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Fraxinellone-mediated targeting of cathepsin B leakage from lysosomes induces ferroptosis in fibroblasts to inhibit hypertrophic scar formation

Biology Direct volume 20, Article number: 17 (2025) Cite this article

Abstract

Background

Hypertrophic scar (HS) is a common fibrotic skin disorder characterized by the excessive deposition of extracellular matrix (ECM). Fibroblasts are the most important effector cells involved in HS formation. Currently no satisfactory treatment has been developed.

Methods

The impact of fraxinellone (FRA) on the proliferation and migration capacity of human hypertrophic scar-derived fibroblasts (HSFs) was assessed by EdU proliferation, wound healing and transwell assays. Quantitative real-time PCR (qRT‒PCR), Western blot (WB), immunofluorescence staining and collagen gel contraction assays were performed to evaluate the collagen production and activation capacity of HSFs. Oxford Nanopore Technologies long-read RNA sequencing (ONT long-read RNA-seq) revealed the occurrence of ferroptosis in HSF and ferroptosis executioner-cathepsin B (CTSB). The mechanisms underlying FRA-induced HSF ferroptosis were examined through fluorescence staining, qRT‒PCR, WB and molecular docking study. The therapeutic efficacy of FRA was further validated in vivo using a rabbit ear scar model.

Results

FRA treatment significantly suppressed the proliferation, migration, collagen production and activation capacity of HSFs. ONT long-read RNA-seq discovered that FRA modulated the expression of transcripts related to ferroptosis and lysosomes. Mechanistically, FRA treatment reduced the protein expression level of glutathione peroxidase 4 (GPX4) and induced the release of CTSB from lysosomes into the cytoplasm. CTSB further induced ferroptosis via spermidine/spermine-N1-acetyltransferase (SAT1)-mediated lipid peroxidation, mitochondrial damage and mitogen-activated protein kinase (MAPK) signalling pathway activation, eventually affecting the function of HSFs. Moreover, FRA treatment attenuated the formation of HS in rabbit ears via CTSB-mediated ferroptosis. The antifibrotic effects of FRA were abrogated by pretreatment with a CTSB inhibitor (CA-074-me).

Conclusions

This study reveals that FRA ameliorates HS by inducing CTSB leakage from lysosomes, causing SAT1-mediated lipid peroxidation, mitochondrial damage and MAPK signalling pathway activation, thus mediating HSF ferroptosis. Therefore, FRA could be a promising therapeutic agent for treating HS.

Introduction

Hypertrophic scar (HS) is a common fibrotic skin disorder that mainly develops from skin injuries deep into the dermis, such as burns, surgeries and other traumatic lesions, and presents as a tough and hyperaemic bulge accompanied by varying degrees of itching or pain [1]. HS not only affects appearance but also leads to skin dysfunction, which causes physical and psychological impairments and seriously affects the quality of life of patients [2]. Currently, several clinical interventions are recommended for the treatment of HS, such as surgical resection, intralesional corticosteroid injection, pressure and laser therapy [3]; however, these treatment strategies fail to satisfy clinical needs due to the high recurrence rate and limited therapeutic efficacy [4].

Although various cell types have been implicated in the pathogenesis of HS, the most important effector cells are fibroblasts [1]. In response to numerous pathological stimuli, including mechanical stress, chronic inflammation, and transforming growth factor-β (TGF-β) signalling, fibroblast activation occurs, resulting in invasive proliferation and the excessive secretion of type I and type III collagen, thus leading to disordered deposition of the extracellular matrix (ECM) [5,6,7]. Furthermore, the differentiation of fibroblasts to myofibroblasts results in scar contracture, eventually leading to the pathological remodelling of the skin architecture and functional impairment [1]. Thus, preventing persistent fibroblast activation and suppressing myofibroblast function appears to be a promising approach for preventing HS [8, 9].

Fraxinellone (FRA) (structure shown in Fig. 1A), a type of limonoid isolated from the root bark of Dictamnus dasycarpus, possesses a wide range of biological properties, such as anti-inflammatory [10], anticancer [11] and neuroprotective functions [12]. However, the antifibrotic effect of FRA on HS has not been reported, and the exact molecular mechanism involved remains elusive.

Fig. 1
figure 1

FRA suppressed the proliferation and migration of HSFs in vitro. (A) Chemical structure diagram of FRA. (B) HSFs and NDFs were treated with different concentrations of FRA for different durations and evaluated by a CCK-8 assay (n = 4). (C) EdU (green) proliferation assay for HSFs after incubation with various concentrations of FRA (100 µM, 200 µM and 300 µM) for 48 h (scale bar = 200 μm). (D) Quantitative analysis of EdU-positive cells. (E) Representative images of the wound healing assay of HSFs treated with or without FRA (300 µM) for 0, 6, 12 and 24 h (scale bar = 200 μm). The red lines indicate the wound boundaries. (F) The quantitative results are presented as the relative migration area, with 0% applied to the area measured at 0 h. (G) Representative images of the transwell assay of HSFs after treatment with FRA for 48 h (scale bar = 200 μm). (H) Quantitative results of the number of invaded cells per field. The data are shown as means ± SDs (n = 3 independent experiments unless otherwise specified). *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant

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As a novel form of programmed cell death, ferroptosis is characterized by iron-dependent lipid peroxidation-mediated plasma membrane rupture [13]. Morphologically, ferroptotic cells exhibit mitochondrial disorder, including atrophy, densification and the absence of cristae [14]. Mechanistically, ferroptosis has several key features, such as iron overload, the excessive accumulation of lipid peroxides and the downregulated expression of glutathione peroxidase 4 (GPX4), a ferroptosis-protective gene [15]. Apart from the above essential factors, several studies have shown that lysosomes and their internal hydrolases, such as cathepsin B (CTSB), are also involved in the execution of ferroptosis [16, 17]. CTSB, a lysosomal cysteine protease, plays a crucial role in autophagy and apoptosis [18]; however, research on its role in ferroptosis is limited. Although the underlying molecular mechanisms of ferroptosis have not been completely elucidated, ferroptosis is closely related to the development and control of various fibrotic diseases [19,20,21,22]. Triggering ferroptosis in myofibroblasts may be effective in treating scarring [4, 23].

In this study, we found that FRA induced CTSB leakage from lysosomes and CTSB could induce ferroptosis via spermidine/spermine-N1-acetyltransferase (SAT1)-mediated lipid peroxidation, mitochondrial damage and mitogen-activated protein kinase (MAPK) signalling pathway activation to affect the behaviour of fibroblasts. In vivo validation studies were conducted using a rabbit ear model, and the results showed that FRA mitigated abnormal scar formation and improved collagen fiber arrangement. Collectively, our findings indicate that FRA can induce fibroblasts ferroptosis via CTSB to attenuate HS formation.

Materials and methods

Cell isolation and culture

Primary human hypertrophic scar-derived fibroblasts (HSFs) and normal skin-derived fibroblasts (NDFs) were isolated following the protocol described in our previous study [24]. The isolated cells were cultured in complete high-glucose Dulbecco’s modified Eagle medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin streptomycin (Gibco, USA). Cells passaged to the 3rd -6th generations were used for all experiments.

Cell counting kit-8 (CCK-8) assay

Cell viability was evaluated using a CCK-8 assay kit (Beyotime, China). Briefly, HSFs or NDFs were seeded in 96-well plates, after receiving the indicated treatment, the culture medium was replaced with fresh medium containing 10% CCK-8 reagent, and the cells were incubated at 37 °C for 1 h. Then, the absorbance at 450 nm was measured by a microplate reader.

5-ethynyl-20-deoxyuridine (EdU) proliferation assay

Cell proliferation was detected using a BeyoClick™ EdU-488 Cell Proliferation Kit (Beyotime, China). In short, HSFs were incubated with EdU for 2 h, fixed with 4% paraformaldehyde (PFA), and incubated with an EdU Click reaction solution for 30 min. After the cell nuclei were stained with DAPI, fluorescence signals were captured with a Zeiss 710 laser-scanning microscope (Zeiss, USA).

Wound healing assay

A wound healing assay was used to assess the migration of HSFs. HSFs were seeded in 6-well plates to near confluence. A scratch wound was made in the middle of each monolayer using a sterilized 200-µl pipette tip. The HSFs were then treated with FRA. The scratched monolayers were photographed at 0, 6, 12 and 24 h. Finally, ImageJ software was used to measure the wound area.

Transwell assay

A transwell assay was performed to assess the migration ability of HSFs using an 8 mm polycarbonate membrane transwell chamber (Merck, USA). HSFs were added to the upper chamber and treated with FRA, while DMEM with 10% FBS was added to the lower chamber. After 24 h, the cells that migrated to the bottom surface of the membrane were fixed with 4% PFA for 5 min and stained with crystal violet for 10 min. Then, the number of migrated cells in six randomly selected fields was quantified under a microscope (Nikon, Japan).

RNA purification and quantitative real-time PCR (qRT‒PCR)

Total RNA from HSFs was isolated using TRIzol reagent (Invitrogen). cDNA was synthesized using a Prime Script RT Master Mix kit (Takara, Japan). The primers used in this study are listed in the Supplementary materials, Table S1. qRT‒PCR was performed with a TB Green Premix Ex Taq™ kit (Takara, Japan). The messenger RNA (mRNA) expression levels of target genes were calculated by the 2−ΔΔCt method and normalized to the expression level of glyceraldehyde phosphate dehydrogenase (GAPDH).

Western blot (WB)

HSFs were lysed in radioimmunoprecipitation assay lysis buffer (Beyotime, China) for 30 min on ice. The proteins were separated via SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore, USA). The primary antibodies used in this study are listed in the Supplementary materials, Table S2. Protein expression levels were quantified with ImageJ software.

Immunofluorescence staining

HSFs were seeded in confocal culture dishes and subjected to the corresponding treatments before they were fixed with 4% PFA for 15 min, permeabilized with 0.5% Triton X-100 (Sigma, USA) for 20 min and blocked with 5% donkey serum (Jackson, USA) for 1 h. After overnight incubation with the primary antibody at 4 °C, the cells were incubated with secondary antibodies at room temperature for 1 h. Followed by DAPI staining, Images were captured under a confocal fluorescence microscope (Leica). The mean fluorescence intensity was calculated using ImageJ software, i.e., integrated fluorescence intensity in the protein expression region divided by the area of the protein expression region.

Collagen gel contraction assay

HSFs were resuspended in 500 µl of collagen gel suspension (Shengyou, China) and seeded in 24-well plates. The plates were incubated at 37 °C for 30 min to allow collagen gel polymerization. Then 500 µl of culture medium was added to each well. The gels were slowly detached from the wells over time. The area of each collagen gel was photographed after 3 days and then quantified using ImageJ software.

Nanopore long-read RNA sequencing

HSFs treated with or without FRA (300 µM) for 48 h were subjected to ONT long-read RNA-seq. Three independent replicates per group were established. After RNA preparation and library construction, cDNA samples were sequenced using a PromethION sequencer (Oxford Nanopore Technologies, Oxford, UK). ONT long-read RNA-seq was performed by Wuhan Benagen Technology Co., Ltd. (Wuhan, China). The method we used for multiple comparison correction was Benjamini-Hochberg method, and the corrected P value, i.e., P-adjust, was used for the determination of differentially expressed genes (DEGs) and differentially expressed transcripts (DETs). Novel genes and transcripts were identified using gffcompare (version 0.12.1). Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) and gene set enrichment analysis (GSEA) were performed for all DEGs and DETs.

FerroOrange and C11-BODIPY staining

Total intracellular ferrous ion and lipid peroxides were assessed using a FerroOrange probe (Dojindo, Japan) and a C11-BODIPY probe (Invitrogen, USA). After receiving the indicated treatments, HSFs seeded in confocal culture dishes were incubated with FerroOrange working solution (1 µM) or C11-BODIPY working solution (10 µM) in the dark at 37 °C for 30 min. Then the HSFs were visualized under a confocal fluorescence microscope. The mean fluorescence intensity was calculated using ImageJ software.

Reactive oxygen species (ROS) assay

Intracellular ROS levels were visualized using a DCFH-DA fluorescent probe (Beyotime, China) [25]. Briefly, after the corresponding treatments, HSFs were cultured in medium with 10 µM DCFH-DA in the dark at 37 °C for 20 min. Confocal fluorescence microscopy was used to acquire images. The mean fluorescence intensity was quantified using ImageJ software.

MitoTracker staining

Mitochondrial morphology was evaluated using a MitoTracker Red fluorescent probe (Beyotime, China). After the indicated treatments, HSFs were stained with MitoTracker working solution (200 nM) in the dark at 37 °C for 30 min. Fluorescence images were captured using a confocal fluorescence microscope. Mitochondrial morphology was quantified using ImageJ software.

Mitochondrial membrane potential assay

A JC-1 fluorescent probe (Beyotime, China) was used to assess the mitochondrial membrane potential. After the indicated interventions, JC-1 staining working solution was added and HSFs were cultured in the dark at 37 °C for 20 min. Subsequently, the HSFs were imaged under a confocal fluorescence microscope. The mean fluorescence intensity was calculated using ImageJ software. The quantitative methods of all the above fluorescence experiments were as follows, i.e., integrated fluorescence intensity in the fluorescence region divided by the area of the fluorescence region to obtain relative fluorescence intensity.

Transmission electron microscopy (TEM)

TEM was conducted to evaluate mitochondrial ultrastructure. After the indicated treatments, the HSFs were fixed with 2.5% glutaraldehyde at 4 °C overnight. Next, the samples were dehydrated, embedded in epoxy resin, and incubated at 60 °C for 3 days. Ultrathin sections were prepared and stained with 2% uranyl acetate and lead citrate, and the mitochondrial ultrastructure was evaluated using a transmission electron microscope (Hitachi, Japan).

Malonaldehyde (MDA) level measurement

Intracellular MDA levels were measured using an MDA assay kit (Beyotime, China). After the indicated interventions, HSFs were lysed and added with the MDA working solution, then the mixed solution was heated at 100 °C for 15 min. After centrifugation at 1000 rpm at room temperature for 10 min, the supernatant was collected, and a microplate reader was used to measure the absorbance at 532 nm.

Molecular docking study

The binding of FRA to CTSB was assessed using molecular docking analysis. The three-dimensional structure of the FRA compound (CAS No. 28808-62-0) was prepared with the LigPrep module (Schrödinger, USA). The crystal structure of human CTSB (PDB ID: 8B4T) was prepared by the Protein Preparation Wizard (Schrödinger, USA). Residues Gln23, Cys29, His110, His111, His199 and Trp221 of CTSB were selected to generate the receptor grid. In silico molecular docking was performed using the standard precision module in Glide (Schrödinger, USA), the best pose was chosen for analysis, and structural figures were generated by UCSF Chimera.

Animal model

Eighteen male New Zealand white rabbits (6–8 weeks old, 2.0–2.5 kg) were used to assess the effect of FRA on scar formation. Briefly, the rabbits were anaesthetized by intravenous injection of pentobarbital (30 mg/kg). After shaving and disinfecting, four full-thickness wounds on the ventral side of each ear were created using an 8-mm biopsy punch, the perichondrium was completely removed using a scalpel, and the underlying cartilage was kept intact. Eighteen rabbits were randomly divided into three treatment groups and the treatments were administered starting on the 2nd day after surgery. Each wound on the left ear of the rabbits was injected subcutaneously with 100 µl of saline (0.9%) as a control, and each wound on the right ear was injected with 100 µl of FRA (100 µM or 300 µM) or FRA (300 µM) plus ferrostatin-1 (Fer-1, 2 µM) every other day until the 14th day after surgery. Both the saline, FRA and FRA plus Fer-1 were injected at the peripheries of each wound [8, 24, 26].

On the 14th and 28th days after surgery, three rabbits from each treatment group were randomly selected and sacrificed. The whole scar tissue and surrounding normal skin samples were harvested and fixed in 4% PFA.

Histological and immunohistochemistry (IHC) analysis

Fixed specimens were embedded in paraffin and then cut into 5 μm-thick sections. Hematoxylin and eosin (H&E) staining was conducted to assess scar formation, Masson’s trichrome and Sirius red staining were performed to investigate the distribution of the collagen fiber, while IHC was used to assess the expression levels of Ki-67, α-smooth muscle actin (α-SMA), GPX4, and CTSB. Images of stained sections were captured with a microscope, and the staining was quantified by ImageJ software. The scar elevation index (SEI) was calculated using the following formula: SEI = H/H0, where H represents the height of the scar, which was calculated from the highest point of the epithelium to the cartilage surface in the scar tissue, and H0 represents the height of the surrounding normal tissue, which was measured from the stratum corneum to the cartilage surface in the normal tissue.

Statistical analysis

Statistical analysis was carried out using GraphPad Prism 9.0. All the data are expressed as means ± standard deviations (SDs). Student’s t test was used to analyse the significance of differences between two groups, and one-way analysis of variance (ANOVA) was performed to analyse the variance among multiple groups. Sample sizes of cell and animal experiments were determined according to power analysis and our previous studies [8, 24], and on the basis of power analysis, sample sizes of immunohistochemistry were determined according to another study [27]. P < 0.05 was considered to indicate a significant difference (*P < 0.05, **P < 0.01, ***P < 0.001) between groups.

Results

FRA suppressed the proliferation and migration of HSFs in vitro

To investigate the effect of FRA on scar hyperplasia, a CCK-8 assay was performed, and we found that FRA treatment inhibited HSFs growth in a dose- and time-dependent manner but resulted in negligible change in cell proliferation of NDFs (Fig. 1B). Based on the above results, we chose concentrations ranging from 100 to 300 µM for subsequent experiments. An EdU proliferation assay showed that compared with the control (CTL) group, as the FRA dose increased from 100 to 300 µM, the percentage of EdU-positive cells decreased significantly (Fig. 1C-D). Next, a wound healing assay was used to assess whether FRA can abrogate HSFs migration. As shown in Fig. 1E-F, FRA significantly decreased the wound migration area of HSFs at 6, 12 and 24 h. Moreover, the transwell assay indicated that FRA markedly inhibited the migration of HSFs (Fig. 1G-H). Taken together, these results supported the antiproliferative and antimigratory effects of FRA on HSFs in vitro.

FRA affected collagen metabolism and HSFs activation in vitro

HS is characterized by the excessive deposition of collagen, including collagen I and collagen III, which is mainly synthesized and secreted by fibroblasts [1, 28]. As shown in Fig. 2A, FRA downregulated the mRNA expression of COL1A1, COL1A2 and COL3A1 in a concentration-dependent manner. WB and immunofluorescence staining results showed that FRA markedly decreased the protein levels of COL 1 and COL 3 (Fig. 2B-E). Matrix metalloproteinases (MMPs) and their inhibitors, i.e., tissue inhibitors of metalloproteinases (TIMPs), which participate in collagen degradation, are involved in the pathogenesis of HS [29]. As shown in Fig. S1A, FRA markedly upregulated MMP1, MMP2 and MMP11 mRNA expression. Interestingly, we found that the mRNA expression of TIMP1 and TIMP2 was upregulated, which might represent negative feedback to inhibit the upregulation of MMP expression. Moreover, FRA had no significant effect on the mRNA levels of MMP3 and MMP9 but induced the upregulation of MMP13 mRNA expression at 300 µM. WB analysis showed that FRA did not increase the protein levels of MMP9 and MMP13 (Fig. S1B-C). Activated fibroblasts, also known as myofibroblasts, contribute to collagen deposition and cicatricial contraction [30]. High expression of α-SMA was detected in myofibroblasts, qRT‒PCR, WB and immunofluorescence staining assays demonstrated that FRA significantly downregulated the TGF‒β1 (5 ng/ml)-induced increase in α-SMA mRNA and protein levels in a dose-dependent manner (Fig. 2F-J). Furthermore, the collagen gel contraction assay indicated that the enhancement of TGF-β1-induced HSFs contraction was notably attenuated with FRA treatment (Fig. 2K-L). In summary, these results indicated that FRA could affect the collagen metabolism of HSFs and suppress their activation.

Fig. 2
figure 2

FRA affected collagen metabolism and HSFs activation in vitro. (A) qRT‒PCR results of the mRNA levels of COL1A1, COL1A2 and COL3A1 in HSFs after treatment with FRA (100 µM, 200 µM and 300 µM) for 48 h. GAPDH served as the control. (B) The protein levels of COL 1 and COL 3 in HSFs incubated with FRA for 48 h were determined by WB analysis. (C) Quantification of COL 1 and COL 3 expression based on WB analysis. (D) Immunofluorescence staining for COL1A1 and COL3 in HSFs after treatment with FRA for 48 h (scale bar = 50 μm). (E) The statistical results of the relative fluorescence intensity of COL1A1 and COL3. (F) α-SMA mRNA expression in HSFs after incubation with TGF-β1 (5 ng/ml) and different concentrations of FRA (0 µM, 100 µM, 200 µM and 300 µM) for 48 h was determined by qRT‒PCR. (G) WB analysis of α-SMA expression in HSFs subjected to the indicated treatments for 48 h. (H) Quantification of the α-SMA protein level based on WB analysis. (I) Immunofluorescence staining for α-SMA and F-actin in HSFs treated with TGF-β1 (5 ng/ml) and FRA (0 µM or 300 µM) for 48 h (scale bar = 50 μm). (J) Quantification of the relative fluorescence intensity of α-SMA. (K) Representative images of the collagen gel contraction assay in different treatment groups on day 3. The dashed white lines indicate the areas of collagen gel. (L) Quantitative results of the collagen gel contraction assay. The data are presented as means ± SDs (n = 3 independent experiments). **P < 0.01, ***P < 0.001

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FRA activated ferroptosis in HSFs

To further explore the molecular mechanism underlying the antifibrotic effect of FRA, we performed Oxford Nanopore Technologies long-read RNA sequencing (ONT long-read RNA-seq) to identify DEGs. The experimental design and the process of HSFs preparation are schematically shown in Fig. 3A. Principal component analysis (PCA) and sample correlation analysis suggested that the expression of mRNAs significantly differed between HSFs treated with or without FRA (Fig. S2A-B). There were 576 significantly downregulated genes and 291 significantly upregulated genes (P. adjust < 0.05 and fold change > 2) in the FRA-treated cells, as illustrated in the volcano plot and heatmap (Fig. S2C-D). Furthermore, KEGG analysis indicated that the DEGs were enriched in the TGF-β signalling pathway, Hippo signalling pathway and ECM-receptor interaction (Fig. 3B), which are related to organ fibrosis [7, 31]. GO analysis also demonstrated that FRA treatment was associated with antifibrosis effects (Fig. 3C). Interestingly, KEGG analysis and GSEA revealed that ferroptosis was significantly activated in FRA-treated HSFs (Fig. 3B, D). We clustered the DEGs closely related to ferroptosis and generated a heatmap of the results (Fig. 3E). And the relationship among ferroptosis, ECM-related DEGs and enriched pathways was illustrated in a chord graph (Fig. 3F). In view of the above results, we speculated that ferroptosis occurred in FRA-treated HSFs.

Fig. 3
figure 3

FRA activated ferroptosis in HSFs. (A) Schematic illustration of the experimental design and the process of HSFs preparation for ONT long-read RNA-seq. (B) KEGG pathway enrichment analysis of the DEGs between the CTL and FRA (300 µM for 48 h)-treated groups. The red boxes indicate the signalling pathways of interest. (C) GO analysis of the DEGs in different groups. The red boxes indicate the signalling pathways of interest. (D) GSEA plot of the ferroptosis pathway based on ONT long-read RNA-seq. (E) Heatmap of ferroptosis-related DEGs between the CTL and FRA-treated groups. Red: high expression levels. Blue: low expression levels. (F) Chord graph of the relationships between the DEGs and enrichment pathways

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FRA affected HSFs behaviour by inducing ferroptosis

To test the above hypothesis, we first performed a CCK-8 assay and found that FRA-induced changes in cell viability were blocked by Fer-1 (Fig. S3A). Then qRT‒PCR was performed to verify the expression of ferroptosis-related molecules detected by RNA sequencing. As shown in Fig. 4A and Fig. S3B, the mRNA expression of the investigated genes was significantly upregulated after 24 h of FRA treatment and gradually decreased over time. After 48 h of FRA treatment, the mRNA levels of almost all the investigated genes were obviously higher than those in the CTL group, a finding that was consistent with the RNA sequencing results. Interestingly, the mRNA expression of GPX4 increased in the first two days after treatment, but the WB results showed that the protein level of GPX4 markedly decreased after 48 h. In addition, the protein level of heme oxygenase-1 (HO-1) increased, suggesting the occurrence of ferroptosis (Fig. 4B-C). Next, the level of MDA, the final product of lipid peroxidation, in FRA-treated HSFs was significantly elevated (Fig. 4D), while C11-BODIPY staining showed that FRA increased lipid peroxide accumulation in a dose-dependent manner (Fig. 4E, G). FerroOrange staining revealed ferrous iron overload in HSFs, and the fluorescence intensity increased sharply as the FRA concentration increased (Fig. 4E, G). Next, MitoTracker staining and JC-1 staining indicated that HSFs displayed a decrease in mitochondrial volume and membrane potential after FRA treatment (Fig. 4F, H). TEM revealed shrunken mitochondria accompanied by membrane rupture, decreased cristae and obvious vacuoles in the FRA-treated HSFs (Fig. 4I). Moreover, the treatment of HSFs with FRA also increased intracellular ROS levels in a concentration-dependent manner (Fig. S3C-D), while immunofluorescence staining showed that the protein level of GPX4 decreased markedly as the concentration of FRA increased (Fig. S3E-F). Taken together, our data indicated that FRA could induce ferroptosis in HSFs by decreasing GPX4 protein levels, accelerating ferrous iron overload, promoting lipid peroxide and ROS accumulation, and destroying mitochondrial structure and function.

Fig. 4
figure 4

FRA affected HSFs behaviour by inducing ferroptosis. (A) The mRNA levels of GPX4, HO-1, SAT1 and SLC7A11 in HSFs treated with FRA (300 µM) for 24, 48 and 96 h were measured using qRT‒PCR. GAPDH was used as the internal reference gene. (B) WB results of the expression of GPX4 and HO-1 in HSFs incubated with or without FRA (300 µM) for 48 h. (C) Statistical analysis of the relative protein expression of GPX4 and HO-1 in HSFs. (D) MDA levels in different groups at 48 h were quantitatively evaluated by an MDA assay kit. (E) HSFs were treated with FRA (100 µM, 200 µM and 300 µM) for 48 h. Representative images of FerroOrange staining (scale bar = 50 μm) and C11-BODIPY staining (scale bar = 50 μm). (F) Representative images of MitoTracker staining (scale bar = 50 μm) and JC-1 staining (scale bar = 50 μm). Quantitative analysis of the relative fluorescence intensity of ferrous iron, lipid peroxide (G), MitoTracker Red area and the aggregate/monomer ratio (H). (I) TEM images of the mitochondrial ultrastructure in HSFs treated with or without FRA for 48 h (scale bar = 1 μm for the upper images; scale bar = 250 nm for the lower images). The results are expressed as the means ± SDs (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant

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CTSB was identified as a crucial gene in FRA-induced HSFs ferroptosis

To explore the potential mechanism of ferroptosis mediated by FRA in HSFs, we used ONT long-read RNA-seq to identify transcripts. The overall results were shown in the Supplementary materials, Table S3. A total of 263,375 transcripts and 64,596 genes were detected; among them, 10,481 transcripts and 1842 genes were identified as novel (Fig. S4A). Nearly 40% of the detected genes had two or more transcripts (Fig. S4B). There was a significant difference in transcript expression between the CTL and FRA-treated groups, as illustrated in Fig. S4C-D. We found 650 transcripts showing upregulated expression and 981 transcripts showing downregulated expression (P. adjust < 0.05 and fold change > 2) (Fig. S4E-F). Next, KEGG analysis and GSEA revealed that in addition to the ferroptosis pathway, apoptosis, autophagy and lysosome pathways were activated (Fig. 5A, Fig. S5A). Given the correlation between apoptosis, autophagy, ferroptosis and lysosomes [32, 33], we speculated that lysosomal proteins might be involved in ferroptosis induction, which was confirmed by GO analysis (Fig. 5B). Next, we analysed lysosomal protein related transcripts and found that most transcripts belonged to CTSB, a cysteine proteolytic enzyme. Furthermore, the CTSB transcripts were clustered and represented by a heatmap (Fig. S5B). A chord graph was used to show the relationships between CTSB transcripts and enriched pathways (Fig. S5C). And the expression of the CTSB gene and the six transcripts accounting for the highest proportion of CTSB was shown in Fig. S6A. The mRNA level of ENST00000534149, which was the transcript accounting for the highest proportion of CTSB, was measured by qRT‒PCR, and the result was consistent with that of RNA sequencing (Fig. S6B). Finally, pie charts were developed to show the relative contribution of each DET to CTSB (Fig. S6C). The CTSB transcript expression data were shown in the Supplementary materials, Table S4. These data demonstrated that transcript analysis provided more detailed and accurate information than did gene expression analysis. To investigate the relationship between FRA and CTSB, we performed a molecular docking study and found that FRA could form hydrogen bonds with specific amino acid residues of CTSB, including His111, His112, and His200, and FRA had an affinity of– 5.56 kcal/mol for CTSB (Fig. 5C), suggesting that FRA may bind to CTSB and stabilize its protein structure, thereby increasing the expression of CTSB protein and promoting its leakage from lysosome. Next, we performed immunofluorescence and WB to verify this hypothesis. As shown in Fig. 5D, after 48 h of FRA treatment, the punctate staining of CTSB, which is typical of a lysosomal distribution, changed to a diffuse cytoplasmic distribution, suggesting the occurrence of lysosomal membrane permeabilization, and the expression of CTSB also increased (Fig. 5E), which was consistent with the results of WB that the protein levels of pro-CTSB and CTSB were increased (Fig. 5F-G). In addition to mitochondrial damage, CTSB is tightly associated with lipid peroxidation [34]; As shown in Fig. 5F-G, the expression of SAT1 increased after 48 h of FRA treatment; however, there was no significant difference in the expression of the acyl-CoA synthetase long-chain family 4 (ACSL4) protein, demonstrating that CTSB induced lipid peroxidation through SAT1. The mRNA expression of CTSB significantly increased after 24 h of FRA treatment and gradually decreased over time (Fig. 5H). Previous studies have reported that MAPK family members, which include extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), are involved in ferroptosis progression, CTSB-induced necroptosis and apoptosis [35,36,37]. As shown in Fig. 5I-J, FRA treatment dose-dependently promoted the phosphorylation of ERK, JNK and p38, and FRA-induced changes of HSFs viabilities and lipid peroxides production were blocked by PD98059 (inhibitor of ERK), SP600125 (inhibitor of JNK) and SB203580 (inhibitor of p38) (Fig. S7A-C), suggesting MAPK signalling pathway was involved in CTSB-mediated ferroptosis. The above results demonstrated that FRA could mediate CTSB leakage from lysosomes and that CTSB could subsequently induce ferroptosis via SAT1-mediated lipid peroxidation and MAPK signalling pathway activation.

Fig. 5
figure 5

CTSB was identified as a crucial gene in FRA-induced HSFs ferroptosis. (A) KEGG pathway enrichment analysis of the DETs between the CTL and FRA (300 µM for 48 h)-treated groups. The red boxes indicate the signalling pathways of interest. (B) GO analysis of the DETs in different groups. The red boxes indicate the signalling pathways of interest. (C) Structural model of CTSB complexed with FRA. In the close-up view, the hydrogen bonds formed between the compound and the protein are depicted as dashed black lines, and the His111, His112, and His200 residues are involved in hydrogen bond interactions. (D) Immunofluorescence staining of CTSB and LAMP1 in HSFs incubated with or without FRA for 48 h (scale bar = 50 μm). (E) Quantification of the relative fluorescence intensity of CTSB. (F) The protein levels of ACSL4, SAT1, pro-CTSB and CTSB in HSFs treated with or without FRA for 48 h were analysed by WB. (G) Quantification of ACSL4, SAT1, pro-CTSB and CTSB protein levels based on WB analysis. (H) The mRNA expression of CTSB in HSFs treated with FRA (300 µM) for 24, 48 and 96 h was measured by qRT‒PCR. GAPDH was used as the internal reference gene. (I) WB analysis of the levels of MAPK signalling pathway proteins, including p-ERK/ERK, p‐JNK/JNK and p‐p38/p38, in HSFs treated with various concentrations of FRA (100 µM, 200 µM and 300 µM) for 48 h. (J) The p‐ERK/ERK, p‐JNK/JNK and p‐p38/p38 protein ratios were quantified based on WB analysis. The results are expressed as the means ± SDs (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant

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FRA alleviated HS formation through ferroptosis in vivo

On the basis of the above results, we established a rabbit ear HS model to investigate the in vivo therapeutic effect of FRA. Treatment schedule and experimental design were illustrated in Fig. 6A-B. Figure 6C showed photographs of the scars at different time intervals. On the 14th day after surgery, the wounds were mostly re-epithelialized, and on the 28th day, HS was observed, with a raised surface and a dark red colour in the CTL group. Instead, the surfaces of the scars became smoother and the colour was lighter in the FRA (100 µM) group. And a superior therapeutic effect was observed in the FRA (300 µM) group, as the surfaces of the scars were flatter, and the scar boundaries became blurred. However, after the application of Fer-1 (2 µM), the formation of scars was more serious than that of the single FRA treatment group. H&E staining revealed that the thickness of the HS tissue dramatically decreased after FRA treatment, but Fer-1 could reverse this effect (Fig. 6D). This trend was further verified by the SEI (Fig. 6E). Furthermore, Masson’s trichrome and Sirius red staining showed that collagen fiber was dense and misaligned, with more irregular deposition on day 28 than on day 14 in the CTL group. In contrast, scars treated with FRA exhibited obvious decreases in collagen deposition and relatively regular collagen distribution on both day 14 and day 28, and these changes were blocked by Fer-1 (Fig. 6D-E).

Fig. 6
figure 6

FRA alleviated HS formation through ferroptosis in vivo. (A) Schedule of the different stages of the in vivo study. (B) Experimental schematic of FRA mitigated scar formation in a rabbit ear HS model. (C) Images of the wounds and the scars formed on day 0, 14 and 28 after treatment with various concentrations of FRA (100 µM and 300 µM) or FRA (300 µM) plus Fer-1 (2 µM) (scale bar = 18 mm). (D) Representative H&E staining, Masson Trichrome staining, and Sirius Red staining images of the wounds and the scars formed on day 14 and day 28 after different treatments. Scale bar, 1 mm in H&E staining images, 500 μm in Masson Trichrome staining images (top), 200 μm in Masson Trichrome staining images (enlarged) and 100 μm in Sirius Red staining images. (E) Quantification of the SEI, collagen density (%) based on Masson Trichrome staining and collagen density (%) based on Sirius Red staining on day 14 and day 28. (F) Immunohistochemical staining of Ki-67, α-SMA, GPX4 and CTSB on day 14 and day 28 after different treatments. Scale bar, 500 μm in the top images and 200 μm in the enlarged images. (G). Quantification of Ki-67-positive cells and relative expression levels of α-SMA, GPX4 and CTSB on day 14 and day 28 (n = 10, arbitrary units). The results are presented as the means ± SDs (n = 3 independent experiments unless otherwise specified). *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant

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At the molecular level, we examined the levels of proteins related to the proliferation, activation and ferroptosis of fibroblasts by IHC. The number of Ki-67-positive cells significantly decreased after FRA treatment. However, only FRA (300 µM) group on day 28 exhibited a marked decrease in the α-SMA expression level, moreover, FRA obviously decreased GPX4 expression on both day 14 and day 28, and all these effects were rescued by Fer-1 treatment (Fig. 6F-G). The expression level of CTSB markedly increased with the application of FRA both on day 14 and day 28, and the administration of Fer-1 did not affect the expression of CTSB (Fig. 6F-G). Taken together, these data suggested that FRA could mitigate scar formation and that the therapeutic effects of FRA on HS could be attributed to CTSB-mediated fibroblasts ferroptosis.

CTSB inhibition attenuated the effect of FRA on HSFs ferroptosis in vitro

To elucidate the functional role of CTSB in the induction of HSFs ferroptosis and amelioration of fibrosis, we used CA-074-me, a specific CTSB inhibitor [38]. As shown in Fig. 7A, FRA-induced diffuse cytoplasmic CTSB immunostaining changed to punctate staining after pretreatment with CA-074-me for 2 h, and the increase in CTSB expression was reversed (Fig. 7B). Next, the upregulation of CTSB and SAT1 protein expression was reversed by CA-074-me, but the increase in the expression of pro-CTSB was augmented by CA-074-me. Moreover, neither FRA treatment nor CA-074-me pretreatment followed by FRA treatment had an obvious effect on the expression of ACSL4. And the depletion of GPX4 protein by FRA was further aggravated by pretreatment with CA-074-me (Fig. 7C, Fig. S8A). In addition, the mRNA levels of CTSB and SAT1 exhibited the same trend (Fig. 7D). The FRA-induced increases in MDA production, lipid peroxidation and ferrous iron accumulation were also strongly blocked by CA-074-me (Fig. 7E-G), and the decreases in mitochondrial volume and membrane potential were significantly reversed (Fig. 7H-I). Furthermore, the FRA-mediated increase in ROS levels was altered by CA-074-me (Fig. S8B-C). CA-074-me pretreatment also abrogated the phosphorylation of ERK, JNK, and p38 induced by FRA (Fig. S8D-E). Finally, the collagen gel contraction assay indicated that CA-074-me pretreatment attenuated the FRA-induced inhibition of HSFs contraction (Fig. 7J-K). And WB analysis revealed that the decrease in the expression of α-SMA, COL1 and COL3 induced by FRA treatment was reversed by the application of CA-074-me (Fig. 7L, Fig. S8A). The above results indicated that CA-074-me indeed reversed FRA-mediated ferroptosis to abrogate the antifibrotic effects of FRA and that resistance to ferroptosis after CTSB inhibition was independent of GPX4.

Fig. 7
figure 7

CTSB inhibition attenuated the effect of FRA on HSFs ferroptosis in vitro. (A) Immunofluorescence staining of CTSB and LAMP1 in HSFs pretreated with or without CA-074-me (20 µM) for 2 h and then incubated with FRA (300 µM) for 48 h (scale bar = 50 μm). (B) Quantification of the relative fluorescence intensity of CTSB. (C) The protein levels of Pro-CTSB, CTSB, ACSL4, SAT1 and GPX4 under the indicated treatments were determined by WB. (D) The mRNA levels of SAT1 and CTSB in HSFs subjected to the indicated treatments were determined by qRT‒PCR. GAPDH was used as the internal reference gene. (E) MDA concentration in HSFs under the indicated treatments was assessed using an MDA assay kit. (F) Representative images of FerroOrange staining (scale bar = 50 μm) and C11-BODIPY staining (scale bar = 50 μm) in different treatment groups. (G) Quantitative analysis of the relative fluorescence intensity of ferrous iron and lipid peroxide. (H) Representative images of MitoTracker staining (scale bar = 50 μm) and JC-1 staining (scale bar = 50 μm). (I) Quantitative analysis of the relative fluorescence intensity of the MitoTracker Red area and the aggregate/monomer ratio. (J) Representative images of the collagen gel contraction assay of HSFs under the indicated treatments on day 3. The dashed white lines indicate the areas of collagen gel. (K) Quantitative results of the collagen gel contraction assay. (L) WB results of the expression of α-SMA, COL1 and COL3 in HSFs after the indicated treatments. The results are expressed as the means ± SDs (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001

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Discussion

HS is an aberrant scar that develops from an abnormal healing process after skin injury. During the pathological process of HS, activated fibroblasts, also called myofibroblasts, play vital roles in collagen secretion and scar contracture [30]. Accumulating evidence has demonstrated that the induction of ferroptosis in vital effector cells exhibits promising antifibrotic therapeutic efficacy [4, 23, 39]. Moreover, Gao et al. suggested that iron distribution disorders, which are characterized by iron deficiency in hepatocytes and iron accumulation in hepatic stellate cells, contribute to liver lipogenesis and fibrosis [40]. Another recent study revealed that iron accumulation occurs in multiple types of fibrotic diseases and that iron uptake promotes the proliferation and activation of lung fibroblasts [41]. These studies confirm that fibrosis-driven cells in fibrotic tissues may be in an iron-rich state. And research on iron metabolism in HS tissue has confirmed that more elemental iron is deposited in human HS tissues than in normal skin [42, 43]. In addition, compared with fibroblasts from normal skin, myofibroblasts from HS tissues exhibit an increase in iron uptake and a decrease in iron efflux, contributing to an intracellular iron-rich environment and thus increasing the ferroptotic sensitivity of cells [4]. Therefore, targeting ferroptosis induction in myofibroblasts is emerging as a novel therapeutic strategy for reversing aberrant scarring, and natural compounds show great potential in this aspect. As a natural compound isolated from the root bark of Dictamnus dasycarpus, the anti-fibrotic effects of FRA have been evaluated in various organ fibrosis. Wu et al. revealed that FRA can inhibit the activation of hepatic stellate cells by downregulating CUG-binding protein 1 (CUGBP1) expression levels to alleviate liver fibrosis [44]; it is also effective in the treatment of renal fibrosis through a similar mechanism [45]; another study suggested that FRA can ameliorate intestinal fibrosis in mice by inhibiting the TGF-β/Smad2/3 signalling pathway and disrupting the interaction between HSP47 and collagen [46]. Our study firstly confirms that FRA can affect HSFs behaviours such as collagen metabolism by inducing ferroptosis (Figs. 4 and 7), this reflects the complexity of the mechanisms by which FRA exerts anti-fibrosis and affects collagen metabolism in different organs, and together with other natural compounds that play a similar role, it further validates the potential of the induction of ferroptosis in fibroblasts to inhibit collagen secretion and fibrosis progression [47, 48]. Interestingly, 48 h of FRA treatment decreased the protein expression of GPX4, but the opposite effect was observed at the transcription level (Fig. 4). GPX4 is regarded as a central regulator of ferroptosis, and ferroptosis requires the downregulation of GPX4 protein expression or inactivation of GPX4, triggering the accumulation of lipid peroxides [15]. Because FRA treatment decreased GPX4 protein expression but increased GPX4 mRNA levels, the decrease in GPX4 protein levels could be attributed to post-transcriptional events. In general, post-transcriptional modifications that mediate protein degradation mainly include ubiquitin proteasome system and autophagy lysosomal pathway [49]. Ubiquitin proteasome system involves the covalent coupling of ubiquitin to lysine residues on the target protein, and then the marked protein was degraded via the proteasome [50]. Many small molecules or natural products have been identified to induce ferroptosis of cancer cells through the ubiquitin-mediated degradation of GPX4 [51, 52]. And autophagy lysosomal pathway includes three major types such as microautophagy, chaperone-mediated autophagy and macroautophagy, all these pathways eventually lead to the delivery of the target protein to lysosome for further degradation [53]. Many studies have shown that targeting this pathway to induce GPX4 degradation can also mediate ferroptosis in cancer cells [49, 54]. The above studies further emphasize the important role of GPX4 in preventing ferroptosis, and studies about the anticancer mechanisms of small molecules also reflect the broad prospect of inducing ferroptosis of major pathogenic cells by targeting ubiquitin or autophagy-mediated GPX4 degradation in the treatment of related diseases. Our study found that FRA might mediate GPX4 degradation through post-transcriptional modification to induce fibroblasts ferroptosis, which reflects the possibility of FRA for the treatment of hypertrophic scar, and due to the degradation of the GPX4 protein, the GPX4 mRNA expression level increased, as in a negative feedback loop. However, the main mechanism of FRA-mediated GPX4 degradation needs further research.

We showed that FRA induced ferroptosis in HSFs to interfere with HS progression, but how does FRA trigger ferroptosis? To elucidate the underlying mechanism, we performed ONT long-read RNA-seq to identify DETs. Due to the complexity of posttranscriptional modifications, the same gene can produce different transcripts in different states [55]. The expression of some transcripts may not be consistent with that of their host gene [56, 57]. Therefore, it is necessary to carry out transcript-level studies. Currently, long-read RNA-seq, which can accurately detect full-length RNA transcripts, provides an opportunity to reveal the diversity of spliced isoforms [58]. Several studies have reported the complexity of the transcriptome using this technology [57, 59, 60]. Our findings suggested that CTSB, a lysosomal protein-coding gene, had 22 DETs. Then, we evaluated the expression and fraction of the DETs of CTSB and found that the expression of all DETs was upregulated, a finding that was consistent with the protein levels of CTSB (Fig. 5) (Fig. S6).

CTSB, a cysteine proteolytic enzyme primarily located in lysosomal compartments, plays a vital role with other proteases in lysosomal proteolysis metabolism under physiological conditions [61]. Under pathological conditions such as inflammation or infection, the expression of CTSB increases, accompanied by increased lysosomal membrane permeabilization, resulting in the leakage of CTSB into the cytoplasm to mediate various types of programmed cell death, especially autophagy and apoptosis [18]. Recently, studies have begun to explore the role of CTSB in ferroptosis in different cells. In myeloid cells, Xu et al. showed that M2-type macrophages are more sensitive to hemin-induced ferroptosis than are M1-type macrophages and that CA-074-me inhibits ferroptosis in M2-type macrophages to ensure their survival, thus resulting in M2 macrophage polarization to promote neurological function recovery after spinal cord injury [16]. Additionally, Lu et al. reported that CTSB plays a vital role in the progression of ferroptosis in microglia following intracerebral haemorrhage, thus triggering secondary injury [17]. However, the role of CTSB in ferroptosis of HSFs is still elusive. In this study, in vitro and in vivo data showed that FRA could induce the upregulation of CTSB expression and the release of CTSB from lysosomes, thus leading to ferroptosis, and that CA-074-me could reverse this process (Figs. 5, 6 and 7). However, the specific mechanism of CTSB-induced ferroptosis needs to be further clarified.

Previous studies have focused on the relationships among lipid peroxidation, mitochondrial damage and CTSB. Nagakannan et al. reported that during ferroptosis, lysosomal membrane lipid peroxidation induces lysosomal membrane permeabilization, mediating the release of CTSB into the cytoplasm, causing lipid peroxidation of the organelle membrane, aggravating lysosomal membrane leakage, and ultimately leading to mitochondrial damage. CA-074-me disrupts this vicious cycle, and the protective effects of CA-074-me are independent of glutathione (GSH) and GPX4 [34]. These data indicate that lipid peroxidation is the most important factor in ferroptosis and that lysosomal destruction and lipid peroxidation are downstream of GSH depletion and GPX4 inhibition. In view of the above results, we assessed the expression of lipid peroxidation-related proteins and found that the protein levels of ACSL4, a lipid metabolism enzyme that increases the sensitivity of cells to lipid peroxidation by catalysing the synthesis of long-chain polyunsaturated CoAs [62], did not change obviously and that the mRNA and protein levels of SAT1, which is involved in p53-mediated ferroptosis by promoting the production of lipid peroxides [63], significantly increased; notably, these changes together with mitochondrial damage could be reversed by pretreatment with CA-074-me. Moreover, our data showed that the protective effects of CA-074-me were independent of GPX4, as GPX4 protein depletion was further augmented in CA-074-me-pretreated HSFs (Figs. 5 and 7). Interestingly, our experimental results (Fig. 7) together with previous studies indicated that CA-074-me can inhibit the increase of CTSB protein expression and lysosomal membrane permeabilization induced by various stimuli [34, 38, 64, 65]. As a protein inhibitor, Nagakannan et al. indicated that CA-074-me mainly inhibits CTSB lysosomal leakage and activation [66]. According to our results and related literatures, most stimuli including FRA can interact with CTSB to stabilize its structure or to promote its leakage, thus disrupting cell homeostasis and further increasing CTSB protein expression and lysosomal leakage, therefore, the inhibitory effect of CA-074-me on CTSB protein expression was caused by its stabilization of lysosomal membrane and inhibition of CTSB activity. MAPKs, a serine/threonine protein kinase family containing JNK, ERK, and p38, play a vital role in regulating various cellular activities [35]. Several studies have shown that MAPKs are involved in ferroptosis. Zhao et al. reported that erastin dose-dependently promotes the phosphorylation of ERK, JNK, and p38 in ATDC5 chondrocytes, which indicates that the activation of the MAPK signalling pathway plays a role in the development of ferroptosis [67]. Yang et al. discovered that the activation of MAPKs induces the upregulation of HO-1 expression, thus leading to ferroptosis in osteocytes [68]. However, whether MAPKs are associated with FRA-induced CTSB release-mediated ferroptosis is unclear. Our results showed that FRA promoted the phosphorylation of ERK, JNK, and p38 in a dose-dependent manner and FRA-induced changes of HSFs viabilities and lipid peroxides production were blocked by MAPKs inhibitor; moreover, FRA induced MAPKs activation was blocked by CA-074-me, suggesting that MAPKs participate in CTSB-mediated ferroptosis (Fig. 5) (Fig. S7, 8). Finally, pretreatment with CA-074-me reversed the antifibrotic effects of FRA on HSFs, indicating that CTSB-induced ferroptosis plays an important role in the alleviation of HS (Fig. 7). Taken together, these data suggest that FRA-mediated CTSB leakage into the cytoplasm induces ferroptosis via SAT1-mediated lipid peroxidation, mitochondrial damage and the activation of the MAPK signalling pathway, thus affecting fibroblasts activities to achieve antifibrotic effects.

Fig. 8
figure 8

Schematic illustration of the study. (A) FRA affects fibroblasts behaviour by inducing ferroptosis to ameliorate hypertrophic scar. (B) ONT long-read RNA-seq reveals that FRA induces ferroptosis of fibroblasts by CTSB. (C) FRA can mediate CTSB leakage from lysosomes in fibroblasts, and CTSB induces ferroptosis via SAT1-mediated lipid peroxidation, mitochondrial damage and MAPK signalling pathway activation. Schematic plots were created in BioRender.com

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At present, the most commonly used agents for hypertrophic scar are corticosteroids, in order not to affect wound repair, corticosteroids are generally applied to mature hypertrophic scar, rather than in the early stage of injury to prevent the formation of hypertrophic scar [3]. As for ferroptosis inducers such as RSL3, in addition to inducing over-activated fibroblasts ferroptosis, it can also affect the activities of HUVEC or other cells [69], so the application of RSL3 in the early stage after injury is not conducive to wound repair. And our in vivo study confirmed that FRA could inhibit the formation of hypertrophic scar in rabbit ears by inducing over-activated fibroblasts ferroptosis (Fig. 6), and in vitro experiment showed that FRA had little effects on normal skin-derived fibroblasts (Fig. 1). So compared with corticosteroids and RSL3, FRA can be used in the early stage after injury to inhibit the formation of hypertrophic scar, the effect is relatively stable and the toxicity is relatively small. Although our study used rabbit ear scar model to verify the therapeutic effects of FRA, the following problems still need to be further explored in order to apply FRA to human body. In view of the different anti-fibrosis mechanisms of FRA in different organs [44,45,46], this indicates that FRA may have a variety of targets, even if it is applied locally in the dermis at the scar, it may also affect cells other than fibroblasts through different targets to produce local adverse reactions. In addition, due to the different locations, hardness and thickness of hypertrophic scar, the absorption, distribution and metabolism of FRA after local injection will also change to some extent, which may lead to local failure to reach effective concentration or excessive absorption of FRA into blood vessels, resulting in adverse reactions. What’s more, the effective concentration of FRA in human body needs to be further explored to avoid adverse reactions caused by high drug concentration.

This study has several limitations. We demonstrated that CTSB is essential for FRA-induced ferroptosis, however, the specific targets of CTSB and FRA have not been identified. Moreover, the interactions of CTSB with other classical ferroptosis biomarkers such as GPX4 are still elusive. In the future, we will focus on the above points, and we will also further explore the clinical application value of FRA.

Conclusions

In summary, we elucidated the molecular mechanism by which FRA exerts antifibrotic effects through the induction of ferroptosis both in vitro and in vivo. Our results showed that CTSB was involved in FRA-induced ferroptosis in HSFs and that the inhibition of CTSB by CA-074-me could alleviate ferroptosis in HSFs by mitigating SAT1-mediated lipid peroxidation, mitochondrial damage and MAPK signalling pathway activation, eventually reversing the antifibrotic effects of FRA.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

HS:

Hypertrophic scar

FRA:

Fraxinellone

HSFs:

Hypertrophic scar-derived fibroblasts

ONT long-read RNA-seq:

Oxford Nanopore Technologies long-read RNA sequencing

GPX4:

Glutathione peroxidase 4

CTSB:

Cathepsin B

SAT1:

Spermidine/spermine-N1-acetyltransferase

MAPK:

Mitogen-activated protein kinase

ECM:

Extracellular matrix

DEG:

Differentially expressed gene

DET:

Differentially expressed transcript

ROS:

Reactive oxygen species

MDA:

Malonaldehyde

ACSL4:

Acyl-CoA synthetase long-chain family 4

ERK:

Extracellular signal-regulated kinase

JNK:

c-Jun N-terminal kinase

p38 MAPK:

p38 mitogen-activated protein kinase

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Acknowledgements

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Funding

This work was supported by Shanghai Ninth People’s Hospital, Cross-research fund project (JYJC202236), Shanghai Ninth People’s Hospital, the Natural Science Foundation of Shanghai Municipal (Grant No. 21ZR1437000), the Health and Family Planning Commission of Shanghai Municipality (2023ZDFC0303) and Shanghai Plastic Surgery Research Center of Shanghai Priority Research Center (2023ZZ02023).

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  1. Wei Xu, Hao Lv and Yaxin Xue contributed equally to this work.

Authors and Affiliations

  1. Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai, 200011, China

    Wei Xu, Hao Lv, Yaxin Xue, Danyang Zhao & Dong Han

  2. Shanghai Institute for Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai, 200011, China

    Wei Xu, Hao Lv, Yaxin Xue, Chuandong Wang, Danyang Zhao & Dong Han

  3. Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedic Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai, 200011, China

    Xiaofeng Shi, Shaotian Fu & Xiaojun Li

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Contributions

W.X., H.L. and Y.X. contributed equally to this work. W.X., H.L. and Y.X. were responsible for research design, cell experiments, data analyses, figure preparation and writing manuscript. X.S. and S.F: contributed to animal experiment. X.S. and X.L. contributed to cell experiments. C.W., D.Z. and D.H.: contributed to research design, methodology, writing reviewing and editing this paper. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Chuandong Wang, Danyang Zhao or Dong Han.

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Ethics approval and consent to participate

HS and normal skin samples were collected from patients at the Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital. Written informed consent was obtained from patients or their legal guardians in accordance with the Declaration of Helsinki and it was approved by the Human Research Ethics Committee of Shanghai Jiao Tong University School of Medicine affiliated ninth people’s hospital (SH9H-2022-T143-2). None of the patients had received any treatment prior to surgery. The animal experiments were approved by the Animal Ethical Committee of Shanghai Jiao Tong University Nongsheng Biotechnology Co., Ltd. (JDLL 20230908-1).

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Xu, W., Lv, H., Xue, Y. et al. Fraxinellone-mediated targeting of cathepsin B leakage from lysosomes induces ferroptosis in fibroblasts to inhibit hypertrophic scar formation. Biol Direct 20, 17 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13062-025-00610-5

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Biology Direct

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