plates (3 × 10⁴/mL and 100 μl/well) and treated with 0, 2, 4, 6, 8 or 10 μM PD. Five replicate wells were used for each concentration. After cells adhered and stabilized, the corresponding concentration of PD was added for 0, 12, 24, 48 and 72 h. CCK-8 solution (10 μl; Dojindo, Japan) was then added to each well and incubated for 2 h at 37 °C in the dark. Cell viability was quantified by measuring the absorbance at 450 nm with a microplate reader (Biotek, Vermont, USA).

HSFs were seeded in 6-well plates at a density of 5 × 10⁵/well in CM. When the cells reached confluence, the medium was replaced with DMEM containing 1% serum for 12 h. A scratch wound with a uniform width was made, and the floating cells were washed away with sterile phosphate-buffered saline (PBS). The HSFs in each well were treated with 0, 2, 4, 6 or 8 μM PD and cultured for 24 h. The size of the scratch was recorded with an inverted microscope (Nikon, Japan) at 0, 6, 12 and 24 h [39].

HSFs (1 × 10⁵/mL and 200 μl of DMEM) were added to the upper chamber of Millicell Hanging Cell Culture Inserts (Millipore, USA), and 700 μl of CM containing different concentrations of PD (0, 2, 4 or 8 μM) was added to the lower chamber. After 24 h of incubation, the chamber was removed and fixed with 4% paraformaldehyde for 5 min, gently washed 3 times and stained with crystal violet for 10 min. Cells in the upper layer of the chamber were gently wiped away with cotton swabs, and cells in the lower layer of the chamber were quantified by taking pictures under a microscope (Nikon, Japan).

After cells adhered and stabilized, they were treated with 0, 2, 4, 6 or 8 μM PD for 48 h. Cells were then digested with trypsin and washed with cold PBS. Apoptosis was detected using the Alexa Fluor^® 488 annexin V/dead cell apoptosis kit (Invitrogen, USA) and flow cytometry (Beckman, USA), and the results were analyzed by FlowJo software version 10 (FlowJo, LLC, Ashland, Oregon, USA).

HSFs (1 × 10⁵/well) were seeded on cover slips and treated with PD (0 μM), TGF-β1 (5 ng/mL) or TGF-β1 (5 ng/mL) + PD (8 μM) for 48 h. Cells were then fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 (Sigma, USA) for 20 min and blocked with 5% goat serum (Gibco, USA) for 1 h. Slides were incubated with rabbit monoclonal anti-α-SMA antibodies (Abcam, UK) at 4 °C overnight, and on the next day, they were incubated with Alexa Fluor 488 goat anti-rabbit IgG secondary antibodies (Abcam, UK) for 1 h at room temperature. Slides were then sealed with DAPI Fluoromount-G (SouthernBiotech, USA), and a confocal microscope (Nikon, Japan) was used to analyze the fluorescence intensity.

Animal experiments and surgical operations were performed in accordance with the “Guide for Care of Laboratory Animals” outlined by the National Ministry of Science and were approved by the Animal Ethical Committee of Jiagan Biotechnology Co., Ltd (Approval number: JGLL-20200620). Twelve New Zealand white rabbits (6–8 weeks old and weighing 2.5–3.0 kg) were used, and all animals were raised and processed using standard procedures.

Anesthesia and analgesia were induced before the operation. Atropine sulfate (0.05 mg) was subcutaneously injected, and 5 mg/kg Zoletil^® 50 (Virbac SA) was injected into the ear vein 15 min later. The auxiliary analgesic and muscle relaxant, xylazine hydrochloride (1 mg/kg), was then injected intramuscularly. An 8 mm biopsy punch was used to establish four full-thickness dermal defects on the ventral sides of both ears, and the perichondrium was removed to completely expose the cartilage. At 24 h post-operation, each wound on the left ear of the rabbit was injected with normal saline as a control, while the right ear was injected with different concentrations of PD as follows: low concentration group (8 μM in 100 μl, n = 6) and high concentration group (40 μM in 100 μl, n = 6). The injections were performed in four equal locations around the wounds.

Animals were fed normally after the surgery. After 14 or 28 days of continuous PD administration, animals were sacrificed using air embolization under anesthesia state, and full-thickness scars were obtained. Calipers were used to measure the maximum protuberant heights of HSs and normal skin. Wounds were evaluated by the scar elevation index (SEI) [6], which is the ratio of the total thickness of the wound to that of normal tissue.

After the samples were fixed with 4% paraformaldehyde for 24 h, they were rinsed with running water. After dehydration in graded ethanol, the paraffin-embedded tissue was cut into 5 μm-thick sections. Tissue sections were stained with Sirius red stain (Fluka, Switzerland) and observed with a polarized upright microscope (Nikon, Japan). ImageJ software was used to measure and analyze the collagen fibers.

Immunohistochemistry (IHC) was performed to examine proliferating cell nuclear antigen (PCNA) and α-SMA levels. After deparaffinizing the section, pepsin (Sigma, USA) was used for antigen retrieval at 37 °C for 30 min. Then, 3% H₂O₂ was applied to block endogenous peroxidase activity. Mouse monoclonal anti-PCNA antibodies and anti-α-SMA antibodies (Abcam, UK) were added and incubated at 4 °C overnight. Sections were then incubated with goat anti-mouse IgG-HRP (Invitrogen, USA) for 1 h at room temperature. Finally, sections were stained with a DAB detection kit (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin.

To study the inhibitory effects of PD on fibrosis and collagen deposition, HSFs were treated with 0, 2, 4, 6 or 8 μM PD for 3 days. The differences in fibrosis-related genes were analyzed by qRT-PCR and Western blotting, respectively. The qRT-PCR results showed that PD dose-dependently downregulated the mRNA levels of Col I, Col III and α-SMA, and the effect was significant at 6 or 8 μM PD (Fig. 1B–D) (p < 0.05). This trend was also verified by Western blotting (Fig. 1E). In response to 6 μM PD, the protein expression levels of Col I and Col III were significantly downregulated (Fig. 1F and G) (p < 0.05). However, the protein expression of α-SMA only showed a declining trend in response to 8 μM PD (Fig. 1H) (p < 0.05).

The effects of different concentrations of PD on HSFs were analyzed by CCK-8 assays. As shown by the cell proliferation curves, PD inhibited cell proliferation in a dose-dependent manner at concentrations ranging from 6 to 10 μM (Fig. 2A) (p < 0.05). Next, the HSF migration affected by PD were examined by wound healing and Transwell assays. The results of the wound healing assay showed that the migration of PD-treated HSFs was inhibited at 12 and 24 h (Fig. 2B). After quantitative analysis, the wound healing percentages in the PD-treated groups were significantly lower than those in the control group (Fig. 2C) (p < 0.05). The Transwell results indicated that the cell migration in the PD treatment group was decreased compared to that of the control group (Fig. 2D) as indicated by the quantitative analysis (Fig. 2E) (p < 0.05). Flow cytometric analysis showed that PD promoted HSFs apoptosis (Fig. 2F), and the proportion of apoptotic cells gradually increased from 2.7 to 31.84% with increasing concentrations of PD (Fig. 2G) (p < 0.05).

Studies have shown that HSs show relatively high expression of α-SMA; and TGF-β1 can induce α-SMA expression in HSs [19]. To study the effect of PD on the function of TGF-β1 in HSFs, cells were treated with PD (0 μM), TGF-β1 (5 ng/mL), TGF-β1 (5 ng/mL) + PD (2 μM), TGF-β1 (5 ng/mL) + PD (4 μM), TGF-β1 (5 ng/mL) + PD (6 μM), or TGF-β1 (5 ng/mL) + PD (8 μM), and the results showed that PD inhibited the effect of TGF-β1 on α-SMA activation. Immunofluorescence staining (Fig. 3A) showed that the expression of α-SMA induced by TGF-β1 was significantly reduced by high concentrations of PD. qRT-PCR and Western blotting (Fig. 3B and C) showed similar results, and the effects were most significant at 8 μM PD (Fig. 3D) (p < 0.05).

The effect of PD was evaluated on a rabbit ear scar model in vivo. The images of the scars and the SEI score were recorded on days 14 and 28 (Fig. 4A). The results showed that compared with the control group, scars treated with a high concentration of PD (40 μM) were alleviated, and the SEI was also significantly reduced (Fig. 4B) (p < 0.05). The number of PCNA-positive cells in the scar samples from the high concentration (40 μM) PD treatment group was significantly reduced on days 14 and 28 (Fig. 4C). A significant difference was observed after 28 days of treatment rather than 14 days (Fig. 4D) (p < 0.05). The Sirius red staining showed a distribution of Col I in the scar samples (Fig. 4E), which was significantly greater on day 28 compared to day 14. However, Col I was markedly inhibited after treatment with low and high concentrations of PD (Fig. 4F) (p < 0.05). Compared to the control group (Fig. 4G), IHC staining and quantitative analysis of α-SMA showed that HSFs proliferation and angiogenesis in the scar area were significantly decreased in the high concentration PD group (Fig. 4H) (p < 0.05).

To explore the molecular mechanism of the effects of PD on the proliferation, migration and apoptosis of HSFs, the expression of related proteins was analyzed. Firstly, PD inhibited the activation of the Smad2/3 pathway in HSFs (Fig. 5A). With increasing concentrations of PD, the phosphorylation of Smad2/3 gradually decreased (Fig. 5B–C) (p < 0.05), and the expression of TGF-β RI also decreased in a dose-dependent manner (Fig. 5D) (p < 0.05). However, at high concentration (10 μM) of PD, the protein expression of TGF-β1 was inhibited significantly (Fig. 5E) (p < 0.05). In addition, the mRNA expression of TGF-β1 increased slightly at low PD concentration and decreased at high PD concentration (p < 0.05), which was consistent with the protein expression of TGF-β1 (Fig. 5F). Moreover, PD inhibited the protein expression of MMP2 (Fig. 5G), which is an important protein related to cell migration, but the inhibition of MMP2 did not change significantly with the increase of PD concentration (Fig. 5H) (p < 0.05). Then the protein expression of caspase 3, caspase 9 and Bcl-2, which are important proteins related to cell apoptosis, were analyzed (Fig. 5I). The result showed that the protein expression of caspase 3 and caspase 9 increased slowly with increasing concentrations of PD (Fig. 5M–N) (p < 0.05). Interestingly, barely detectable levels of Bcl-2 were found at low PD concentration, but these levels increased significantly at high PD concentration (Fig. 5L) (p < 0.05). At the same time, the expression of p65 decreased at high PD concentration (Fig. 5J) (p < 0.05), but the expression of AKT was not affected (Fig. 5K).