The CTGF exon five sequence was retrieved from the NCBI database, and ASOs targeting this sequence were designed. The gamper design method was used, incorporating modifications such as MOE, LNA, and PS backbone to enhance the stability, affinity, and specificity of the ASOs. All ASO sequences were synthesized by Tsingke Biotech Co., Ltd., with the specific sequences listed in Table 1. The ASOs were dissolved in sterile PBS without Ca²⁺ and Mg²⁺, centrifuged (10,000 rpm at room temperature for 1–2 min), and filtered through a 0.22 μm filter.

Human skin tissues from scar patients and normal controls were collected from Department of Plastic and Burn Surgery, Tianjin First Central Hospital. Keloid patients were diagnosed based on standard criteria. The clinical characteristics of the subjects are shown in Table 2.

Paraffin-embedded tissue sections were deparaffinized and subjected to antigen retrieval with sodium citrate solution (Solarbio). Sections were blocked and incubated overnight at 4 °C with primary antibody (CTGF, Cell Signaling Technology), followed by incubation with secondary antibodies (Affinity) at room temperature for 1 h. Sections were mounted with DAPI-containing anti-fade medium (YEASEN) and imaged using a confocal microscope (ZEISS LSM800).

Cells were seeded into 96-well plates, and after 24 h of incubation, cells were pretreated with various concentrations (0,10,50,100,250,500,1000 nM) of ASO#1, Scrambled-ASO, MOE-ASO#1, LNA-ASO#1. Then, 10 µL of CCK-8 solution (YEASEN) was added to each well, and the cells were incubated for an additional 2 h. The absorbance (OD value) at 450 nm was measured using a microplate reader.

Skin tissues were homogenized in saline to create a 10% homogenate. Hydroxyproline content was measured using a hydroxyproline assay kit (Solarbio) according to the manufacturer’s instructions.

After 24 h of cell treatment, EdU reagent (Beyotime) was added and incubated for 2 h. Cells were washed, fixed, and incubated with Click reaction solution for 30 min. EdU detection and Hoechst 33,342 staining were performed, and images were captured using a fluorescence microscope (ZEISS LSM800).

Skin samples were collected from euthanized mice and digested with trypsin (Solarbio) overnight at 4 °C. Dermal layers were then incubated with collagenase (Solarbio) at 37 °C, followed by DNase treatment. The samples were centrifuged to isolate fibroblasts.

At 70% confluence, cells were transfected with ASOs using Lipo8000(Beyotime) according to the kit instructions. The cells were then incubated for 48 h before proceeding with subsequent experiments.

All FAM-labeled ASOs were synthesized by Tsingke Biotech Co., Ltd. FAM-labeled ASOs were transfected into cells using Lipo8000 (Beyotime) and incubated for 1, 3, 5, and 7 days. At the end of each incubation period, cells were fixed with 4% paraformaldehyde. Cells were stained with DAPI(YEASEN) followed by three washes with PBS, and FAM fluorescence was observed using a confocal microscope (ZEISS LSM800).

Rabbit ear scar samples were cut from the most elevated point, and the vertical distance from the highest point of the scar to the cartilage surface and from the ventral skin surface to the cartilage surface was measured. SEI was calculated as SEI = (vertical distance from the highest point of the scar to the cartilage surface)/(vertical distance from the ventral skin surface to the cartilage surface).

Total RNA was extracted from cell or skin tissue samples using TRIzol (Solarbio). cDNA was synthesized using a reverse transcription kit. Quantitative PCR was performed using SYBR Green premix (YEASEN), and analysis was conducted on a real-time PCR system. The primer sequences used in qRT-PCR is shown in Table 3. PCR conditions were: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 30 s. GAPDH was used as the reference gene, and relative gene expression was calculated using the 2^−ΔΔCT method.

Cell or skin tissue samples were lysed in RIPA buffer (Beyotime) and centrifuged to extract total protein. Protein samples were separated by SDS-PAGE, transferred to PVDF membranes (Millipore), blocked, and incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies at room temperature. Protein detection was performed using an ECL kit (YEASEN) and visualized. For Western blotting analysis, the following primary antibodies were used: CTGF, Col-1α, Col-3α,α-SMA, Fn and β-tubulin (Cell Signaling Technology); TGF-β1,p-Smad2/3, Smad2/3, p-AKT, AKT, p-ERK, ERK, p-PI3K and PI3K (Affinity).

Draw three parallel horizontal lines in the center of a 12-well plate. Once KF cells reach confluency, create vertical scratches with a yellow pipette tip. Wash dislodged cells with PBS and incubate in drug-containing medium. Collect samples at 24 h and capture images at three fixed positions for each group. Analyze using ImageJ. Healing Rate = (Initial Scratch Area - Scratch Area at Time t)/Initial Scratch Area × 100%.

Data were analyzed using GraphPad Prism 9.0 and are presented as mean ± SD. The normality of data distribution within each group was assessed using the Shapiro-Wilk test. Comparisons between two groups were performed using the independent samples t-test, and comparisons among multiple groups were made using one-way ANOVA, followed by Tukey’s post-hoc test for pairwise comparisons. p < 0.05 was considered statistically significant.

Immunofluorescence analysis (Figure 1A) using human pathological scars confirmed marked CTGF overexpression, supporting its therapeutic potential (Khoo et al., 2006; Qiu et al., 2024). Based on this finding, we rationally designed 11 antisense oligonucleotides (ASOs) targeting exon five of CTGF for systematic evaluation (Figure 1B).

Given that fibroblasts are key effector cells in scar pathology (Dong et al., 2022; Talbott et al., 2022), we isolated primary fibroblasts from mouse skin (PSF) for preliminary ASO screening (Li et al., 2021). The experimental results showed that upon transfecting these fibroblasts with ASOs, the scrambled ASO treatment group did not exhibit a significant reduction in CTGF protein expression (Supplementary Figures S1A-C). In contrast, ASO#1 demonstrated excellent inhibitory effects, achieving a half-maximal inhibitory concentration (IC50) of 50 nM with a strong dose-dependent response (Figures 1C,D). To further evaluate the time-dependent effects, we measured the impact of 50 nM ASO#1 on CTGF protein expression levels at different time points (12, 24, 48, 72, and 96 h). The results (Supplementary Figure S2) showed that CTGF protein expression was significantly reduced 48 h after transfection. This indicates that ASO#1 achieved significant inhibition at the 48-h time point, providing a critical reference for subsequent studies.

During scar formation, the synthesis and deposition of Col-1α increase significantly, influencing scar tissue development (Sharma et al., 2022; Xu et al., 2022). To further assess the inhibitory effect of ASOs on fibroblast activation, we measured protein levels of Col-1α. Notably, consistent with CTGF expression, CTGF-ASO#1 exhibited dose-dependent inhibition of type I collagen (Figures 1C,E).

Unmodified nucleic acid drugs have significant drawbacks, including instability in vivo, rapid degradation by nucleases upon entering the bloodstream, and quick clearance by the kidneys, resulting in a short half-life (Chan et al., 2006; Ramasamy et al., 2022). The Gamper design method optimizes ASOs for better targeting efficiency and specificity. As shown in Figure 2A, we modified the initial sequences with MOE and LNA (Bockstahler et al., 2022; Zhu et al., 2022). We transfected the modified CTGF-ASOs into PSF to observe their impact on CTGF protein expression. Compared to unmodified sequences, MOE and LNA modifications significantly improved the inhibitory effect on CTGF expression (Figure 2B).

Further fluorescence analysis using FAM-labeled ASOs showed that the fluorescence of unmodified ASO sequences almost disappeared by day 5 post-transfection, whereas the fluorescence of modified CTGF-ASOs persisted through day 7 (Figure 2C). This suggests that modified CTGF-ASOs remain in the cells longer, likely due to increased resistance to ribonuclease degradation caused by the modifications.

To explore the impact of modified ASOs on PSF cell proliferation, we conducted EdU assays. The results (Figure 2D) confirmed our expectations: both MOE and LNA-modified CTGF-ASOs significantly inhibited PSF cell proliferation. Notably, the LNA-modified ASO required a significantly lower concentration than the MOE-modified ASO to achieve the same level of cell proliferation inhibition. To exclude the cytotoxicity caused by high concentrations, we performed the CCK-8 assay to evaluate the cytotoxicity of ASO#1, MOE-ASO#1, LNA-ASO#1 and scrambled ASO at concentrations of 10, 50, 100, 250, 500, and 1,000 nM, as shown in Supplementary Figures S3A-D. The cell viability of ASO#1 or scrambled control at the tested concentrations did not show significant reduction, indicating that no cytotoxicity was observed under these conditions.

Collagen, the most abundant protein in the skin, is crucial for maintaining skin structure and function (Epstein and Munderloh, 1978; Huang et al., 2024). We focused on the mRNA expression levels of type I and III collagen. The results (Figures 2E–I,L) showed that MOE and LNA-modified CTGF-ASOs dose-dependently inhibited the expression of Col-1α, Col-3α mRNA and protein. While also inhibited the expression of α-SMA, and fibronectin (Fn) mRNA and protein in PSF Cells. Additionally, LNA-modified ASOs demonstrated superior inhibitory effects at the same molar concentrations compared to MOE-modified ASOs (Figures 2E–H).

We also examined the effects of MOE and LNA-modified CTGF-ASOs on the TGF-β1/Smad and Non-Smad signaling pathways, which regulate fibrosis. TGF-β1 is a key regulator of fibrosis, influencing its progression through these pathways (Hao et al., 2023). The results (Figures 2J,K,M,N) indicated that MOE and LNA-modified CTGF-ASOs significantly inhibited the phosphorylation levels of Smad 2/3, PI3K, AKT, and ERK in the cells.

In summary, the MOE and LNA modifications enhanced the stability and efficacy of CTGF-ASOs, effectively inhibiting PSF cell proliferation, ECM protein expression, and key fibrosis-related signaling pathways. These findings highlight the therapeutic potential of modified CTGF-ASOs in scar treatment.

To evaluate the biological function of ASOs in vivo, we established a rabbit ear scar model (Figure 3A); (Ren et al., 2013). By day 28 post-surgery, prominent hypertrophic scars developed on the rabbit ears, characterized by raised, firm, and reddish scar tissue in the model group (Figure 3B; Supplementary Figure S4A). HE staining (Figure 3C; Supplementary Figure S4B) revealed thickened epidermis, numerous new capillaries, inflammatory cells, and fibroblasts in the dermis of the scar tissue. Masson staining (Figure 3D) showed disorganized, nodular, or whorled collagen fibers.

After 4 weeks of treatment with MOE-ASO#1 or LNA-ASO#1, the height of the scar protrusions decreased, and the color lightened. HE staining showed reduced dermal thickness and fewer inflammatory cells and fibroblasts post-treatment. Masson staining indicated that collagen fibers, which appeared dense and disordered in the model group, became more organized following treatment. Additionally, hydroxyproline content was lower in the treated groups compared to the model group (Figure 3E). To assess the anti-inflammatory effect of CTGF-ASO in scars, we measured IL-6 levels in the scar tissue, finding that CTGF-ASO treatment reduced IL-6 production (Figure 3F). In the comparison of the efficacy between MOE-ASO#1 and LNA-ASO#1, LNA-ASO#1 is more effective in treating scar proliferation.

qPCR and Western blot analysis revealed that both MOE and LNA-ASO#1 treatments significantly downregulated the expression of CTGF, type I collagen, type III collagen, α-SMA and Fn proteins in the scar tissue (Figures 3G–L) and LNA-ASO#1 is more effective.

To gain a more comprehensive understanding of the mechanism, we further examined the expression levels of proteins in the TGF-β1/Smad and Non-Smad signaling pathways. The phosphorylation levels of Smad 2/3, PI3K, AKT, and ERK were significantly inhibited by ASO treatment, consistent with the trends observed in ECM protein levels (Figures 3M,N).

In addition, we established a mouse hypertrophic scar model as shown in Figure 4A (Li et al., 2019). Scar formation was induced by applying surface tension to a silicone ring sutured onto the wound. On day 14 post-surgery, wounds were healed, and intralesional treatments began. Data were collected on day 42 post-surgery. As seen in Figure 4B and Supplementary Figure S5A, the model group displayed large scar areas, whereas the treatment groups exhibited significantly reduced scar areas and new hair growth.

Histological analysis using HE, Masson and Sirius Red Staining was performed on the scar tissue sections to observe morphological changes and collagen structure. As shown in Figures 4C,D, Supplementary Figure S5B, the model group scars lacked hair follicles, sebaceous glands, and other dermal appendages. After 4 weeks of treatment with MOE-ASO#1 or LNA-ASO#1, the scar areas were reduced, and skin morphology resembled normal tissue, with the reappearance of hair follicles and sebaceous glands. Hydroxyproline content was lower in the treated groups compared to the model group (Figure 4F). To assess the anti-inflammatory effects of CTGF-ASO in scars, IL-6 levels were measured in the scar tissue, and CTGF-ASO treatment reduced IL-6 production (Figure 4E).

Further analysis showed significant downregulation of CTGF, type I collagen, type III collagen, and α-SMA mRNA and protein expression in mouse scar tissue following ASO treatment (Figures 4G–L). In the comparison of the efficacy between MOE-ASO#1 and LNA-ASO#1, LNA-ASO#1 is more effective in treating scar proliferation (Figures 4G–K).

Mechanistic studies revealed significant changes in the TGF-β1/Smad and Non-Smad signaling pathways. ASO treatment inhibited TGF-β1-induced Smad signaling, as indicated by reduced phosphorylation levels of Smad 2/3. Additionally, the PI3K, AKT, and ERK signaling pathways were effectively inhibited (Figures 4M,N). These findings confirm the potent antifibrotic activity of MOE and LNA-ASO#1.

To more accurately simulate the effects of MOE/LNA-ASO#1 in humans, we established a nude mouse keloid xenograft model (Li et al., 2019). This model preserves the complex interactions between human cells in both the epidermis and dermis, making it a close approximation of in vivo human tissue studies. The results showed that treatment with MOE/LNA-ASO#1 significantly reduced the volume and weight of the keloid tissues compared to untreated controls (Figures 5A,B; Supplementary Figure S6). The weight of keloids treated with LNA-ASO#1 was lower than those treated with MOE-ASO#1. Additionally, hydroxyproline content was lower in the treated groups than in the model group (Figure 5C), indicating reduced collagen content. To assess the anti-inflammatory effects of CTGF-ASO in scars, IL-6 levels were measured in the keloid and MOE/LNA-ASO#1 treatment reduced IL-6 production (Figure 5D). And, LNA-ASO#1 was more effective than MOE-ASO#1 in treating keloids.

Further gene expression analysis of the keloid tissues revealed that ASO treatment dose-dependently reduced the expression of CTGF, α-SMA, Col-1α, Col-3α, and Fn (Figures 5E–J). This indicates that ASOs inhibited the production of ECM proteins in keloid tissues. Additionally, the phosphorylation levels of Smad 2/3, PI3K, AKT, and ERK were significantly reduced following ASO treatment, demonstrating effective inhibition of these signaling pathways (Figures 5K,L). These results suggest that ASOs downregulate the expression of fibrosis-related pathogenic genes in skin fibroblasts by inhibiting the TGF-β1/Smad and non-Smad signaling pathways.

To further validate the inhibitory effects of ASOs on keloid fibroblast (KF) proliferation and migration, we established an ex-vivo keloid culture model. In the untreated group, KF cells migrated out from the edge of the keloid explants and spread across the culture dish within a week. In contrast, MOE/LNA-ASO#1 treatment effectively inhibited KF cell proliferation and migration (Figures 5M,N).