Caspase-9 activates β-catenin signaling to promote pulmonary fibrosis

Abstract

Background

Pulmonary fibrosis (PF) is a progressive interstitial lung disease marked by extracellular matrix accumulation and epithelial damage, with limited therapeutic options. Alveolar epithelial cell apoptosis is a key pathological hallmark of PF, but the upstream regulators driving this process remain unclear. Caspase-9, a central initiator of the intrinsic apoptotic pathway, has been implicated in fibrotic diseases across multiple organs. However, its role in lung fibrosis and its molecular interactions are not fully elucidated.

Methods

Caspase-9 expression was analyzed in human PF lung tissues, bleomycin (BLM)-induced mouse models, and TGF-β1-treated MLE-12 alveolar epithelial cells. Functional studies included pharmacological inhibition, siRNA knockdown, and overexpression of Caspase-9. Fibrosis and apoptosis were assessed using Western blot, qPCR, immunohistochemistry, TUNEL, and electron microscopy. Interaction with β-catenin was examined via co-localization, modulation, and rescue experiments.

Results

Caspase-9 and cleaved-Caspase-9 were significantly upregulated in fibrotic lungs and TGF-β1-stimulated epithelial cells. Caspase-9 inhibition reduced collagen deposition, improved lung architecture, and suppressed pro-fibrotic markers in mice. In MLE-12 cells, Caspase-9 knockdown attenuated TGF-β1-induced apoptosis, restored E-cadherin, and downregulated fibrotic genes. Conversely, Caspase-9 overexpression aggravated fibrosis and apoptosis. Mechanistically, Caspase-9 interacted with β-catenin, enhanced its nuclear accumulation, and promoted downstream fibrotic signaling. β-catenin silencing reversed Caspase-9-induced fibrosis, while β-catenin activation nullified the protective effects of Caspase-9 inhibition both in vitro and in vivo. These results identify a functional Caspase-9/β-catenin axis in PF progression.

Conclusions

Caspase-9 drives pulmonary fibrosis by promoting epithelial apoptosis and activating β-catenin signaling. Targeting the Caspase-9/β-catenin axis may offer a promising therapeutic strategy for PF.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-025-07020-1.

Introduction

Pulmonary fibrosis (PF) is a debilitating and life-threatening interstitial lung disease characterized by excessive deposition of extracellular matrix (ECM), distortion of alveolar architecture, and a progressive decline in pulmonary function [1–3]. Despite advances in our understanding of the underlying pathophysiology, the clinical management of PF remains a major challenge. Current pharmacological interventions, including antifibrotic agents such as pirfenidone and nintedanib, can slow disease progression to some extent but are unable to reverse established fibrosis or significantly improve long-term outcomes [4, 5]. Consequently, there is an urgent need to explore novel molecular targets and develop more effective therapeutic strategies to halt or even reverse the fibrotic process.

Among the diverse pathological mechanisms implicated in PF, apoptosis has emerged as a central feature [6]. Apoptosis, or programmed cell death, plays a critical role in maintaining tissue homeostasis. However, in the context of PF, excessive or dysregulated apoptosis-particularly of alveolar epithelial cells-disrupts the epithelial barrier, facilitates fibroblast activation, and promotes chronic fibrogenic signaling, ultimately leading to aberrant tissue remodeling and scarring [6–8]. Understanding the upstream regulators of apoptosis in fibrotic lungs is therefore essential to decipher the initiation and progression of this disease. Caspases, a family of cysteine-dependent aspartate-directed proteases, are the principal executors of apoptosis [9]. Among them, caspase-9 functions as a key initiator of the intrinsic (mitochondrial) apoptotic pathway. Upon mitochondrial outer membrane permeabilization, cytochrome c is released into the cytosol and binds to Apaf-1, leading to the formation of the apoptosome and subsequent activation of caspase-9. This activation triggers a cascade of downstream effector caspases (such as caspase-3 and − 7), culminating in controlled cellular disassembly [10–12]. Given the critical contribution of epithelial apoptosis to PF, elucidating the role of caspase-9 in lung fibrogenesis is of particular relevance.

Recent studies have begun to uncover a potential link between caspase-9 and fibrotic diseases across various organ systems [13–16]. Increased expression and activation of caspase-9 have been observed in experimental models of renal, hepatic, and cardiac fibrosis [13–16]. For instance, in murine models of kidney disease, caspase-9 activation contributes to tubular epithelial apoptosis and drives interstitial matrix accumulation, exacerbating renal fibrosis [13]. Similarly, in models of liver fibrosis, caspase-9-dependent hepatocyte apoptosis has been shown to activate hepatic stellate cells and promote collagen deposition [14, 16]. These observations suggest that caspase-9 may not merely be a passive bystander during tissue injury, but rather a key pathological mediator driving fibrotic remodeling, potentially through both apoptosis-dependent and -independent mechanisms. Given the conserved role of caspase-9 in fibrogenesis across organs, it is plausible that this pathway may also contribute significantly to pulmonary fibrosis. However, the molecular mechanisms linking caspase-9 activity to pro-fibrotic signaling in the lung remain incompletely understood.

In this study, we sought to investigate the functional role of caspase-9 in pulmonary fibrosis and delineate the downstream mechanisms involved. Using both bleomycin-induced mouse models and TGF-β-stimulated alveolar epithelial cells, we examined the expression, activity, and pathological significance of caspase-9. Furthermore, we evaluated the therapeutic potential of pharmacological caspase-9 inhibition and explored its impact on β-catenin-mediated pro-fibrotic signaling. Our findings identify caspase-9 as a key upstream regulator of pulmonary fibrosis and provide a rationale for targeting the caspase-9/β-catenin axis as a novel strategy to mitigate fibrotic lung disease.

Materials and methods

Clinical specimens

Lung tissue samples were collected from 20 patients (10 non-fibrotic controls and 10 pulmonary fibrosis cases) at the Second Xiangya Hospital of Central South University. The diagnosis of PF was based on the American Thoracic Society (ATS)/European Respiratory Society (ERS) consensus diagnostic criteria [17]. The human study was approved by the Ethics Committee of The Second Xiangya Hospital of Central South University (Approved protocol number: 2013001). Prior to their inclusion in the study, all participants were recruited with written informed consent.

Bleomycin-induced pulmonary fibrosis in mice

C57BL/6 mice (Hunan SJA Laboratory Animal Co., Ltd.) were maintained under controlled conditions (22 ± 2 °C, 70% humidity, 12-h light/dark cycle) with ad libitum access to standard chow and water. Pulmonary fibrosis was induced via intratracheal administration of bleomycin (5 mg/kg dissolved in 50 µL saline; Nippon Kayaku), following a previously described protocol [18]. When mice were intratracheally instilled with bleomycin, they were intraperitoneally injected with Z-LEHD-FMK TFA (a Caspase-9 inhibitor, 10 mg/kg, Selleck) or WAY-262,611 (a β-catenin agonist ,20 mg/kg, Selleck) or a negative control (NC). The injections were continued for 21 days. The survival status of the mice was monitored during this period, and the mice were sacrificed at the end of the 21-day treatment. Animals were euthanized on day 21 post-injection for lung tissue collection. All procedures were conducted under protocols approved by the Animal Care Ethics Committee of The Second Xiangya Hospital of Central South University (approval number: 2020265).

Cell culture, transfection, and treatment

The MLE-12 cell line, which consists of mouse alveolar type II epithelial cells [19] was acquired from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (product number ZQ0470). These cells were cultured in an environment with a 5% CO₂ atmosphere at a temperature of 37 °C. Plasmids, namely oe-NC, NC-siRNA, oe-Caspase-9, Caspase-9-siRNA, oe-β-catenin, and β-catenin-siRNA, were introduced into the MLE-12 cells using the Lipofectamine 2000 kit (catalogue number 2028090, Invitrogen). Subsequently, the MLE-12 cells were incubated with 10 ng/mL of TGF-β1 for 48 h to imitate pulmonary fibrosis (PF) [18, 20].

Tissue staining

For histological evaluation, lung tissues from both human patients and animal models were harvested and processed using hematoxylin-eosin (HE) and Masson trichrome staining techniques. For HE Staining, tissue sections were first sequentially stained with hematoxylin (Abiowell, AWI0001) and eosin (Abiowell, AWI029) to visualize cellular morphology and architectural changes. This classic histological method allowed assessment of alveolar structure, inflammatory cell infiltration, and epithelial-mesenchymal alterations characteristic of pulmonary fibrosis. For Masson Trichrome Staining, tissue sections were processed using a Masson trichrome kit (Abiowell, AWI0253) to specifically quantify collagen deposition. This stain differentiates collagen (blue) from muscle (red) and cytoplasm (pink), enabling objective evaluation of fibrotic severity through semiquantitative analysis of collagen-positive areas.

Immunohistochemistry (IHC) and immunocytochemistry (ICC)

We used IHC and ICC to detect relative cytokines in lung tissues and MLE-12 cells, respectively. Tissue and Cell Pretreatment: Lung tissue sections were first subjected to hot antigen retrieval, then treated with 1% periodic acid solution for 10 min to inactivate endogenous enzymes. MLE-12 cells, after fixation, were incubated with 3% H₂O2₂ for 10 min for the same purpose. Antibody Incubation. The sections and cells were incubated overnight at 4 °C with primary antibodies: collagen I (1:100, AB316222), collagen III (1:100, AB184993), α-SMA (1:300, AB124964), fibronectin (1:200, AB2413), β-Catenin (1:100, 51067-2-AP), Caspase-9 (1:400, 10380-1-AP), and cleaved-Caspase-9 (1:100, Asp315), SFTPC (1:200,10774-1-AP). Signal Detection?  After washing, they were incubated with the secondary antibody HRP goat anti-rabbit IgG (AWS0005) at room temperature for 30 min, followed by DAB working solution (ZLI-9018). Finally, images were observed and collected using a Motic BA210T microscope.

Western blot

Total protein was extracted from lung tissues and cells using RIPA buffer (Abiowell, AWB0136), separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk and probed overnight at 4 °C with primary antibodies against Caspase-9 (1:1000, Immunoway PT0299R), cleaved-Caspase-9 (1:1000, Immunoway Asp315), Caspase3 (1:5000, Cell Signaling Technology 9662), cleaved-Caspase3 (1:1000, Cell Signaling Technology 9664), fibronectin (1:2000, Abcam AB2413), α-SMA (1:2000,Abcam AB124964),vimentin (1:5000, Proteintech 10366-1-AP), E-cadherin (1:5000, Proteintech 20874-1-AP), collagen I (1:1000, Zenbio 343277), collagen III (1:1000, Zenbio R23957), TGF-β1 (1:2000, Proteintech HZ-1011-GMP), β-Catenin (1:2000, Proteintech 51067-2-AP), BAX(1:5000, Proteintech 50599-2-Ig), BCL-2(1:1000, Huabio ET1702-53), and loading controls GAPDH (1:5000, Proteintech 66516-1-Ig) and β-Tubulin (1:5000, Proteintech 80762-1-RR).Following washing, HRP-conjugated secondary antibodies (goat anti-mouse IgG 1:5000, Proteintech SA00001-1; goat anti-rabbit IgG 1:6000, Proteintech SA00001-2) were applied for 1 h at room temperature. Protein bands were visualized using SuperECL Plus (Abiowell, AWB0005) and imaged with a ChemiScope6100 (CLiNX), with densitometry analysis normalized to GAPDH.

Quantitative realtime PCR (qRTPCR)

Total RNA was extracted from lung tissues and cells using TRIzol (15596026, Thermo). We took 50 pg-5 µg of the isolated total RNA and reverse-transcribed it into cDNA with the HiFiScript cDNA Synthesis Kit (CW2569, CoWin Biosciences). Next, the cDNA samples were amplified using the UltraSYBR Mixture (CW2601, CoWin Biosciences) on a QuantStudio 1 Real-Time PCR system (Thermo). Finally, the relative expression levels of the target genes were calculated with GAPDH as the reference gene.

TUNEL assay

Apoptosis in MLE-12 cells and rat lung tissues was detected using a TUNEL Apoptosis Detection kit (YEASEN, 40306ES50). Cells were permeabilized with Proteinase K (37 °C, 15 min), incubated with TdT reaction mix (TdT enzyme + biotin- 11-dUTP) for 1 h, and stained with streptavidin-TRITC. Lung tissues underwent antigen retrieval, Proteinase K treatment, and incubation with TdT reaction mix (FITC-12-dUTP + TdT enzyme). All samples were counterstained with DAPI (Abiowell, AWC0291) and imaged under a fluorescence microscope, with TUNEL-positive cells quantified using ImageJ.

Transmission electron microscope(TEM) status analysis

Lung epithelial cells were fixed with 2.5% glutaraldehyde (Abiowell, AWI0097) and 1% osmium tetroxide (TED PELLA, 18456), then sequentially dehydrated in ethanol, infiltrated with propylene oxide (MERYER, M25514), and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate (Zhongjingkeyi, GZ02625) and lead citrate before mitochondrial morphology was examined under a transmission electron microscope (TEM, JEOL JEM1400).

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8.0. Student’s t - test evaluated differences between two groups, while one - way ANOVA with Tukey’s post hoc test compared multiple groups. Statistical significance was defined as P < 0.05.

Results

Both Caspase-9 and cleaved-Caspase-9 were induced in human fibrotic lung tissues

Caspase-9 and cleaved-Caspase-9 are key mediators of apoptosis and have been implicated in lung fibrosis. In this study, we first examined their expression in human fibrotic lung tissues. Pathological assessment confirmed the presence of fibrosis (Figure S1). Western blot analysis showed significantly increased protein levels of Caspase-9 and cleaved-Caspase-9 in fibrotic lungs compared to normal controls (Fig. 1A–B). Consistently, qPCR revealed elevated Caspase-9 mRNA levels in fibrotic samples (Fig. 1C), suggesting transcriptional upregulation. Immunofluorescence further demonstrated stronger staining intensity of both proteins in fibrotic regions, confirming their increased expression and localization in diseased tissue (Fig. 1D–E). The upregulation of caspase-9 suggests it may be functionally associated with the progression of PF, warranting further investigation into its potential role in pathogenesis.

Fig. 1.

Both Caspase-9 and cleaved-Caspase-9 were induced in human fibrotic lung tissues. A Western blot analysis of caspase-9 (CASP9) and cleaved caspase-9 (cCASP9) expression in pulmonary fibrosis (PF) samples. GAPDH served as the loading control. B Densitometric quantification of CASP9 and cCASP9 normalized to GAPDH. Protein ratios in the control group were set to 1.0, with experimental groups normalized accordingly. Data are presented as mean ± SD (n = 10). Statistical significance was determined using Student’s t-test (*p < 0.05). C Quantitative real-time PCR (qPCR) analysis of caspase-9 mRNA expression, GAPDH served as the loading control. Data are expressed as mean ± SD (n = 10), *P < 0.05 indicates a significant difference. D–E Immunofluorescence staining for CASP9 (D) and cCASP9 (E) in PF tissues. Surfactant Protein C (Sftpc) is a well-characterized marker predominantly synthesized and secreted by type II alveolar epithelial cells, and it plays a critical role in maintaining alveolar homeostasis and normal physiological function. In our study, we performed Sftpc staining specifically to serve as a marker for type II alveolar epithelial cells. Scale bar: 50 μm

Both Caspase-9 and cleaved Caspase-9 were induced in TGF-β induced MLE-12 cells

To investigate the role of Caspase-9 in pulmonary fibrosis at the cellular level, we established a TGF-β1-induced MLE-12 cell model. Western blot analysis confirmed successful model induction (Figure S2). Caspase-9 and cleaved-Caspase-9 expression peaked at 10 ng/ml TGF-β1 for 48 h (Fig. 2A–D). qPCR analysis showed corresponding increases in Caspase-9 mRNA levels (Fig. 2E), indicating transcriptional upregulation. Immunofluorescence further demonstrated enhanced expression and localization of Caspase-9 and its cleaved form in TGF-β1-treated cells compared to controls (Fig. 2F–G). The upregulation of Caspase 9 shows a correlation with fibrotic responses, providing a basis for further exploring its potential role in such processes.

Fig. 2.

Both Caspase-9 and cleaved Caspase-9 were induced in TGF-β induced MLE-12 Cells. To investigate Caspase-9’s role in epithelial fibrosis, MLE-12 cells were exposed to TGF-β at increasing concentrations (0–10 ng/mL) and durations (0–48 h). After treatment, cell morphology was recorded, and lysates were collected for immunoblot analysis. Western blot analysis A, C visualized the expression levels of CASP9 and cCASP9, using β - Tubulin as the loading control. Densitometric quantification B, D of CASP9 and cCASP9 was carried out with the ratio of CASP9/β-Tubulin and cCASP9/β-Tubulin in the control group set as 1.0, and values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 5), and statistical significance was determined, where *p < 0.05 indicates a significant difference. E qPCR showed the expression of CASP9 during TGF-β treatment, also presented as mean ± SD (n = 5) with *P < 0.05 for significant difference. Immunofluorescence images F–G displayed the expression of CASP9 and cCASP9 with a scale bar of 50 μm. Overall, these results demonstrate the promotion of fibrosis by Caspase-9 in MLE-12 cells under TGF - β treatment

Inhibition of Caspase-9 attenuates fibrosis and apoptosis in alveolar epithelial cells

To elucidate the functional role of Caspase-9 in pulmonary fibrosis, we assessed whether its inhibition could mitigate fibrotic and apoptotic responses in alveolar epithelial cells. An siRNA with high knockdown efficiency was selected and validated by Western blot and qPCR (Fig. 3A–C). We further demonstrated via Western blot and immunofluorescence staining analysis that Caspase-9 siRNA can significantly inhibit the expression of both Caspase-9 and activated Caspase-9, regardless of TGF-β treatment (Fig. 3D–G). Upon TGF-β1 stimulation, MLE-12 cells exhibited elevated expression of fibrosis markers such as fibronectin (FN), collagen I (Col I), collagen III (Col III) and vimentin, indicating a fibrotic phenotype. However, Caspase-9 knockdown significantly reversed these changes, as confirmed by reduced protein levels in WB analysis (Fig. 3D–E). In addition, our results further demonstrated that under TGF-β stimulation, the expression of E-cadherin (E-cad) is inhibited. However, caspase-9-targeting siRNA can attenuate this inhibitory effect, ultimately leading to a reduction in extracellular matrix (ECM) deposition (Fig. 3D–E). Taking together, these results suggest a pro-fibrotic role of Caspase-9.

Fig. 3.

Inhibition of Caspase-9 Alleviates Fibrosis in MLE-12 Cells. MLE-12 cells were transfected with Caspase-9-siRNA or NC-siRNA for 24 h, followed by treatment with or without 10ng/ml TGF-β for 48 h. A Western blot analysis visualized the expression levels of CASP9 and GAPDH, using β-Tubulin as the loading control. Densitometric quantification B of CASP9 and GAPDH was carried out with the ratio of CASP9/β-Tubulin and GAPDH/β-Tubulin in the control group set as 1.0, and values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 5), and statistical significance was determined, where *p < 0.05 indicates a significant difference. C qPCR analysis showing the expression of CASP9. Data are expressed as mean ± SD (n = 5), *P < 0.05 indicates a significant difference. D Western blot analysis was carried out to visualize the expression levels of Fibronectin, E-cadherin(E-cad), collagen III, collagen I, vimentin, CASP9 and cCASP9; GAPDH as the loading control. E Densitometric quantification of Fibronectin, E-cadherin, collagen III, collagen I, vimentin, CASP9 and cCASP9. In the NC group, the ratio of Fibronectin/GAPDH, collagen III/GAPDH, collagen I/GAPDH, vimentin/GAPDH, CASP9/GAPDH and cCASP9/GAPDH was set as 1.0; In the Caspase9 siRNA + TGF-βgroup, the E-cadherin/GAPDH was set as 1.0. All the values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 5). *p < 0.05 indicates a significant difference; F–G Immunofluorescence images showing the expression of CASP9 and cCASP9. scale bar: 50 μm

In parallel, we investigated the impact of Caspase-9 on apoptosis. TGF-β1 treatment led to a marked increase in cleaved caspase-3 expression, while Caspase-9 inhibition significantly attenuated this effect (Fig. 4A–C), indicating its involvement in apoptotic signaling. Furthermore, our Western blot analysis revealed a significant downregulation of Bcl-2/Bax expression ratio in NC siRNA + TGF-β1 group, and Caspase-9 inhibition alleviated this downregulation significantly (Fig. 4D–E). Consistently, TUNEL staining revealed a reduced number of apoptotic cells following Caspase-9 knockdown (Fig. 4G–F). Ultrastructural analysis by TEM further confirmed decreased apoptotic features, including chromatin condensation and apoptotic body formation (Fig. 4H). These results demonstrate that Caspase-9 contributes to both fibrotic and apoptotic responses in alveolar epithelial cells, and its inhibition confers protective effects, highlighting its potential as a therapeutic target in pulmonary fibrosis.

Fig. 4.

Inhibition of Caspase-9 Alleviates apoptosis in MLE-12 Cells. MLE-12 cells were transfected with Caspase-9-siRNA or NC-siRNA for 24 h, followed by treatment with or without 10ng/ml TGF-β for 48 h. A Western blot analysis visualized the expression levels of CASP3 and cCASP3, using β-Tubulin as the loading control. B–C Densitometric quantification of CASP3 and cCASP3 was carried out with the ratio of CASP3/β-Tubulin and cCASP3/β-Tubulin in the control group set as 1.0, and values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 5), and statistical significance was determined, where *p < 0.05 indicates a significant difference. D Western blot analysis visualized the expression levels of BCL-2 and BAX, using β-Tubulin as the loading control. E Statistical analysis of the Bcl-2/Bax expression ratio, All data are presented as mean ± SD (n = 5), and statistical significance was determined, where *p < 0.05 indicates a significant difference. G–F Apoptosis was quantified by counting TUNEL-positive cells. scale bar: 50 μm. H TEM was used to observe MLE-12 cells under different treatments. The scale bar :500 nm

Overexpression of Caspase-9 enhances pulmonary fibrosis and apoptosis

To further elucidate the functional role of Caspase-9 in pulmonary fibrosis, we conducted gain-of-function experiments by overexpressing Caspase-9 in MLE-12 cells. The goal was to determine whether Caspase-9 upregulation could exacerbate the fibrotic response, particularly under TGF-β1 stimulation. Western blotting and qPCR confirmed efficient overexpression of Caspase-9 at both protein and mRNA levels (Fig. 5A–C), establishing a robust Caspase-9-overexpressing model. Through Western blot and immunofluorescence analyses, we further found that Caspase-9 overexpression leads to a significant increase in both total and activated Caspase-9 levels, irrespective of TGF-β treatment (Fig. 3D–G). When stimulated with TGF-β1, MLE-12 cells showed heightened expression of fibrotic markers including fibronectin (FN), collagen I (Col I), collagen III (Col III) and vimentin, reflecting a fibrotic phenotype. Notably, Caspase-9 overexpression exacerbated these changes, as evidenced by elevated protein levels in Western blot analyses (Fig. 5D–E). Additionally, our results revealed that TGF-β stimulation suppresses E-cadherin (E-cad) expression, and this suppressive effect is further strengthened by Caspase-9 overexpression, ultimately resulting in increased ECM deposition (Fig. 5D–E). Collectively, these findings further support that Caspase-9 promotes pulmonary fibrosis. We next explored whether Caspase-9 overexpression could enhance apoptosis. TGF-β1 treatment led to a marked increase in cleaved caspase-3 expression, a key mediator of apoptosis. This effect was significantly intensified by Caspase-9 overexpression (Fig. 6A–C), suggesting that Caspase-9 promotes apoptotic signaling in pulmonary epithelial cells. Furthermore, our Western blot analysis revealed a significant downregulation of Bcl-2/Bax expression ratio in OE-NC + TGF-β1 group, and Caspase-9 overexpression further promoted this downregulation (Fig. 6D–E). TUNEL assay results further demonstrated a higher number of TUNEL-positive cells in the Caspase-9 overexpression group compared to the TGF-β1-only group, confirming enhanced apoptosis (Fig. 6G–F). Transmission electron microscopy (TEM) provided additional evidence, revealing classic apoptotic features such as chromatin condensation and nuclear fragmentation in cells overexpressing Caspase-9 (Fig. 6H). Collectively, these findings indicate that Caspase-9 overexpression not only amplifies pulmonary fibrosis but also enhances apoptosis, emphasizing its pivotal role in mediating cell death and fibrotic progression in pulmonary diseases.

Fig. 5.

Overexpression of Caspase-9 enhance lung fibrosis in MLE-12 Cells. MLE-12 cells were transfected with OE-Caspase-9 or OE-NC for 24 h, followed by treatment with or without 10ng/ml TGF-β for 48 h. A Western blot analysis visualized the expression levels of CASP9, GAPDH as the loading control. Densitometric quantification B of CASP9 was carried out with the ratio of CASP9/GAPDH in the control group set as 1.0, and values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 5), and statistical significance was determined, where *p < 0.05 indicates a significant difference. C qPCR analysis showing the expression of CASP9. Data are expressed as mean ± SD (n = 5), *P < 0.05 indicates a significant difference. D Western blot analysis was carried out to visualize the expression levels of Fibronectin, E-cadherin (E-cad), collagen III, collagen I, vimentin, CASP9 and cCASP9; GAPDH as the loading control. E Densitometric quantification of Fibronectin, E-cadherin, collagen III, collagen I, vimentin, CASP9 and cCASP9. In the OE-NC group, the ratio of Fibronectin/GAPDH, collagen III/GAPDH, collagen I/GAPDH, vimentin/GAPDH, CASP9 /GAPDH and cCASP9/GAPDH was set as 1.0; In the OE-caspase9 + TGF-βgroup, the E-cadherin/GAPDH was set as 1.0. All the values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 5). *p < 0.05 indicates a significant difference. F–G Immunofluorescence images showing the expression of CASP9 and cCASP9. scale bar:50 μm

Fig. 6.

Overexpression of Caspase-9 increase lung apoptosis in MLE-12 Cells. MLE-12 cells were transfected with OE-Caspase-9 or OE-NC for 24 h, followed by treatment with or without 10ng/ml TGF-β for 48 h. A Western blot analysis visualized the expression levels of CASP3 and cCASP3, using β-Tubulin as the loading control. B–C Densitometric quantification of CASP3 and cCASP3 was carried out with the ratio of CASP3/β-Tubulin and cCASP3/β-Tubulin in the control group set as 1.0, and values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 5), and statistical significance was determined, where *p < 0.05 indicates a significant difference. D Western blot analysis visualized the expression levels of BCL-2 and BAX, using β-Tubulin as the loading control. E Statistical analysis of the Bcl-2/Bax expression ratio, All data are presented as mean ± SD (n = 5), and statistical significance was determined, where *p < 0.05 indicates a significant difference.G–F Apoptosis was quantified by counting TUNEL-positive cells. Scale bar:50 μm. HTEM was used to observe MLE-12 cells under different treatments. The scale bar :500 nm

Caspase-9 Inhibition attenuates pulmonary fibrosis and apoptosis in mice

To assess the role of Caspase-9 in vivo, we established a bleomycin (BLM)-induced pulmonary fibrosis model in mice. Western blot, histological staining (HE, Masson), and immunohistochemistry confirmed successful fibrosis induction by day 21 (Figure S3A–C). Notably, both Caspase-9 and cleaved-Caspase-9 were significantly upregulated in fibrotic lungs (Fig. 7A–C), as further validated by qPCR (Fig. 7D) and co-immunofluorescence with Sftpc, indicating activation primarily in alveolar epithelial cells (Figure S3D). To evaluate the therapeutic potential of targeting Caspase-9, BLM-treated mice received a Caspase-9 inhibitor. Fibrosis markers, including fibronectin, collagen I/III, and α-SMA, were markedly reduced in caspase9 inhibitor-treated lungs as shown by Western blot and qPCR (Fig. 7E–G). Histological analysis (HE, Masson; Fig. 7H) and macroscopic examination (Fig. 7J) revealed attenuated lung remodeling, decreased tissue stiffness, and less congestion. Immunofluorescence and immunohistochemistry (Fig. 7H–I) confirmed downregulation of Caspase-9/cleaved-Caspase-9 in alveolar regions, supporting the anti-fibrotic effect of Caspase-9 inhibition. We next examined apoptosis in lung tissues. Western blot analysis revealed that cleaved-caspase 3 levels were significantly elevated following BLM treatment but were markedly reduced upon Caspase-9 inhibition (Fig. 8A–C), suggesting suppressed apoptotic activity. Western blot analysis revealed a significant downregulation of Bcl-2/Bax expression ratio in BLM-treated mice groups compared with Control group, and Caspase-9 inhibition alleviated this downregulation significantly (Fig. 8D–E). TUNEL staining (Fig. 8G–F) further showed a substantial decrease in apoptotic cells in the inhibitor group. Ultrastructural analysis by electron microscopy (Fig. 8H) confirmed reduced apoptotic features, including chromatin condensation and apoptotic body formation. Collectively, these data demonstrate that Caspase-9 inhibition mitigates BLM-induced pulmonary fibrosis and epithelial apoptosis in mice, supporting its potential as a therapeutic target.

Fig. 7.

Inhibiting Caspase-9 could attenuate BLM-induced lung fibrosis in mice. Eight-week-old C57BL/6J mice were given a single intratracheal instillation of either bleomycin (3.5 mg/kg) or normal saline. In the bleomycin-instilled mice, Z-LEHD-FMK TFA (a Caspase-9 inhibitor, 10 mg/kg) or a negative control (NC) was intraperitoneally injected daily until the day before sacrifice, with a typical experimental period of 21 days. A Western blot analysis visualized the expression levels of CASP9 and cCASP9, using β-Tubulin as the loading control. Densitometric quantification (B, C) of CASP9 and cCASP9 was carried out with the ratio of CASP9/β-Tubulin and cCASP9/β-Tubulin in the control group set as 1.0, and values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 5), and statistical significance was determined, where *p < 0.05 indicates a significant difference. D qPCR showed the expression of CASP9 during BLM treatment, also presented as mean ± SD (n = 5) with *P < 0.05 for significant difference. E Western blot analysis was carried out to visualize the expression levels of Fibronectin, collagen III, collagen I, α-SMA, CASP9 and cCASP9; GAPDH as the loading control. F Densitometric quantification of Fibronectin, collagen III, collagen I, α-SMA, CASP9 and cCASP9. In the NC group, the ratio of Fibronectin/GAPDH, collagen III/GAPDH, collagen I/GAPDH, α-SMA/GAPDH, CASP9 and cCASP9/GAPDH was set as 1.0, and the values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 4). *p < 0.05 indicates a significant difference. G qPCR showed the expression of CASP9 during BLM and Z-LEHD-FMK TFA treatment, also presented as mean ± SD (n = 5) with *P < 0.05 for significant difference. H representative images of HE staining, scale bar: 100 μm; Masson staining, scale bar:100 μm; Immunohistochemical images of Fibronectin, collagen III, collagen I, α -SMA, scale bar:100 μm. I Immunofluorescence images showing the expression of CASP9 and cCASP9. Surfactant Protein C (Sftpc) is a well-characterized marker predominantly synthesized and secreted by type II alveolar epithelial cells, and it plays a critical role in maintaining alveolar homeostasis and normal physiological function. In our study, we performed Sftpc staining specifically to serve as a marker for type II alveolar epithelial cells. scale bar: 50 μm. J Macroscopic examination of lung tissues

Fig. 8.

Inhibiting of Caspase-9 attenuate BLM-induced lung apoptosis in mice. Eight-week-old C57BL/6J mice were given a single intratracheal instillation of either bleomycin (3.5 mg/kg) or normal saline. In the bleomycin-instilled mice, Z-LEHD-FMK TFA (a Caspase-9 inhibitor, 10 mg/kg) or a negative control (NC) was intraperitoneally injected daily until the day before sacrifice, with a typical experimental period of 21 days. A Western blot analysis visualized the expression levels of CASP3 and cCASP3, using GAPDH as the loading control. B–C Densitometric quantification of CASP3 and cCASP3 was carried out with the ratio of CASP3/GAPDH and cCASP3/GAPDH in the control group set as 1.0, and values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 5), and statistical significance was determined, where *p < 0.05 indicates a significant difference. D Western blot analysis visualized the expression levels of BCL-2 and BAX, using GAPDH as the loading control. E Statistical analysis of the Bcl-2/Bax expression ratio, All data are presented as mean ± SD (n = 5), and statistical significance was determined, where *p < 0.05 indicates a significant difference. G–F TUNEL-positive cell quantification evaluated apoptotic activity. scale bar: 50 μm. H TEM was used to observe Lung tissue under different treatments. The scale bar:2 μm

The Caspase-9/cleaved-Caspase-9 and β-catenin interaction mediates pulmonary fibrosis

To investigate the molecular mechanism underlying Caspase-9-induced fibrosis, we examined protein interactions and found a direct association between Caspase-9/cleaved-Caspase-9 and β-catenin in human and mouse fibrotic lung tissues (Fig. 9A–D). This interaction suggests a functional partnership in the fibrotic process. Consistent with this, β-catenin expression was significantly elevated in both human and mouse fibrotic tissues, as well as in TGF-β1-treated MLE-12 cells, highlighting its conserved role in pulmonary fibrosis (Fig. 9E–J). We further explored the relationship between Caspase-9 and β-catenin through rescue experiments. Overexpression of Caspase-9 in TGF-β1-treated MLE-12 cells led to increased fibrosis markers (FN, collagen I, collagen III, α-SMA). However, silencing β-catenin with siRNA abrogated this fibrotic response. Conversely, activating β-catenin in Caspase-9-inhibited cells restored the fibrotic phenotype, suggesting that Caspase-9 acts upstream of β-catenin in the fibrotic pathway (Fig. 9K–L). Immunofluorescence staining (Fig. 9M–N) confirmed the colocalization of Caspase-9 and β-catenin in alveolar epithelial cells, supporting the functional data. These findings establish a novel mechanism where the interaction between Caspase-9 and β-catenin amplifies fibrogenic signaling in pulmonary epithelial cells. Targeting this axis may provide a therapeutic strategy for fibrotic lung diseases.

Fig. 9.

Caspase-9 Regulates Pulmonary Fibrosis via β-Catenin Signaling. MLE-12 cells were transfected with OE-Caspase-9, OE-β-catenin or β-catenin-siRNA for 24 h, followed by treatment with or without 10ng/ml TGF-β for 48 h.A–B co-immunoprecipitation of Caspase-9 and β-catenin in MLE-12 cells treated with TGF-β1.C–D co-immunoprecipitation of cleaved-Caspase-9 and β-catenin in MLE-12 cells treated with TGF-β1.E–J Western blot analysis visualized the expression levels of β-catenin in human and mouse tissues as well as MLE-12 cells, GAPDH served as the loading control. Densitometric quantification of β-catenin was carried out with the ratio of β-catenin/GAPDH in the control group set as 1.0, and values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 5), and statistical significance was determined, where *p < 0.05 indicates a significant difference. K. Western blot analysis was carried out to visualize the expression levels of Fibronectin, collagen III, collagen I, α-SMA; GAPDH as the loading control. L Densitometric quantification of Fibronectin, collagen III, collagen I, α-SMA. In the control group, the ratio of Fibronectin/GAPDH, collagen III/GAPDH, collagen I/GAPDH, α-SMA/GAPDH was set as 1.0, and the values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 4). *p < 0.05, compared with control group; # p < 0.05,compared with control + TGF-β group;@ p < 0.05,compared with oe-caspase9 + TGF-β group; % p < 0.05,compared with oe-caspase9 + β-catenin-siRNA + TGF-β group. M–N Immunofluorescence images showing the expression of CASP9 and cCASP9. scale bar: 50 μm

Then, we have performed rescue experiments in our murine model to assess the impact on physiological parameters of lung fibrosis. Western blot and Immunohistochemical analysis revealed that fibrosis markers significantly increased in BLM-treated mice, including Fibronectin, collagen I, collagen III and α-SMA, while intraperitoneal injection of caspase 9 inhibitor markedly reduced these markers (Fig. 10A–C). Consistent with molecular findings, HE and Masson staining showed similar results (Fig. 10C). BLM-treated mice had severe alveolar destruction and collagen deposition. Caspase 9 inhibitor alleviated structural abnormalities, reduced collagen, confirming its anti-fibrotic effect. Importantly, to validate the role of β-catenin in this process, we then conducted additional experiments where β-catenin was activated concurrently with caspase-9 inhibition. Under these conditions, the fibrotic markers and pathological scores were re-elevated, indicating that the protective effect of caspase-9 inhibition was abrogated by β-catenin activation. These in vivo rescue data further confirm that caspase-9 promotes pulmonary fibrosis, at least in part, through β-catenin-mediated signaling.

Fig. 10.

Caspase-9 Regulates BLM-induced lung fibrosis in mice via β-Catenin Signaling. Eight-week-old C57BL/6J mice were given a single intratracheal instillation of either bleomycin (3.5 mg/kg) or normal saline. In the bleomycin-instilled mice, Z-LEHD-FMK TFA (a Caspase-9 inhibitor, 10 mg/kg), WAY-262,611 (a β-catenin agonist,20 mg/kg), or a negative control (NC) was intraperitoneally injected daily until the day before sacrifice, with a typical experimental period of 21 days. A Western blot analysis was carried out to visualize the expression levels of Fibronectin, collagen III, collagen I, β-catenin, α-SMA, CASP9 and cCASP9; β-Tubulin as the loading control. B Densitometric quantification of Fibronectin, collagen III, collagen I, α-SMA, CASP9 and cCASP9. In the NC group, the ratio of Fibronectin/β-Tubulin, collagen III/β-Tubulin, collagen I/β-Tubulin, β-catenin/β-Tubulin α-SMA/β-Tubulin, CASP9 and cCASP9/β-Tubulin was set as 1.0, and the values of other experimental groups were normalized accordingly. All data are presented as mean ± SD (n = 4). *p < 0.05 indicates a significant difference. C representative images of HE staining, scale bar: 100 μm; Masson staining, scale bar:100 μm; Immunohistochemical images of Fibronectin, collagen III, collagen I, α -SMA, scale bar:100 μm

Discussion

PF remains one of the most challenging and life-threatening lung diseases, with limited therapeutic options available for patients [21, 22]. Despite the availability of antifibrotic drugs such as pirfenidone and nintedanib, these treatments primarily slow the progression of the disease without reversing established fibrosis or improving long-term outcomes significantly [17, 23, 24]. Therefore, understanding the molecular mechanisms driving PF is essential for the development of more effective therapeutic strategies. This study focuses on exploring the role of Caspase-9 in pulmonary fibrosis, specifically its interaction with β-catenin, a key player in the Wnt/β-catenin signaling pathway. Our results provide significant insights into the contribution of Caspase-9 to fibrotic processes in the lung, highlighting its potential as a novel therapeutic target.

Caspase-9 is a central initiator of apoptosis through the mitochondrial pathway [25, 26]. Upon activation, it leads to the cleavage of effector caspases, such as Caspase-3, initiating a cascade that results in cell death [25, 26]. In the context of PF, dysregulated apoptosis, especially of alveolar epithelial cells, plays a critical role in the disruption of lung architecture and promotes fibroblast activation, a hallmark of fibrosis [27, 28]. Previous studies have shown that Caspase-9 is upregulated in fibrotic tissues across various organs, including the liver, kidney, and heart, where it contributes to both apoptotic and non-apoptotic fibrotic responses [13–16]. Our findings align with this body of work, as we observed increased expression of Caspase-9 and its active form in both human and mouse fibrotic lung tissues. These results suggest that Caspase-9 not only plays a role in cell death but also contributes to the pro-fibrotic microenvironment.

Moreover, in vitro studies using the TGF-β1-treated MLE-12 cell model revealed that Caspase-9 inhibition attenuates both fibrosis and apoptosis, indicating that Caspase-9 is a key mediator of both processes in alveolar epithelial cells. This observation is consistent with previous research suggesting that caspases may drive fibrogenesis through both apoptotic and non-apoptotic mechanisms, with Caspase-9 being a critical player [13]. Furthermore, Caspase-9 overexpression amplified fibrotic markers and apoptosis, further emphasizing its role in promoting fibrotic progression. These findings provide compelling evidence for the involvement of Caspase-9 in driving both fibrosis and cell death in the lung, which could contribute to the pathogenesis of PF.

A significant part of our study was dedicated to understanding the molecular mechanisms by which Caspase-9 promotes pulmonary fibrosis. We discovered a direct interaction between Caspase-9 and β-catenin, a key mediator of the Wnt/β-catenin signaling pathway [29]. This interaction was observed both in human and mouse fibrotic tissues, and its functional significance was confirmed through various experimental approaches. Silencing β-catenin reversed the pro-fibrotic effects of Caspase-9 overexpression, while pharmacological activation of β-catenin in Caspase-9-inhibited cells restored fibrosis markers. These findings suggest that Caspase-9 acts upstream of β-catenin to amplify fibrogenic signaling in pulmonary epithelial cells, highlighting the Caspase-9/β-catenin axis as a critical pathway in pulmonary fibrosis.

The Wnt/β-catenin signaling pathway is known to regulate a variety of cellular processes, including cell proliferation, differentiation, and apoptosis [30–32]. In the context of fibrosis, aberrant activation of β-catenin has been implicated in the regulation of fibrosis-related gene expression and ECM deposition [18, 31]. Several studies have demonstrated the role of β-catenin in promoting fibrosis in various organs, including the liver and kidney [32, 33]. For instance, β-catenin activation in hepatic stellate cells contributes to liver fibrosis [33] and β-catenin signaling has been shown to enhance renal fibrosis in response to injury [32]. Our findings suggest that Caspase-9-induced β-catenin activation is a key mechanism in promoting pulmonary fibrosis, providing a new perspective on how cell death and signaling pathways converge to drive fibrosis in the lung.

Our results are in line with previous studies exploring the role of Caspase-9 in fibrotic diseases [13–16] yet they extend the current understanding by identifying a novel Caspase-9/β-catenin interaction in pulmonary fibrosis. For example, previous work has established that TGF-β1 is a potent inducer of fibrosis, and its role in activating apoptotic and fibrotic pathways has been well documented [18]. However, the specific contribution of Caspase-9 in this context was less clear. Our study provides direct evidence that Caspase-9 is not only involved in apoptosis but also plays a central role in driving fibrosis through the β-catenin signaling pathway. Additionally, while the role of β-catenin in fibrosis has been well established, our study highlights a new dimension by showing that Caspase-9 can directly regulate β-catenin activation. This finding offers a novel therapeutic target, as inhibiting the Caspase-9/β-catenin axis may provide a dual approach to reduce both cell death and fibrosis in pulmonary diseases.

In summary, our study identifies Caspase-9 as a key regulator of pulmonary fibrosis through its interaction with β-catenin. We demonstrate that Caspase-9 activates β-catenin signaling, which in turn amplifies fibrotic responses in alveolar epithelial cells. These findings provide a novel mechanistic insight into the pathogenesis of pulmonary fibrosis and open up new therapeutic avenues for targeting the Caspase-9/β-catenin axis. Although challenges remain in translating these findings into clinical practice, our research lays the groundwork for future studies aimed at developing targeted therapies for fibrotic lung diseases.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Z.X. conceptualized the study; J.W., Y.Y., B.Q., and Y.C. conducted experiments; L.G., H.C., and R.H. performed data analysis and provided methodology advice; J.W. drafted the manuscript.

Funding

This study was supported by a grant from Natural Science Foundation of China (82070069).

Data availability

No datasets were generated or analyzed in this study. Data supporting the findings are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The human study was approved by the Ethics Committee of The Second Xiangya Hospital of Central South University (Approved protocol number: 2013001). Prior to their inclusion in the study, all participants were recruited with written informed consent. The animal study was conducted under protocols approved by the Animal Care Ethics Committee of The Second Xiangya Hospital of Central South University (approval number: 2020265).

Competing interests

All authors declare no competing interests.