An active ingredient isolated from Ganoderma lucidum promotes burn wound healing via TRPV1/SMAD signaling

GLSO preparation

Spores were collected from the fruit body of G. lucidum, which grew in the Dabie Mountain of An-Hui Province, and treated with wall-breaking procedures at good-manufacturing-practice (GMP) manufacturing facilities of Guangdong Yuewei Edible Fungi Technology Co. Ltd., Guangzhou, China. The lipid fraction of the spores was isolated by an extremely high-pressure machine using liquid CO₂. The main constituent compounds of GLSO were tested by high-performance liquid chromatography (HPLC) analysis. The methods were provided in our previous publications [10, 16].

Animals and experiments

Animals

All animal studies were conducted in accordance with the Guidelines for the Use and Care of Laboratory Animals of the National Institutes of Health and the Animal Welfare Act Regulations. Each in vivo experiment was approved by the Ethics Committee of Guangdong Institute of Microbiology. The mice (strain ICR mice) were purchased from the Jinan Pengyue Experimental Animal Breeding Company (Shandong, China). Male mice aged 7–8 weeks with body weight 32 ± 3 g, were used for the studies. Each mouse was maintained in a constant environment (20–22° C, 12-h light-dark cycle) with a normal diet, and individually housed in a cage to prevent further damage to the wounds. In this study, the experimental protocols were approved by the Committee of the Guangdong Institute of Microbiology Animal Center (Permit Number: SYXK(YUE)-2021-0156).

Wound creation and treatments

The burn wound creation on the mouse skin and the procedure to handle the mice have been described by us, previously [10]. In brief, the mice were randomly categorized into three groups (n=9 for each group): sham group, control group, and GLSO treated. Mice were anesthetized by intraperitoneal injection and shaved at the middle back prior to introducing the burn wound. Mice in the sham group underwent shaving only. Mice in the control group underwent skin wounding, while those in the GLSO group received GLSO treatment upon the burn wound to test the effect of GLSO on wound repair. A homemade device consisting of a hollow rubber tube with 2 cm internal diameter was used to produce burn wound in the animal by placing it in direct contact with the skin in the shaved area. Following wounding with boiling water, 0.1 mL pure GLSO was applied on the wound twice daily for up to 5 days in the GLSO-treated group. The mice were, then, sacrificed by cervical dislocation at days 1, 3, and 5 (n=3 per group per day) to collect wounded skin and serum samples at these time points. Subsequently, each wounded skin sample was divided into three parts for further analyses.

Tissue analysis

The tissues were subjected to Sirius Red staining. To do so, the collected tissue samples contained the unwounded normal skin as well as the wounded skin area to allow the identification of the wound edge for subsequent analysis. The tissues were fixed with formalin and cut into 3-μm sections for Sirius Red staining following an already established lab protocol [10]. Images were captured using a polarizing microscope (Nikon, Tokyo, Japan). The ratio of collagen I to collagen III was calculated using Image-pro Plus 6.0 (Media Cybernetics, MD, USA) by scanning the number of clearly stained pixels for the corresponding collagen-stained area.

To perform immunofluorescent staining, all tissues covering the normal unwounded skin area and the wounded area were fixed with formalin, embedded in paraffin, and cut into 3-μm sections. These sections were deparaffinized through a graded series of dimethylbenzene and ethanol. Antigens were retrieved by incubation in citric acid buffer (pH 6.0, ab93678, Abcam) at 100° C for 20 min. The sections were incubated with 3% H₂O₂ for 25 min to block endogenous peroxidase activity. Cell samples were fixed with 4% paraformaldehyde for 30 min and treated with permeabilizing agent 0.2% Triton X-100 (301G056, Solarbio, Beijing, China) for 10 min. The skin tissues and cell samples were incubated with 10% goat serum albumin at room temperature for 25 min to block non-specific binding. Subsequently, these sections were incubated with primary antibody to SMAD 2/3 (diluted in PBS, 1:200, CST, 8685s) and TRPV1 (diluted in PBS, 1:100, abs134462, Absin) at 4° C overnight. Following washing with PBS thrice, the sections were incubated with goat anti-rabbit immunoglobulin-G immunofluorescence antibody (diluted in PBS, 1:200, 4412S, Cell Signaling Technology, MA, USA) at room temperature for 50 min. The sections were washed again, and incubated with 4’,6-diamidino-2-phenylindole (DAPI, G1012, Servicebio, Wuhan, China) at room temperature for 5 min. Images were captured using an immunofluorescence inverted microscope (EVOS™ FL Auto 2 Imaging System, Shanghai, China).

Cell activity assays

Human keratinocyte line HaCaT cells were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). The cells were cultured in Dulbecco’s modified eagle medium (DMEM) (C11095500BT, Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS, 10099-141, Gibco) at 37° C in a humidified 5% CO₂ atmosphere.

In cell proliferation assay, HaCaT cells (1×10⁴ cells per well) were seeded in 24-well tissue culture plates in DMEM supplemented with 3% FBS for 24 h. Except for the control group, the cells were divided into four groups and treated with different GLSO concentrations: 20, 40, 60, and 80 μg/mL for 24, 48, and 72 h. At each time point, the cells were counted using a cell counter (IC 1000, Countstar, Shanghai, China). The optimal GLSO concentration for cell proliferation was determined in each of the four groups.

To test the effects of TRPV1 on cell proliferation, HaCaT cells (1×10⁴ cells per well) were plated and pre-incubated with TRPV1 inhibitor (0.0258 μg mL^-1 SB705498, Absin, Shanghai, China) for 3 and 16 h. The cells were subsequently treated with 60 μg/ml GLSO for 48 h. Finally, the cells were divided into two parts; one part was fixed and observed under a standard optical microscope, followed by cell number determination, while the other part was used for immunofluorescence assay for TRPV1 and SMAD 2/3 signaling.

In cell migration assay, the in vitro culture scratch assay was adopted as a wound healing model to test the effect of GLSO on HaCaT migration/wound healing process. The cells (1×10⁶ cells per well) were grown in six-well tissue culture plates for 24 h. Following cell adherence, vertical and horizontal cross-shape scratches were created in each well by scraping the cell layer with a 10-μL pipette tip. The wells were washed three times with phosphate-buffered saline (PBS) to clear any detached cells, and divided into four groups. Apart from the control group, the cells were treated with different GLSO concentrations: 20, 60, and 80 μg per ml for 24, 48, 72, and 96 h. Cell migration in the scratch area was photographed and the closure rates of scratch area were calculated using Image-pro Plus 6.0.

Network pharmacology analysis

We performed a network analysis to identify potential targets of GLSO. First, possible GLSO targets were searched using an extensive literature. Second, the two-dimensional (2D) molecular structures of the main compounds characterized by HPLC were searched on PubChem, and the molecular similarity match tool based on the simplified molecular input line entry specification (SMILES) in SwissTargetPrediction (P<0.05) was used to identify the potential proteins targeted by GLSO. Third, we used the keyword “burn” to search relative targets of burn in the Online Mendelian Inheritance in Man (OMIM) database and GeneCards. Fourth, an interaction network for the main compounds and putative GLSO targets for burn treatment was constructed based on their interaction data using protein-protein interaction (PPI) by STRING (Version 11.0), and visualized using Cytoscape software (Version 3.7.2, National Resource for Network Biology, USA). The degree, betweenness centrality, and closeness centrality were analyzed using the Network Analyzer plugin in Cytoscape, which were then applied to determine the topological importance of nodes in the network. Finally, the putative targets of GLSO were used in Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis by Metascape to elucidate the important and related protein pathways.

Western blot assay

For western blotting, frozen skin tissues were ground in liquid nitrogen and lysed with ice-cold Radio-immunoprecipitation Assay (RIPA) buffer (R0278, Sigma, Shanghai, China). The supernatant was collected after centrifugation of the tissue lysate at 12,000 rpm in a refrigerated centrifuge (5804R, Eppendorf, Shanghai, China) at 4° C for 10 min, followed by the measurement of protein concentrations using a BCA assay kit (Thermo Fisher Scientific, NY, USA). Protein samples (40 μg per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Millipore Immobilon, Darmstadt, Germany). The membranes were blocked with 5% skimmed milk in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature for 2 h, and incubated with primary antibodies diluted in PBS against SMAD2/3 (8685s, CST, Shanghai, China), p-SMAD2/3 (8828s, CST), and β-actin (ab52866, Abcam, Shanghai, China) at 4° C overnight. These membranes were, then, washed three times with TBST for 10 min each and incubated with horseradish peroxidase-conjugated secondary antibody (ab6721, Abcam) at room temperature for 1 h. Protein signals were detected using electro-chemiluminescence (ECL) reagent (Thermo Fisher Scientific).

SMAD 2/3 nuclear importation on lipopolysaccharide (LPS) stimulation

HaCaT cells were stimulated with LPS to verify the mechanism of GLSO in accelerating skin wound healing by SMAD 2/3 signaling. HaCaT cells (1×10⁴ cells per well) were seeded in 24-well tissue culture plates for 24 h. Cells were divided into four groups; two of which were treated with 1 μg/ml LPS for 16 h. Subsequently, the GLSO (no pre-LPS) and GLSO+LPS groups were treated with 60 μg/ml GLSO. After 24 h, the cells were collected for immunofluorescence assay to examine SMAD 2/3 nuclear translocation in each group, as described below.

Statistical analysis

The results were presented as the mean ± standard deviation (SD). Normal distribution and statistical comparisons between groups were determined by one-way analysis of variance and least-significant difference test using SPSS version 19.0 (IBM, NY, USA). For statistical testing, the variance between each group was defined using the probability value P and values of P < 0.05 were considered to be statistically significant.

GLSO accelerated collagen fiber regeneration in skin burn wounds

In this study, we isolated the bioactive component of Ganoderma lucidum and examined its roles in promotion of burn wound repair. The procedure is shown in the diagram (Figure 1). Sirius Red staining was performed to determine the collagen I to III ratio in skin wounds treated with GLSO. Treated with picrosirius solution (0.1% Sirius Red in saturated aqueous picric acid, pH 2.0), the tissue sections would display collagen I as red-orange, while collagen III appearing as pale-green shades. As observed under a polarizing microscope, the Sirius Red stained sections showed little difference on day 1 and day 5 of the skin in the sham group (Figure 2A). In the GLSO treated group, there was more collagen I content (red area) than collagen III content (green area) in the GLSO group relative to the untreated (control) group (Figure 2B). Quantitative analysis of Sirius Red staining indicated that the ratio of collagen I to III in the GLSO group was significantly increased compared with the control group (Figure 2C).

Figure 1. Workflow for GLSO treatment of skin burn injury.

Figure 2. The effect of GLSO on the ratio of collagen I to collagen III in skin burn in mice. (A) Sirius Red staining of the sham mouse skin observed using a polarizing microscope. Scale bar=200 μm. (B) Sirius Red staining of the mouse skin treated with or without GLSO. Scale bar=200 μm. (C) Quantitative analysis of the ratio of collagen I to collagen III was performed (n=3 per group). The data are presented as the mean ± SD. ^*P<0.05 versus sham.

GLSO promoted HaCaT cell proliferation and migration

HaCaT cells are human epidermal cells and are widely used to verify the effect of medicines. In the present study, we found that HaCaT cell proliferation was significantly increased 48 h after starting treatment of the wound with 60 μg/ml GLSO relative to the control group (Figure 3A, 3B). The cell migration assay also indicated that 60 μg/ml GLSO decreased the scratch distance and significantly increased the wound closure compared with the control group (Figure 3C, 3D).

Figure 3. The proliferation and migration effects of GLSO on HaCaT cells. (A) Images of HaCaT cell growth with or without GLSO treatment. (B) Quantitative analysis of HaCaT cell proliferation with GLSO at different time points. (C) Images of HaCaT cell migration with or without GLSO treatment. Scale bar, 1 mm. (D) Quantitative analysis of wound closure by HaCaT cell migration with GLSO treatment. The data are presented as the mean ± SD. ^*P<0.05, ^**P<0.01 versus control.

Network pharmacology analysis about GLSO on burn related targets

Trilinolein, 1,2-linolein-3-olein, 1,2-linolein-3-palmitin, 1,2-olein-3-linolein, 1-palmitin-2-olein-3-linolein, triolein, 1,2-olein-3-palmitin, and 1,2-olein-3-stearin are the main compounds of GLSO revealed by HPLC analysis. Their 2D structures are presented in Figure 4. The burn-associated molecules are shown in Figure 5A left panel and the potential GLSO targets are presented in Figure 5A right panel. Among these, no potential targets for trilinolein, 1,2-linolein-3-palmitin, or 1,2-olein-3-stearin were found in the SwissTargetPrediction. However, for the remaining five main GLSO components, 196 candidate targets related to burn injury were found from the database of OMIM and GeneCards. Four targets were found to be overlapping between them (Figure 5A middle), which were TRPV1, cannabinoid receptor 1 (CNR1), prostaglandin-endoperoxide synthase 2(PTGS2), and nitric oxide synthase 2 (NOS2). Regardingly, considerable amount of literature shows the transforming growth factor (TGF)-β signaling pathway to be important in skin wound healing [17]. With a PPI of four coincident targets and the TGF-β signaling pathway, we identified 31 targets as the putative targets of GLSO for burn disease treatment, and found that TRPV1 was an important protein among potential targets of GLSO related to burn disease. The results of the network dataset showed that SMAD2, SMAD3, SMAD4, SMAD7, and TGF-β1 were the most important proteins among the putative targets of GLSO. TGF-β3 was also important but had less influence than the aforementioned proteins (Figures 5B, 4C). Enrichment analysis of putative targets was performed and visualized by KEGG. The analysis showed that the TGF-β signaling pathway was the most important pathway for GLSO activity in burn treatment (Figure 5D). Comprehensively, the network analysis revealed that GLSO possibly improved burn wound healing by interacting with TRPV1 and the TGF-β signaling pathway.

Figure 4. 2D structures of the GLSO main ingredients.

Figure 5. Network pharmacology analysis of GLSO drug-ingredients’ targets. (A) left, Burn disease-associated molecules; right, Potential targets of GLSO; middle, overlap of left and right. (B) Network analyzer analysis of GLSO drug-ingredients’ targets. (C) GLSO-ingredients-disease-targets network. (D) KEGG analysis of potential GLSO targets.

GLSO had a significant effect on SMAD2/3 and TRPV1 in vitro and in vivo

SMAD2 and SMAD3 were the most important proteins, characteristic of the TGF-β signaling pathway. Therefore, we examined SMAD2/3 and TRPV1 levels in the following experiments. Western blot analysis showed that SMAD2/3 phosphorylation levels were significantly increased, whereas SMAD2/3 expression levels were decreased upon GLSO treatment compared with the control group (Figure 6A). The results of immunofluorescence staining showed that GLSO clearly increased TRPV1 expression in skin burn wounds compared with the control group (Figure 6C). HaCaT cells stimulated with GLSO were used to simulate a burn model in vitro. GLSO accelerated SMAD2/3 nuclear translocation, and clearly promoted this translocation upon LPS stimulation (Figure 6B).

Figure 6. Analysis of SMAD2/3 and TRPV1 expression in mouse skin burn. (A) Western blot (upper) and quantitative analyses (lower) of SMAD2/3 and p-SMAD2/3. (B) Immunofluorescence staining of SMAD2/3 nuclear translocation on LPS (1 μg/mL) induction. Scale bar=200 μm. (C) Immunofluorescence staining of TRPV1 expression in skin burn upon GLSO treatment. Scale bar=200 μm. The data are presented as the mean ± SD. ^*P<0.05 versus sham.

GLSO promoted HaCaT cell proliferation by the interaction between SMAD2/3 and TRPV1 signaling

Finally, to verify whether TRPV1 influenced the skin burn wound healing by increasing SMAD2/3 expression, which were the most important signaling proteins in the TGF-β pathway, HaCaT cells were pre-incubated with TRVP1 inhibitor for 3 h or 16 h, and then incubated with GLSO for 48 h (Figure 7A, 7B). The GLSO enhancement of cell proliferation was significantly downregulated by the TRPV1 inhibitor. Immunofluorescence analysis showed that the addition of the TRPV1 inhibitor clearly decreased TRPV1 and SMAD2/3 expression compared with GLSO alone (Figure 7C).

Figure 7. Effect of TRPV1 inhibitor on HaCaT cell proliferation. (A) HaCaT cells were pre-incubated with TRPV1 inhibitor (0.0258 μg/mL) for 3 and 16 h, followed by further incubation with GLSO for 48 h. GLSO-promoted cell proliferation was downregulated by TRPV1 inhibitor. (B) Quantitative analysis of reduced HaCaT cell numbers treated with TRPV1 inhibitor. (C) Immunofluorescence staining for TRPV1 and SMAD2/3 expression treated with TRPV1 inhibitor. Treatment with GLSO increased TRPV1 and SMAD2/3 expression that were down regulated by TRPV1 inhibitor. Scale bar=200 μm. The data are presented as the mean ± SD. *P<0.05, **P<0.01 versus control.