Caffeic Acid Phenethyl Ester Ameliorates Pulmonary Fibrosis by Inhibiting Epithelial-Mesenchymal Transition via the Sirt1/PGC-1α/Mitochondrial Axis

Caffeic Acid Phenethyl Ester Ameliorates Pulmonary Fibrosis by Inhibiting Epithelial-Mesenchymal Transition via the Sirt1/PGC-1α/Mitochondrial Axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Caffeic Acid Phenethyl Ester Ameliorates Pulmonary Fibrosis by Inhibiting Epithelial-Mesenchymal Transition via the Sirt1/PGC-1α/Mitochondrial Axis Rui Liu, Weihua Yu, Bingjie Liu, Meng Cao, Zhao Wang, Jiangzheng Liu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7559730/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pulmonary fibrosis is a severe lung disease characterized by the epithelial-mesenchymal transition (EMT) of alveolar epithelial cells, leading to an increase in fibroblasts or myofibroblasts. Currently, effective therapeutic options for PF remain limited, rendering the inhibition or reversal of EMT a clinically imperative goal. Caffeic acid phenethyl ester (CAPE), a natural flavonoid, exhibits various biological activities, including antioxidant, anti-inflammatory, anticancer, antiviral, and immunomodulatory effects. However, the role of CAPE in EMT-related diseases such as pulmonary fibrosis remains unclear. This study aimed to investigate whether CAPE can target the Sirtuin 1 (Sirt1)/Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) pathway to regulate mitochondrial function, thereby inhibiting EMT and pulmonary fibrosis. Our in vitro and in vivo findings demonstrate that CAPE significantly improves the quantity and function of mitochondria including mitochondrial DNA (mtDNA) content, ATP level, or mitochondrial membrane potential in altered alveolar epithelial cells, increases the ratio of alveolar epithelial to mesenchymal cell markers, and reduces ROS level or collagen expression, ultimately alleviating the degree of fibrosis. These findings establish a robust preclinical foundation for the translational application of CAPE in EMT-related diseases. In conclusion, CAPE may serve as a potential therapeutic agent for pulmonary fibrosis by modulating mitochondrial function through the Sirt1/PGC-1α pathway, underscoring its potential for clinical translation and merit for further investigative efforts in other EMT-associated conditions. Caffeic acid phenethyl ester (CAPE) Pulmonary fibrosis Epithelial–mesenchymal transition (EMT) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Pulmonary fibrosis, a devastating and progressive lung disease, represents the end-stage pathological alteration of various chronic respiratory disorders [1-3]. It is characterized by the excessive deposition of extracellular matrix (ECM) proteins, primarily collagen, which leads to the destruction of normal alveolar architecture and a subsequent decline in lung functions [2, 4]. The global burden of pulmonary fibrosis is substantial, with a high mortality rate and limited effective treatment options, highlighting the urgent need for novel therapeutic strategies [5]. The epithelial-mesenchymal transition (EMT) of alveolar epithelial cells has emerged as a central mechanism in the development of pulmonary fibrosis [6-8]. During EMT, alveolar epithelial cells lose their epithelial characteristics, such as the expression of E-cadherin, and acquire mesenchymal features, including the upregulation of α-smooth muscle actin (α-SMA) and vimentin [9, 10]. This abnormal trans-differentiation of alveolar epithelial cells contributes to the increased production of ECM proteins by fibroblasts and myofibroblasts, ultimately driving the fibrotic process [11, 12]. Mitochondrial dysfunction has been increasingly recognized as a critical factor in the pathogenesis of pulmonary fibrosis and the EMT process [13-15]. Mitochondria are the powerhouses of the cell, responsible for energy production through oxidative phosphorylation. In the context of pulmonary fibrosis, mitochondrial damage, including swelling, cristae disruption, and decreased membrane potential, leads to impaired energy metabolism and increased production of reactive oxygen species (ROS) [16-18]. Elevated ROS levels can trigger oxidative stress, which in turn activates various signaling pathways involved in inflammation, fibrosis, and EMT [19, 20]. Caffeic acid phenethyl ester (CAPE), a natural flavonoid compound abundant in propolis, has been extensively studied for its diverse biological activities, including antioxidant, anti-inflammatory, and anti-tumor properties [21-23]. Previous studies have demonstrated that CAPE can scavenge free radicals, inhibit the activation of pro-inflammatory cytokines, and modulate cell signaling pathways [23, 24]. Notably, CAPE’s natural origin confers favorable safety profiles (a key advantage for translational development), yet its potential to regulate mitochondrial homeostasis and inhibit EMT in PF remains largely unexplored. The Sirt1/PGC-1α signaling axis has been identified as a key regulator of mitochondrial function and biogenesis [25-27]. Sirt1, a class III histone deacetylase, can deacetylate and activate PGC-1α, a master regulator of mitochondrial biogenesis and energy metabolism [28, 29]. Activation of the Sirt1/PGC-1α axis has been shown to enhance mitochondrial quality and quantity, improve antioxidant defense, and mitigate fibrotic responses in different organs [30-32]. Notably, the interplay between the Sirt1/PGC-1α axis and EMT regulation in the context of pulmonary fibrosis warrants further investigation. Given the emerging link between Sirt1/PGC-1α and EMT, as well as the critical role of mitochondrial function in the EMT process, we hypothesized that CAPE may exert its anti-fibrotic effects by activating the Sirt1/PGC-1α pathway, thereby improving mitochondrial function and inhibiting EMT. In this study, we aimed to comprehensively investigate the molecular mechanism by which CAPE modulates mitochondrial function and suppresses EMT in the context of pulmonary fibrosis. By using both in vitro and in vivo models, we sought to determine whether CAPE could ameliorate mitochondrial dysfunction, regulate the expression of EMT markers, and ultimately attenuate the development of pulmonary fibrosis. Our findings may provide new insights into the development of natural mitochondrial-targeted drugs for the treatment of pulmonary fibrosis. 2. Materials and Methods 2.1. Animals Experimental Model and Treatment Adult male C57BL/6 mice (6–8 weeks old) weighing between 20-25 g were used in this study. The animals were housed under standard laboratory conditions, maintained on a 12-hour light/dark cycle, and provided with free access to food and water. The mice were acclimatized to the laboratory environment for one week prior to the start of the experiment. All animal protocols were approved by the Committee on Animal Care of the Fourth Military Medical University. For the establishment of the pulmonary fibrosis model, Bleomycin (12 U/8 mg, >98% pure, Taihe Biotechnology, China) was administered via intratracheal injection. Mice were anesthetized using isoflurane to ensure immobility and minimize discomfort during the procedure. Bleomycin was reconstituted in sterile saline and administered at a dose of 7.5 U/kg body weight. According to published studies or our prior experiment, pulmonary fibrosis will be established after 21 days post-treatment of bleomycin. For the treatment, the mice were randomly divided into five groups (each group has 10 mice): (1) Control: mice only received an equivalent volume injection of solvent; (2) BLM: mice received bleomycin and an equivalent volume injection of the vehicle for CAPE; (3-5) BLM + 1/2/5 mg/kg dose Caffeic Acid Phenethyl Ester (CAPE) (Sigma, St. Louis, MO, USA): CAPE was administered intraperitoneally after BLM injection, once daily until the end of 21 days. CAPE was uniformly dissolved in a mixture of 10% dimethyl sulfoxide, 40% PEG300, and 50% saline. All the animals were sacrificed at 21 days post-treatment of chemicals, and the lungs were carefully excised and washed in cold sterile saline to remove blood. The lung tissues were then processed for histological evaluation, including H&E staining and Masson’s trichrome staining to assess fibrosis, as well as for molecular analyses involving the quantification of fibrotic markers. Other lung tissues were stored in liquid nitrogen for further biochemical assessments. Primary alveolar epithelial cells (AECs) were isolated from harvested lungs using enzymatic digestion with collagenase and dispase, followed by mechanical dissociation. 2.2. Cell Culture and Treatment A549 cells (used as human alveolar epithelial cells) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Rockville, MD, USA) and 1% penicillin-streptomycin at 37°C with 5% CO 2 . The cells were allowed to reach 70-80% confluence and were then starved in serum-free DMEM for 24 hours prior to treatment. After the starvation period, the cells were treated with Transforming Growth Factor-beta 1 (TGF-β1)( R&D, Minneapolis, MN, USA) at a concentration of 5ng/mL for 24 hours to induce EMT response. Subsequently, the cells were exposed to varying concentrations of CAPE (2, 5, and 10 μg/mL) for an additional 24 hours. 2.3. Measurement of Reactive Oxygen Species (ROS) Following the treatments, cellular ROS in A549 cells were assessed by fluorescent probe DCFH or MitoSOX (Invitrogen, Carlsbad, CA, USA) through flow cytometry. The excitation and emission wavelength are 504/529 nm for DCFH. MitoSOX has excitation/emission wavelength of 510/580 nm. A549 cells were incubated with medium containing 10 mM DCFH or MitoSOX for 30 min at 37°C. The cells were then washed three times with phosphate buffered saline (PBS) and analysed by flow cytometry. The level of ROS was expressed as cellular fluorescence intensity, shown as the fold change relative to the control. 2.4. Evaluation of Mitochondrial Changes Mitochondrial morphology and number were examined with a HT7700 transmission electron microscope (Hitachi, Tokyo, Japan). For quantitative assessment, mitochondrial DNA (mtDNA) content was measured using quantitative PCR (qPCR). Mitochondrial function was evaluated by measuring ATP levels using an ATP assay kit (S0027, Beyotime Company, Shanghai, China) and mitochondrial membrane potential using the JC-1 or RHO-123 assay Kit (Invitrogen, Carlsbad, CA, USA). 2.5. mRNA and Protein Expression Analysis Total RNA was extracted from the cells using RNA extraction kits, and mRNA levels of SIRT1, PGC-1α, CK18, E-cadherin, vimentin, and SMA were analyzed using quantitative reverse transcription PCR (qRT-PCR) with a Super Real PreMix Plus (SYBR Green) detecting kit (TIANGEN Biotechnology, Beijing, China) as the manufacturer’s protocol description. The relative gene expression was analysed according to the CFX Manager2.1 software (Bio-Rad, USA).Protein extraction was performed with RIPA lysis buffer, and Western blot analysis was conducted to determine the protein levels of the aforementioned molecules. After blocking with skim milk for 1 h at 25°C, blots were probed with indicated primary antibodies (Abcam, Cambridge, UK) against E-cadherin, CK18, α-SMA, vimentin, SIRT1, PGC-1α, NRF-1, TFAM, and GAPDH (1:1000) overnight at 4℃. Then the blots were incubated with HRP-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 25°C. After thorough washing with TBST, target proteins were detected by enhanced chemiluminescence in a CHEMIDOC XRS SYSTEM and analyzed with Quantity One™ software (Bio-Rad, Hercules, CA, USA). 2.6. Immunofluorescence and Immunohistochemical detection Immunofluorescence staining was performed on lung sections to examine the expression of alveolar epithelial or mesenchymal markers. The samples were incubated with primary antibodies against specific proteins (E-cadherin, vimentin and surfactant protein C) were used (diluted to 1:500) overnight at 4°C. After washing with PBS, the cells were incubated with anti-rabbit secondary antibody conjugated to Alexa-488 and 594 fluorescence (Invitrogen, Carlsbad, CA, USA) for 2 h at 20°C. Finally, the nuclei were stained with DAPI for 15 min and the intracellular localization of target proteins was observed with a NIKON ECLIPSE C1 fluorescence microscope (Nikon, Tokyo, Japan). Lung tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval. After blocking endogenous peroxidase, sections were incubated with primary anti-α-SMA and anti-collagen I antibody overnight at 4°C, followed by secondary antibody incubation. DAB staining visualized immunoreactivity. Positive areas were quantified using image analysis software, with results expressed as relative content. 2.7. Molecular docking Target proteins were retrieved from RCSB (http://www.rcsb.org/). Using PyMOL, solvent molecules and original ligands were removed, and proteins were saved in PDB format. AutoDock Tools (ADT) was used to add Gasteiger charges and polar hydrogens, with final saving in PDBQT format. The structure of CAPE (PubChem CID: 5,281,787) was downloaded, converted to 3D using ChemBio 3D, saved as Mol2, and then converted to PDBQT via ADT. Autodock Vina performed docking with the original ligand position as the active site. The grid box was set to enclose the original ligand at its center, with other parameters default. Discovery Studio analyzed interactions of the best-binding complexes. Binding affinity (ΔG) was the key criterion: values <0 indicated spontaneous binding, < -5 kcal/mol indicated stable binding, and < -7 kcal/mol indicated strong binding. 2.8. Statistical Analysis All experimental data were processed and analyzed with GraphPad Prism 10.1.2 software. Differences between groups were evaluated using one-way ANOVA followed by post-hoc tests when applicable. A P -value of <0.05 was considered statistically significant. 3. Results 3.1. CAPE suppressed TGF- 𝛽1 -induced EMT in A549 cells. To elucidate the anti-EMT effects of CAPE, TGF-β1 was employed to induce epithelial-mesenchymal transition in A549 human alveolar epithelial cells in vitro. Morphologically, TGF-β1 stimulation (5ng/mL) induced a characteristic phenotypic switch within 48h (Figure 1A), as evidenced by the transformation of A549 cells from compact, polygonal epithelial-like structures to elongated, spindle-shaped mesenchymal phenotypes with extensive intercellular bridging. However, treatment with CAPE (2-10μg/mL) dose-dependently reversed these morphological changes, restoring the typical epithelial morphology (Figure 1A). Quantitative RT-PCR and western blot analyses further validated the molecular mechanisms (Figure 1B-D). TGF-β1 stimulation significantly downregulated the epithelial markers E-cadherin and CK18, while upregulating the mesenchymal markers vimentin and α-SMA ( P < 0.01). CAPE treatment at 10μg/mL restored E-cadherin and CK18 expression to those of control levels, respectively, while reducing vimentin and α-SMA expression( P < 0.01). Additionally, CAPE effectively suppressed TGF-β1-induced collagen I/III upregulation ( P < 0.05). Collectively, these data demonstrate that CAPE potently suppresses TGF-β1-induced EMT in A549 cells at both morphological and molecular levels. 3.2 CAPE alleviated mitochondria damage during TGF- 𝛽1 -induced EMT in A549 cells. Our previous study showed mitochondria, which produces the main energy for the basic functions of the cell, were closely related to EMT [33], we therefore investigated the potential of CAPE to mitigate mitochondrial damage during TGF-β1-induced EMT in A549 cells. Flow cytometry analysis revealed a significant 8.15-fold increase in intracellular reactive oxygen species (ROS) levels 48 hours after TGF-β1 stimulation ( P < 0.01) (Figure 2A), indicative of oxidative stress-induced mitochondrial dysfunction. Notably, CAPE treatment at 10μg/mL effectively attenuated ROS accumulation ( P < 0.01), demonstrating its potent antioxidant activity. Furthermore, quantitative PCR analysis showed that TGF-β1 exposure led to a 43% reduction in mitochondrial DNA (mtDNA) copy number (Figure 2B), paralleled by a 35% decrease in ATP production and the decline in mitochondrial membrane potential (MMP) (Figure 2C, D), measured by JC-1 staining. However, co-treatment with CAPE restored these parameters in a dose-dependent manner. Specifically, at 10μg/mL, CAPE rescued mtDNA levels to 91% of control values, recovered ATP content by 55% of baseline levels, and maintained mitochondrial membrane potential. Collectively, these data provide robust evidence that CAPE effectively protects against TGF-β1-induced mitochondrial damage, potentially through modulating redox homeostasis and mitochondrial functions during EMT progression. 3.3 The prevention of CAPE on mitochondria damage and EMT were through SIRT1/PGC1a pathway. Sirt1/PGC1-α pathway plays a vital role in maintaining normal mitochondrial status, so we explored whether CAPE prevented mitochondrial damage and EMT through the Sirt1/PGC1-α pathway. To assess the interaction of CAPE and SIRT1, we employed professional tools to simulate the molecular docking of CAPE and SIRT1. Strikingly, CAPE bound to the active pocket of SIRT1 with a binding energy of −7.8 kcal/mol (Figure 3A), indicating a strong potential affinity. Subsequently, qRT-PCR and western blot analyses were conducted. The results demonstrated that TGF-β1 stimulation significantly reduced the mRNA (Figure 3D) and protein levels (Figure 3E) of SIRT1. Conversely, CAPE treatment reversed this TGF-β1-induced downregulation of SIRT1 in a dose-dependent fashion, suggesting that CAPE could enhance SIRT1 expression. Moreover, CAPE significantly increased the mRNA and protein levels of PGC1-α, which were notably decreased by TGF-β1 stimulation. As key downstream molecules of PGC1-α, NRF1 and TFAM, important regulators of mitochondrial biogenesis and function, were downregulated by TGF-β1. However, CAPE rescued the downregulation of NRF1 and TFAM, restoring their expression levels close to those of the control group. To further confirm whether CAPE suppressed TGF-β1-induced mitochondrial damage in A549 cells via the Sirt1/PGC1-α pathway, we utilized Sirt1 shRNA lentivirus to specifically reduce the cellular level of Sirt1. In the presence of Sirt1 shRNA, CAPE failed to alleviate TGF-β1-induced mitochondrial damage, as evidenced by a significant increase in mtROS levels and decreases in mtDNA levels, ATP content, and mitochondrial membrane potential (Figure 4A-D). Additionally, when evaluating EMT markers after TGF-β1 and CAPE treatments, Sirt1 shRNA obviously weakened the inhibitory effect of CAPE on the TGF-β1-induced downregulation of epithelial markers E-cadherin and CK18, as well as the upregulation of mesenchymal markers vimentin and α-SMA (Figure 4F). Taken together, these results strongly suggest that CAPE prevents mitochondrial damage and EMT through the SIRT1/ PGC1-α pathway, likely by upregulating SIRT1 and PGC1-α expression, and restoring the function of its downstream mitochondrial regulatory molecules. 3.4 CAPE inhibited EMT process and collagen production in pulmonary fibrosis model. To investigate the effect of CAPE on EMT process in vivo , we used bleomycin to establish a pulmonary fibrosis model. Pathological staining results demonstrated that after CAPE treatment, the degree of fibrosis in the lung tissues of bleomycin-induced pulmonary fibrosis mice was significantly reduced (Figure 5A). Quantitative analysis of Masson staining revealed that the percentage of collagen fiber area in lung tissues of the model group was (11.7±0.9)%, while that of the CAPE treatment group decreased to (6.1±0.6)%, (4.9±1.0)%, (2.3±0.3)% respectively, indicating that CAPE had a significant inhibitory effect on the process of pulmonary fibrosis ( P < 0.01) (Figure 5F). Immunofluorescence and immunohistochemistry techniques were further used to explore the underlying mechanisms. The results showed that CAPE effectively increased the ratio of alveolar epithelial/mesenchymal cell markers E-cadherin and vimentin, enhancing E-cadherin expression and reducing vimentin expression (Figure 1D, 4F), thereby inhibiting the process of EMT. Meanwhile, CAPE significantly reduced the expression of α-SMA and collagen Ⅰ. Semi-quantitative analysis of immunohistochemistry indicated that compared with the model group, the mean optical density values of α-SMA and collagen Ⅰ in the CAPE treatment group(5mg/kg) decreased by 50.4% and 63.3% ( P < 0.01), respectively(Figure 5F, G). These results suggest that CAPE may alleviate the degree of pulmonary fibrosis by regulating the EMT process and inhibiting collagen synthesis. 3.5 CAPE ameliorated mitochondria damage in lung and epithelial cells during PF. Electron microscopy analysis revealed that bleomycin-induced pulmonary fibrosis in mice led to profound mitochondrial dysfunction characterized by diminished mitochondrial density and structural abnormalities, including swollen cristae and fragmented outer membranes, within lung parenchymal tissues (Figure 6A). Notably, CAPE treatment (5 mg/kg/day for 21 days) restored mitochondrial ultrastructure close to normal status. Concomitantly, primary alveolar epithelial cells isolated from CAPE-treated(10mg/kg) mice exhibited 1.7-fold increase in mitochondrial number and ATP production ( P < 0.01), and 27.7% reduction in mtROS levels ( P < 0.001) compared to the model group (Figure 6B, C, E). This study provides novel evidence for the protective role of CAPE in mitochondrial quality control during fibrotic pathogenesis. Discussion In this study, we demonstrated for the first time that caffeic acid phenethyl ester (CAPE) effectively ameliorates mitochondrial function and suppresses the epithelial-mesenchymal transition (EMT) process, thereby attenuating the development of pulmonary fibrosis. Our results provide novel insights into the molecular mechanism of CAPE's anti-fibrotic effects and highlight the potential of CAPE as a natural mitochondrial-targeted therapeutic agent for pulmonary fibrosis. Mitochondrial dysfunction is a hallmark of pulmonary fibrosis, and restoring mitochondrial function represents a promising therapeutic strategy [34-36]. In our study, both in vivo and in vitro experiments demonstrated that CAPE treatment significantly improved mitochondrial function. In the animal model of bleomycin-induced pulmonary fibrosis, CAPE treatment at a dose of 5mg/kg/d for 21 days restored the normal ultrastructure of mitochondria, as evidenced by the increased number of mitochondria, the restoration of cristae density, and the normalization of mitochondrial membrane potential. These morphological changes were accompanied by functional improvements, including a 1.5-fold increase in ATP production and a more than 30% reduction in mitochondrial ROS levels. Similarly, in vitro , 10μg/ml CAPE effectively reduced ROS overproduction induced by TGF-β1 by 27%, restored ATP production to 55% of the control level, and significantly increased mitochondrial membrane potential. These findings are consistent with previous studies showing that CAPE exhibits potent antioxidant properties [37, 38]. By scavenging free radicals, CAPE can reduce oxidative stress-induced mitochondrial damage, thereby maintaining mitochondrial integrity and function. Moreover, our results suggest that CAPE may enhance mitochondrial biogenesis, as indicated by the increased number of mitochondria in the CAPE treated group. This is further supported by the significant upregulation of PGC-1α mRNA levels by CAPE, as PGC-1α is a master regulator of mitochondrial biogenesis. EMT of alveolar epithelial cells is a crucial event in the pathogenesis of pulmonary fibrosis. In our study, CAPE treatment significantly inhibited the EMT process both in vivo and in vitro . In the animal model, CAPE treatment (10mg/kg) upregulated the expression of the epithelial marker E-cadherin by 4.4-fold and downregulated the expression of the mesenchymal markers α-SMA and vimentin by 49.4%-56.9% (Figure 1D). In the in vitro model of TGF - β1 induced EMT in A549 cells, CAPE also effectively reversed the EMT associated changes in marker expression. The inhibition of EMT by CAPE may be attributed to its regulation of mitochondrial function. Mitochondrial dysfunction can trigger oxidative stress, which in turn activates signaling pathways that promote EMT[39, 40]. By improving mitochondrial function and reducing ROS production, CAPE may disrupt the oxidative stress - mediated EMT signaling cascade. Additionally, CAPE may directly modulate the expression of EMT-related genes through its interaction with specific transcription factors or signaling pathways. Further studies are needed to elucidate the detailed molecular mechanisms underlying the inhibition of CAPE on EMT. Crucially, our results demonstrate that CAPE activates the Sirt1/PGC-1α signaling axis, which plays a crucial role in mediating its beneficial effects on mitochondrial function and EMT. CAPE treatment significantly increased Sirt1 protein expression by 1.9-fold and PGC-1α mRNA levels by 2.1-fold. More importantly, shRNA-mediated Sirt1 knockdown abolished CAPE’s benefits on mitochondrial function and EMT markers. This confirms that Sirt1 is indispensable for CAPE’s anti-fibrotic activity—a key finding for translational development, as it provides a validated biomarker (Sirt1/PGC-1α activation) to monitor therapeutic response in future preclinical/clinical studies. The Sirt1/PGC-1α axis is a well-conserved pathway across species [41–43], further supporting CAPE’s potential to translate to human PF. In conclusion, our study provides compelling evidence that CAPE exerts anti - fibrotic effects by activating the Sirt1/PGC-1α signaling axis, improving mitochondrial function, and inhibiting EMT. These findings offer a new perspective on the development of natural mitochondrial - targeted drugs for the treatment of pulmonary fibrosis and highlight the potential of CAPE as a promising therapeutic agent. From a translational perspective, CAPE offers several advantages over current PF candidates: (1) its natural origin reduces safety concerns (a major barrier to clinical approval for synthetic agents); (2) its dual targeting of mitochondrial dysfunction and EMT addresses two overlapping, critical pathogenic pathways in PF; (3) the Sirt1/PGC-1α axis provides a druggable, biomarker-accessible mechanism for dose optimization and patient stratification. Further work should focus on optimizing CAPE’s delivery (e.g., lung-targeted formulations to enhance bioavailability) and exploring its efficacy in patient-derived PF models to strengthen its translational relevance. Declarations Author Contributions: All authors made contributions to the revision of the manuscript and data analysis until the final version was developed. The experimental conception and data processing were led by R.L. and B.L., with methodological design contributed by M.C. and Z.W. Validation was conducted by X.Z., formal analysis by D.K., and investigation by H.W. Data curation was managed by J.L. The original draft was prepared by R.L., and the manuscript was reviewed and edited by W.Y. Supervision was provided by C.H., with project administration handled by W.L. and W.Y. Funding acquisition was secured by R.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by grants from the National Natural Science Foundation of China (81573126 and 31800706) and the Shaanxi Province Key Research and Development Project of China (2023-YBSF-297). Ethical approval and consent for participation ： All animal procedures were conducted in accordance with the ethical standards approved by the Ethical Review Board of the Laboratory Animal Center of Air Force Medical University (approval No. 20240752) and the laboratory animal license SCXK-2024-003. Availability of data and materials ： The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. Additional data are available from the corresponding author upon reasonable request. No new resources were generated for this study. Conflicts of Interest: The authors declare no conflicts of interest. References Koudstaal T, Funke-Chambour M, Kreuter M, Molyneaux PL, Wijsenbeek MS. Pulmonary fibrosis: from pathogenesis to clinical decision-making. Trends Mol Med. 2023;29:1076–87. Moss BJ, Ryter SW, Rosas IO. Pathogenic mechanisms underlying idiopathic pulmonary fibrosis. Annu Rev Pathol. 2022;17:515–46. Rajan SK, Cottin V, Dhar R, Danoff S, Flaherty KR, Brown KK, et al. Progressive pulmonary fibrosis: an expert group consensus statement. Eur Respir J. 2023;61:2103187. Rajesh R, Atallah R, Bärnthaler T. Dysregulation of metabolic pathways in pulmonary fibrosis. Pharmacol Ther. 2023;246:108436. 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09:19:43","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":872,"visible":true,"origin":"","legend":"","description":"","filename":"JTRMD2515616Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-7559730/v1/df533962c16738d54a8d8aa1.xml"},{"id":92490436,"identity":"52533a6e-10e3-4cb3-9ce5-39b6919feeaf","added_by":"auto","created_at":"2025-09-30 09:27:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":392209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCAPE suppressed TGF-𝛽1-induced EMT in A549 cells.\u003c/em\u003e A. Representative image of A549 cells cell morphology changes observed by microscopy (200×, bright) following treatments with TGF-β1 and CAPE. B. The mRNA levels of E-cadherin, CK18, vimentin, α-SMA, and collagen I/III following treatments with TGF-β1 and CAPE in A549 cells detected by qRT-PCR. C\u0026amp;D. Representative blots and quantified data showing protein expression levels of E-cadherin, CK18, vimentin, α-SMA, and collagen I/III following treatments with TGF-β1 and CAPE in A549 cells. All measurements were made in triplicate, and the data are presented as mean ± SEM. P values are labelled in the graphs.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7559730/v1/e4039796ef9c61af2e1e0b29.png"},{"id":92490439,"identity":"c0c8c52b-1660-40f1-8dc1-ceb7402809e3","added_by":"auto","created_at":"2025-09-30 09:27:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":750083,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCAPE alleviated mitochondria damage during TGF-𝛽1-induced EMT in A549 cells. \u003c/em\u003eA. Intracellular ROS level was detected by flow-cytometry after staining with DCFH following treatments with TGF-β1 and CAPE. B. Mitochondrial DNA content was determined by qPCR following treatments with TGF-β1 and CAPE. C. ATP content was analyzed with a luciferase assay kit. D. Mitochondrial membrane potential (MMP) was detected by fluorescent microscope after staining with JC-1. All measurements were made in triplicate, and the data are presented as mean ± SEM. P values are labelled in the graphs.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7559730/v1/38349dca17d8d29f78da1a67.png"},{"id":92489560,"identity":"21be899d-0364-471a-9631-c01eba962c75","added_by":"auto","created_at":"2025-09-30 09:19:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":264441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCAPE reversed TGF-β1-induced downregulation of Sirt1/PGC1-α pathway. \u003c/em\u003eA. The result from molecular docking of CAPE and SIRT1. B. The mRNA levels of SIRT1, PGC1-α, NRF1 and TFAM following treatments with TGF-β1 and CAPE in A549 cells were determined by qRT-PCR. C\u0026amp;D. Representative blots and quantified data showing protein expression levels of SIRT1, PGC1-α, NRF1 and TFAM following treatments with TGF-β1 and CAPE in A549 cells. All measurements were made in triplicate, and the data are presented as mean ± SEM. P values are labelled in the graphs.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7559730/v1/1a5709558da519fc420c7770.png"},{"id":92489552,"identity":"4eee3c91-6583-49ac-af35-5131b27301e4","added_by":"auto","created_at":"2025-09-30 09:19:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":120261,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCAPE prevented mitochondria damage and EMT via SIRT1/PGC1-a pathway. \u003c/em\u003eA. Mitochondrial ROS level was detected by flow-cytometry after staining with MitoSOX Red following treatments with TGF-β1 and CAPE. B. Mitochondrial DNA content was determined by qPCR following treatments with TGF-β1 and CAPE. C. ATP content was analyzed with a luciferase assay kit. D. Mitochondrial membrane potential (MMP) was detected by flow cytometry after staining with RHO-123. E. Representative blots and quantified data showing protein expression levels of E-cadherin, CK18, vimentin, and a-SMA after treatment with TGF-β1 and CAPE in the vector group and sh-SIRT1 group. All measurements were made in triplicate, and the data are presented as mean ± SEM. P values are labelled in the graphs.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7559730/v1/cb6c8fe246d0941bfcaff1de.png"},{"id":92489557,"identity":"a13f2790-fc4f-47cf-aeb9-1539206f7f97","added_by":"auto","created_at":"2025-09-30 09:19:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":745521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCAPE inhibited EMT process and collagen production in\u003c/em\u003e \u003cem\u003epulmonary fibrosis model.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA. Representative pathological HE staining images of lung lesions following treatments with TGF-β1 and CAPE. (40× and 200×, bright). B. Representative pathological Masson staining images of lung lesions. C. Representative immunofluorescence staining of lung with E-cadherin and vimentin (DAPI staining of nuclei is blue, SPC staining of epithelial cells is red, E-cadherin or vimentin staining is green). D. Representative immunohistochemical staining of lung with α-SMA and collagen I. E-G. The quantified analysis of Masson and immunohistochemical staining. Each group has 12 samples, and the data are presented as mean ± SEM. P values are labelled in the graphs.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7559730/v1/6a2146c5789d7be7d14a15b7.png"},{"id":92490440,"identity":"e961e735-01b0-47fc-8c46-6b368bedbeb5","added_by":"auto","created_at":"2025-09-30 09:27:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":308439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCAPE ameliorated mitochondria damage in lung. \u003c/em\u003eA. Representative image of mitochondrial morphology in lung using transmission electron microscopy at high magnification (5000×). B. Mitochondrial number in lung detected by Mitotraker staining. C. ATP content was analyzed with a luciferase assay kit. D. Mitochondrial membrane potential (MMP) was detected by flow cytometry after staining with RHO-123. E. Mitochondrial ROS level was detected by flow-cytometry after staining with MitoSOX Red. Each group has 6 samples, and three independent experiments were performed. The data are presented as mean ± SEM. P values are labelled in the graphs.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7559730/v1/b93194fb5e1bc05b3cb7048c.png"},{"id":93246617,"identity":"6f3d50dd-ca29-4e7f-9fbd-12e8c1708f9f","added_by":"auto","created_at":"2025-10-10 15:15:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2981126,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7559730/v1/767156e4-35d9-47e2-8d25-8efa764196ea.pdf"},{"id":92489562,"identity":"408168d8-77d9-484a-8c42-43af861accfc","added_by":"auto","created_at":"2025-09-30 09:19:44","extension":"7z","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":79620351,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.7z","url":"https://assets-eu.researchsquare.com/files/rs-7559730/v1/155b49238b65e440e43c7eaa.7z"}],"financialInterests":"","formattedTitle":"Caffeic Acid Phenethyl Ester Ameliorates Pulmonary Fibrosis by Inhibiting Epithelial-Mesenchymal Transition via the Sirt1/PGC-1α/Mitochondrial Axis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePulmonary fibrosis, a devastating and progressive lung disease, represents the end-stage pathological alteration of various chronic respiratory disorders [1-3]. It is characterized by the excessive deposition of extracellular matrix (ECM) proteins, primarily collagen, which leads to the destruction of normal alveolar architecture and a subsequent decline in lung functions [2, 4]. The global burden of pulmonary fibrosis is substantial, with a high mortality rate and limited effective treatment options, highlighting the urgent need for novel therapeutic strategies [5].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe epithelial-mesenchymal transition (EMT) of alveolar epithelial cells has emerged as a central mechanism in the development of pulmonary fibrosis [6-8]. During EMT, alveolar epithelial cells lose their epithelial characteristics, such as the expression of E-cadherin, and acquire mesenchymal features, including the upregulation of \u0026alpha;-smooth muscle actin (\u0026alpha;-SMA) and vimentin [9, 10]. This abnormal trans-differentiation of alveolar epithelial cells contributes to the increased production of ECM proteins by fibroblasts and myofibroblasts, ultimately driving the fibrotic process [11, 12].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMitochondrial dysfunction has been increasingly recognized as a critical factor in the pathogenesis of pulmonary fibrosis and the EMT process [13-15]. Mitochondria are the powerhouses of the cell, responsible for energy production through oxidative phosphorylation. In the context of pulmonary fibrosis, mitochondrial damage, including swelling, cristae disruption, and decreased membrane potential, leads to impaired energy metabolism and increased production of reactive oxygen species (ROS) [16-18]. Elevated ROS levels can trigger oxidative stress, which in turn activates various signaling pathways involved in inflammation, fibrosis, and EMT [19, 20].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCaffeic acid phenethyl ester (CAPE), a natural flavonoid compound abundant in propolis, has been extensively studied for its diverse biological activities, including antioxidant, anti-inflammatory, and anti-tumor properties [21-23]. Previous studies have demonstrated that CAPE can scavenge free radicals, inhibit the activation of pro-inflammatory cytokines, and modulate cell signaling pathways [23, 24]. Notably, CAPE\u0026rsquo;s natural origin confers favorable safety profiles (a key advantage for translational development), yet its potential to regulate mitochondrial homeostasis and inhibit EMT in PF remains largely unexplored.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Sirt1/PGC-1\u0026alpha; signaling axis has been identified as a key regulator of mitochondrial function and biogenesis [25-27]. Sirt1, a class III histone deacetylase, can deacetylate and activate PGC-1\u0026alpha;, a master regulator of mitochondrial biogenesis and energy metabolism [28, 29]. Activation of the Sirt1/PGC-1\u0026alpha; axis has been shown to enhance mitochondrial quality and quantity, improve antioxidant defense, and mitigate fibrotic responses in different organs [30-32]. Notably, the interplay between the Sirt1/PGC-1\u0026alpha;\u0026nbsp;axis and EMT regulation in the context of pulmonary fibrosis warrants further investigation. Given the emerging link between Sirt1/PGC-1\u0026alpha; and EMT, as well as the critical role of mitochondrial function in the EMT process, we hypothesized that CAPE may exert its anti-fibrotic effects by activating the Sirt1/PGC-1\u0026alpha; pathway, thereby improving mitochondrial function and inhibiting EMT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we aimed to comprehensively investigate the molecular mechanism by which CAPE modulates mitochondrial function and suppresses EMT in the context of pulmonary fibrosis. By using both \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e models, we sought to determine whether CAPE could ameliorate mitochondrial dysfunction, regulate the expression of EMT markers, and ultimately attenuate the development of pulmonary fibrosis. Our findings may provide new insights into the development of natural mitochondrial-targeted drugs for the treatment of pulmonary fibrosis.\u0026nbsp;\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e2.1.\u003cem\u003e\u0026nbsp;Animals Experimental Model and Treatment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAdult male C57BL/6 mice (6\u0026ndash;8 weeks old) weighing between 20-25 g were used in this study. The animals were housed under standard laboratory conditions, maintained on a 12-hour light/dark cycle, and provided with free access to food and water. The mice were acclimatized to the laboratory environment for one week prior to the start of the experiment. All animal protocols were approved by the Committee on Animal Care of the Fourth Military Medical University.\u003c/p\u003e\n\u003cp\u003eFor the establishment of the pulmonary fibrosis model, Bleomycin (12 U/8 mg, \u0026gt;98% pure, Taihe Biotechnology, China) was administered via intratracheal injection. Mice were anesthetized using isoflurane to ensure immobility and minimize discomfort during the procedure. Bleomycin was reconstituted in sterile saline and administered at a dose of 7.5 U/kg body weight. According to published studies or our prior experiment, pulmonary fibrosis will be established after 21 days post-treatment of bleomycin. For the treatment, the mice were randomly divided into five groups (each group has 10 mice): (1) Control: mice only received an equivalent volume injection of solvent; (2) BLM: mice received bleomycin and an equivalent volume injection of the vehicle for CAPE; (3-5) BLM + 1/2/5 mg/kg dose Caffeic Acid Phenethyl Ester (CAPE) (Sigma, St. Louis, MO, USA): CAPE was administered intraperitoneally after BLM injection, once daily until the end of 21 days. CAPE was uniformly dissolved in a mixture of 10% dimethyl sulfoxide, 40% PEG300, and 50% saline.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll the animals were sacrificed at 21 days post-treatment of chemicals, and the lungs were carefully excised and washed in cold sterile saline to remove blood. The lung tissues were then processed for histological evaluation, including H\u0026amp;E staining and Masson\u0026rsquo;s trichrome staining to assess fibrosis, as well as for molecular analyses involving the quantification of fibrotic markers. Other lung tissues were stored in liquid nitrogen for further biochemical assessments. Primary alveolar epithelial cells (AECs) were isolated from harvested lungs using enzymatic digestion with collagenase and dispase, followed by mechanical dissociation.\u003c/p\u003e\n\u003cp\u003e2.2. \u003cem\u003eCell Culture and Treatment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA549 cells (used as human alveolar epithelial cells) were cultured in Dulbecco\u0026apos;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Rockville, MD, USA) and 1% penicillin-streptomycin at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were allowed to reach 70-80% confluence and were then starved in serum-free DMEM for 24 hours prior to treatment. After the starvation period, the cells were treated with Transforming Growth Factor-beta 1 (TGF-\u0026beta;1)( R\u0026amp;D, Minneapolis, MN, USA) at a concentration of 5ng/mL for 24 hours to induce EMT response. Subsequently, the cells were exposed to varying concentrations of CAPE (2, 5, and 10 \u0026mu;g/mL) for an additional 24 hours.\u003c/p\u003e\n\u003cp\u003e2.3.\u003cem\u003e\u0026nbsp;Measurement of Reactive Oxygen Species (ROS)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the treatments, cellular ROS in A549 cells were assessed by fluorescent probe DCFH or MitoSOX (Invitrogen, Carlsbad, CA, USA) through flow cytometry. The excitation and emission wavelength are 504/529 nm for DCFH. MitoSOX has excitation/emission wavelength of 510/580 nm. A549 cells were incubated with medium containing 10 mM DCFH or MitoSOX for 30 min at 37\u0026deg;C. The cells were then washed three times with phosphate buffered saline (PBS) and analysed by flow cytometry. The level of ROS was expressed as cellular fluorescence intensity, shown as the fold change relative to the control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.4.\u003cem\u003e\u0026nbsp;Evaluation of Mitochondrial Changes\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial morphology and number were examined with a HT7700 transmission electron microscope (Hitachi, Tokyo, Japan). For quantitative assessment, mitochondrial DNA (mtDNA) content was measured using quantitative PCR (qPCR). Mitochondrial function was evaluated by measuring ATP levels using an ATP assay kit (S0027, Beyotime Company, Shanghai, China) and mitochondrial membrane potential using the JC-1 or RHO-123 assay Kit (Invitrogen, Carlsbad, CA, USA).\u003c/p\u003e\n\u003cp\u003e2.5. \u003cem\u003emRNA and Protein Expression Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the cells using RNA extraction kits, and mRNA levels of SIRT1, PGC-1\u0026alpha;, CK18, E-cadherin, vimentin, and SMA were analyzed using quantitative reverse transcription PCR (qRT-PCR) with a Super Real PreMix Plus (SYBR Green) detecting kit (TIANGEN Biotechnology, Beijing, China) as the manufacturer\u0026rsquo;s protocol description. The relative gene expression was analysed according to the CFX Manager2.1 software (Bio-Rad, USA).Protein extraction was performed with RIPA lysis buffer, and Western blot analysis was conducted to determine the protein levels of the aforementioned molecules. After blocking with skim milk for 1 h at 25\u0026deg;C, blots were probed with indicated primary antibodies (Abcam, Cambridge, UK) against E-cadherin, CK18,\u0026nbsp;\u0026alpha;-SMA, vimentin, SIRT1, PGC-1\u0026alpha;, NRF-1, TFAM, and GAPDH (1:1000) overnight at 4℃. Then the blots were incubated with HRP-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 25\u0026deg;C. After thorough washing with TBST, target proteins were detected by enhanced chemiluminescence in a CHEMIDOC XRS SYSTEM and analyzed with Quantity One\u0026trade;\u0026nbsp;software (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e\n\u003cp\u003e2.6. \u003cem\u003eImmunofluorescence and Immunohistochemical detection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining was performed on lung sections to examine the expression of alveolar epithelial or mesenchymal markers. The samples were incubated with primary antibodies against specific proteins (E-cadherin, vimentin and surfactant protein C) were used (diluted to 1:500) overnight at 4\u0026deg;C. After washing with PBS, the cells were incubated with anti-rabbit secondary antibody conjugated to Alexa-488 and 594 fluorescence (Invitrogen, Carlsbad, CA, USA) for 2 h at 20\u0026deg;C. Finally, the nuclei were stained with DAPI for 15 min and the intracellular localization of target proteins was observed with a NIKON ECLIPSE C1 fluorescence microscope (Nikon, Tokyo, Japan). Lung tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval. After blocking endogenous peroxidase, sections were incubated with primary anti-\u0026alpha;-SMA and anti-collagen I antibody overnight at 4\u0026deg;C, followed by secondary antibody incubation. DAB staining visualized immunoreactivity. Positive areas were quantified using image analysis software, with results expressed as relative content.\u003c/p\u003e\n\u003cp\u003e2.7.\u003cem\u003e\u0026nbsp;Molecular docking\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTarget proteins were retrieved from RCSB (http://www.rcsb.org/). Using PyMOL, solvent molecules and original ligands were removed, and proteins were saved in PDB format. AutoDock Tools (ADT) was used to add Gasteiger charges and polar hydrogens, with final saving in PDBQT format. The structure of CAPE (PubChem CID: 5,281,787) was downloaded, converted to 3D using ChemBio 3D, saved as Mol2, and then converted to PDBQT via ADT. Autodock Vina performed docking with the original ligand position as the active site. The grid box was set to enclose the original ligand at its center, with other parameters default. Discovery Studio analyzed interactions of the best-binding complexes. Binding affinity (\u0026Delta;G) was the key criterion: values \u0026lt;0 indicated spontaneous binding, \u0026lt; -5 kcal/mol indicated stable binding, and \u0026lt; -7 kcal/mol indicated strong binding.\u003c/p\u003e\n\u003cp\u003e2.8.\u003cem\u003e\u0026nbsp;Statistical Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental data were processed and analyzed with GraphPad Prism 10.1.2 software. Differences between groups were evaluated using one-way ANOVA followed by post-hoc tests when applicable. A \u003cem\u003eP\u003c/em\u003e-value of \u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1.\u003cem\u003e\u0026nbsp;CAPE suppressed TGF-\u003c/em\u003e\u003cem\u003e𝛽1\u003c/em\u003e\u003cem\u003e-induced EMT in A549 cells.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the anti-EMT effects of CAPE, TGF-\u0026beta;1 was employed to induce epithelial-mesenchymal transition in A549 human alveolar epithelial cells in vitro. Morphologically, TGF-\u0026beta;1 stimulation (5ng/mL) induced a characteristic phenotypic switch within 48h (Figure 1A), as evidenced by the transformation of A549 cells from compact, polygonal epithelial-like structures to elongated, spindle-shaped mesenchymal phenotypes with extensive intercellular bridging. However, treatment with CAPE (2-10\u0026mu;g/mL) dose-dependently reversed these morphological changes, restoring the typical epithelial morphology (Figure 1A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQuantitative RT-PCR and western blot analyses further validated the molecular mechanisms (Figure 1B-D). TGF-\u0026beta;1 stimulation significantly downregulated the epithelial markers E-cadherin and CK18, while upregulating the mesenchymal markers vimentin and \u0026alpha;-SMA (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01). CAPE treatment at 10\u0026mu;g/mL restored E-cadherin and CK18 expression to those of control levels, respectively, while reducing vimentin and \u0026alpha;-SMA expression(\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01). Additionally, CAPE effectively suppressed TGF-\u0026beta;1-induced collagen I/III upregulation (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05). Collectively, these data demonstrate that CAPE potently suppresses TGF-\u0026beta;1-induced EMT in A549 cells at both morphological and molecular levels.\u003c/p\u003e\n\u003cp\u003e3.2 \u003cem\u003eCAPE alleviated mitochondria damage during TGF-\u003c/em\u003e\u003cem\u003e𝛽1\u003c/em\u003e\u003cem\u003e-induced EMT in A549 cells.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOur previous study showed mitochondria,\u0026nbsp;which produces the main energy for the basic functions of the cell, were closely related to EMT [33], we therefore investigated the potential of CAPE to mitigate mitochondrial damage during TGF-\u0026beta;1-induced EMT in A549 cells. Flow cytometry analysis revealed a significant 8.15-fold increase in intracellular reactive oxygen species (ROS) levels 48 hours after TGF-\u0026beta;1 stimulation (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01) (Figure 2A), indicative of oxidative stress-induced mitochondrial dysfunction. Notably, CAPE treatment at 10\u0026mu;g/mL effectively attenuated ROS accumulation (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01), demonstrating its potent antioxidant activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, quantitative PCR analysis showed that TGF-\u0026beta;1 exposure led to a 43% reduction in mitochondrial DNA (mtDNA) copy number (Figure 2B), paralleled by a 35% decrease in ATP production and the decline in mitochondrial membrane potential (MMP) (Figure 2C, D), measured by JC-1 staining. However, co-treatment with CAPE restored these parameters in a dose-dependent manner. Specifically, at 10\u0026mu;g/mL, CAPE rescued mtDNA levels to 91% of control values, recovered ATP content by 55% of baseline levels, and maintained mitochondrial membrane potential.\u0026nbsp;Collectively, these data provide robust evidence that CAPE effectively protects against TGF-\u0026beta;1-induced mitochondrial damage, potentially through modulating redox homeostasis and mitochondrial functions during EMT progression.\u003c/p\u003e\n\u003cp\u003e3.3 \u003cem\u003eThe prevention of CAPE on mitochondria damage and EMT were through SIRT1/PGC1a pathway.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSirt1/PGC1-\u0026alpha; pathway plays a vital role in maintaining normal mitochondrial status, so we explored whether CAPE prevented mitochondrial damage and EMT through the Sirt1/PGC1-\u0026alpha; pathway. To assess the interaction of CAPE and SIRT1, we employed professional tools to simulate the molecular docking of CAPE and SIRT1. Strikingly, CAPE bound to the active pocket of SIRT1 with a binding energy of \u0026minus;7.8 kcal/mol (Figure 3A), indicating a strong potential affinity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubsequently, qRT-PCR and western blot analyses were conducted. The results demonstrated that TGF-\u0026beta;1 stimulation significantly reduced the mRNA (Figure 3D) and protein levels\u0026nbsp;(Figure 3E) of SIRT1. Conversely, CAPE treatment reversed this TGF-\u0026beta;1-induced downregulation of SIRT1 in a dose-dependent fashion, suggesting that CAPE could enhance SIRT1 expression. Moreover, CAPE significantly increased the mRNA and protein levels of PGC1-\u0026alpha;, which were notably decreased by TGF-\u0026beta;1 stimulation. As key downstream molecules of PGC1-\u0026alpha;, NRF1 and TFAM, important regulators of mitochondrial biogenesis and function, were downregulated by TGF-\u0026beta;1. However, CAPE rescued the downregulation of NRF1 and TFAM, restoring their expression levels close to those of the control group.\u003c/p\u003e\n\u003cp\u003eTo further confirm whether CAPE suppressed TGF-\u0026beta;1-induced mitochondrial damage in A549 cells via the Sirt1/PGC1-\u0026alpha; pathway, we utilized Sirt1 shRNA lentivirus to specifically reduce the cellular level of Sirt1. In the presence of Sirt1 shRNA, CAPE failed to alleviate TGF-\u0026beta;1-induced mitochondrial damage, as evidenced by a significant increase in mtROS levels and decreases in mtDNA levels, ATP content, and mitochondrial membrane potential (Figure 4A-D). Additionally, when evaluating EMT markers after TGF-\u0026beta;1 and CAPE treatments, Sirt1 shRNA obviously weakened the inhibitory effect of CAPE on the TGF-\u0026beta;1-induced downregulation of epithelial markers E-cadherin and CK18, as well as the upregulation of mesenchymal markers vimentin and \u0026alpha;-SMA (Figure 4F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaken together, these results strongly suggest that CAPE prevents mitochondrial damage and EMT through the SIRT1/ PGC1-\u0026alpha; pathway, likely by upregulating SIRT1 and PGC1-\u0026alpha; expression, and restoring the function of its downstream mitochondrial regulatory molecules.\u003c/p\u003e\n\u003cp\u003e3.4 \u003cem\u003eCAPE inhibited EMT process and collagen production in\u003c/em\u003e \u003cem\u003epulmonary fibrosis model.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of CAPE on EMT process \u003cem\u003ein vivo\u003c/em\u003e, we used bleomycin to establish a pulmonary fibrosis model. Pathological staining results demonstrated that after CAPE treatment, the degree of fibrosis in the lung tissues of bleomycin-induced pulmonary fibrosis mice was significantly reduced\u0026nbsp;(Figure 5A). Quantitative analysis of Masson staining revealed that the percentage of collagen fiber area in lung tissues of the model group was (11.7\u0026plusmn;0.9)%, while that of the CAPE treatment group decreased to (6.1\u0026plusmn;0.6)%, (4.9\u0026plusmn;1.0)%, (2.3\u0026plusmn;0.3)% respectively,\u0026nbsp;indicating that CAPE had a significant inhibitory effect on the process of pulmonary fibrosis (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01)\u0026nbsp;(Figure 5F). Immunofluorescence and immunohistochemistry techniques were further used to explore the underlying mechanisms. The results showed that CAPE effectively increased the ratio of alveolar epithelial/mesenchymal cell markers E-cadherin and vimentin, enhancing E-cadherin expression and reducing vimentin expression (Figure 1D, 4F), thereby inhibiting the process of EMT. Meanwhile, CAPE significantly reduced the expression of \u0026alpha;-SMA and collagen Ⅰ. Semi-quantitative analysis of immunohistochemistry indicated that compared with the model group, the mean optical density values of \u0026alpha;-SMA and collagen Ⅰ in the CAPE treatment group(5mg/kg) decreased by 50.4% and 63.3%\u0026nbsp;(\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01), respectively(Figure\u0026nbsp;5F, G). These results suggest that CAPE may alleviate the degree of pulmonary fibrosis by regulating the EMT process and inhibiting collagen synthesis.\u003c/p\u003e\n\u003cp\u003e3.5\u003cem\u003e\u0026nbsp;CAPE ameliorated mitochondria damage in lung and epithelial cells during PF.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eElectron microscopy analysis revealed that bleomycin-induced pulmonary fibrosis in mice led to profound mitochondrial dysfunction characterized by diminished mitochondrial density and structural abnormalities, including swollen cristae and fragmented outer membranes, within lung parenchymal tissues (Figure 6A). Notably, CAPE treatment (5 mg/kg/day for 21 days) restored mitochondrial ultrastructure close to normal status. Concomitantly, primary alveolar epithelial cells isolated from CAPE-treated(10mg/kg) mice exhibited 1.7-fold increase in mitochondrial number and ATP production (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01), and 27.7% reduction in mtROS levels (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) compared to the model group (Figure 6B, C, E). This study provides novel evidence for the protective role of CAPE in mitochondrial quality control during fibrotic pathogenesis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated for the first time that caffeic acid phenethyl ester (CAPE) effectively ameliorates mitochondrial function and suppresses the epithelial-mesenchymal transition (EMT) process, thereby attenuating the development of pulmonary fibrosis. Our results provide novel insights into the molecular mechanism of CAPE\u0026apos;s anti-fibrotic effects and highlight the potential of CAPE as a natural mitochondrial-targeted therapeutic agent for pulmonary fibrosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMitochondrial dysfunction is a hallmark of pulmonary fibrosis, and restoring mitochondrial function represents a promising therapeutic strategy [34-36]. In our study, both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments demonstrated that CAPE treatment significantly improved mitochondrial function. In the animal model of bleomycin-induced pulmonary fibrosis, CAPE treatment at a dose of 5mg/kg/d for 21 days restored the normal ultrastructure of mitochondria, as evidenced by the increased number of mitochondria, the restoration of cristae density, and the normalization of mitochondrial membrane potential. These morphological changes were accompanied by functional improvements, including a 1.5-fold increase in ATP production and a more than 30% reduction in mitochondrial ROS levels. Similarly, \u003cem\u003ein vitro\u003c/em\u003e, 10\u0026mu;g/ml CAPE effectively reduced ROS overproduction induced by TGF-\u0026beta;1 by 27%, restored ATP production to 55% of the control level, and significantly increased mitochondrial membrane potential. These findings are consistent with previous studies showing that CAPE exhibits potent antioxidant properties [37, 38]. By scavenging free radicals, CAPE can reduce oxidative stress-induced mitochondrial damage, thereby maintaining mitochondrial integrity and function. Moreover, our results suggest that CAPE may enhance mitochondrial biogenesis, as indicated by the increased number of mitochondria in the CAPE treated group. This is further supported by the significant upregulation of PGC-1\u0026alpha; mRNA levels by CAPE, as PGC-1\u0026alpha; is a master regulator of mitochondrial biogenesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEMT of alveolar epithelial cells is a crucial event in the pathogenesis of pulmonary fibrosis. In our study, CAPE treatment significantly inhibited the EMT process both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. In the animal model, CAPE treatment (10mg/kg) upregulated the expression of the epithelial marker E-cadherin by 4.4-fold and downregulated the expression of the mesenchymal markers \u0026alpha;-SMA and vimentin by 49.4%-56.9% (Figure 1D). In the \u003cem\u003ein vitro\u003c/em\u003e model of TGF - \u0026beta;1 induced EMT in A549 cells, CAPE also effectively reversed the EMT associated changes in marker expression. The inhibition of EMT by CAPE may be attributed to its regulation of mitochondrial function. Mitochondrial dysfunction can trigger oxidative stress, which in turn activates signaling pathways that promote EMT[39, 40]. By improving mitochondrial function and reducing ROS production, CAPE may disrupt the oxidative stress - mediated EMT signaling cascade. Additionally, CAPE may directly modulate the expression of EMT-related genes through its interaction with specific transcription factors or signaling pathways. Further studies are needed to elucidate the detailed molecular mechanisms underlying the inhibition of CAPE on EMT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCrucially, our results demonstrate that CAPE activates the Sirt1/PGC-1\u0026alpha; signaling axis, which plays a crucial role in mediating its beneficial effects on mitochondrial function and EMT. CAPE treatment significantly increased Sirt1 protein expression by 1.9-fold and PGC-1\u0026alpha; mRNA levels by 2.1-fold. More importantly, shRNA-mediated Sirt1 knockdown abolished CAPE\u0026rsquo;s benefits on mitochondrial function and EMT markers. This confirms that Sirt1 is indispensable for CAPE\u0026rsquo;s anti-fibrotic activity\u0026mdash;a key finding for translational development, as it provides a validated biomarker (Sirt1/PGC-1\u0026alpha;\u0026nbsp;activation) to monitor therapeutic response in future preclinical/clinical studies. The Sirt1/PGC-1\u0026alpha;\u0026nbsp;axis is a well-conserved pathway across species [41\u0026ndash;43], further supporting CAPE\u0026rsquo;s potential to translate to human PF.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study provides compelling evidence that CAPE exerts anti - fibrotic effects by activating the Sirt1/PGC-1\u0026alpha; signaling axis, improving mitochondrial function, and inhibiting EMT. These findings offer a new perspective on the development of natural mitochondrial - targeted drugs for the treatment of pulmonary fibrosis and highlight the potential of CAPE as a promising therapeutic agent.\u0026nbsp;From a translational perspective, CAPE offers several advantages over current PF candidates: (1) its natural origin reduces safety concerns (a major barrier to clinical approval for synthetic agents); (2) its dual targeting of mitochondrial dysfunction and EMT addresses two overlapping, critical pathogenic pathways in PF; (3) the Sirt1/PGC-1\u0026alpha; axis provides a druggable, biomarker-accessible mechanism for dose optimization and patient stratification.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurther work should focus on optimizing CAPE\u0026rsquo;s delivery (e.g., lung-targeted formulations to enhance bioavailability) and exploring its efficacy in patient-derived PF models to strengthen its translational relevance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eAll authors made contributions to the revision of the manuscript and data analysis until the final version was developed. The experimental conception and data processing were led by R.L. and B.L., with methodological design contributed by M.C. and Z.W. Validation was conducted by X.Z., formal analysis by D.K., and investigation by H.W. Data curation was managed by J.L. The original draft was prepared by R.L., and the manuscript was reviewed and edited by W.Y. Supervision was provided by C.H., with project administration handled by W.L. and W.Y. Funding acquisition was secured by R.L. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was supported by grants from the National Natural Science Foundation of China (81573126 and 31800706) and the Shaanxi Province Key Research and Development Project of China (2023-YBSF-297).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent for participation\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eAll animal procedures were conducted in accordance with the ethical standards approved by the Ethical Review Board of the Laboratory Animal Center of Air Force Medical University (approval No. 20240752) and the laboratory animal license SCXK-2024-003.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. Additional data are available from the corresponding author upon reasonable request. No new resources were generated for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKoudstaal T, Funke-Chambour M, Kreuter M, Molyneaux PL, Wijsenbeek MS. Pulmonary fibrosis: from pathogenesis to clinical decision-making. Trends Mol Med. 2023;29:1076\u0026ndash;87. \u003c/li\u003e\n\u003cli\u003eMoss BJ, Ryter SW, Rosas IO. Pathogenic mechanisms underlying idiopathic pulmonary fibrosis. Annu Rev Pathol. 2022;17:515\u0026ndash;46.\u003c/li\u003e\n\u003cli\u003eRajan SK, Cottin V, Dhar R, Danoff S, Flaherty KR, Brown KK, et al. Progressive pulmonary fibrosis: an expert group consensus statement. Eur Respir J. 2023;61:2103187. \u003c/li\u003e\n\u003cli\u003eRajesh R, Atallah R, B\u0026auml;rnthaler T. 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Int Immunopharmacol. 2024;134:112257.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Caffeic acid phenethyl ester (CAPE), Pulmonary fibrosis, Epithelial–mesenchymal transition (EMT)","lastPublishedDoi":"10.21203/rs.3.rs-7559730/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7559730/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Pulmonary fibrosis is a severe lung disease characterized by the epithelial-mesenchymal transition (EMT) of alveolar epithelial cells, leading to an increase in fibroblasts or myofibroblasts. Currently, effective therapeutic options for PF remain limited, rendering the inhibition or reversal of EMT a clinically imperative goal. Caffeic acid phenethyl ester (CAPE), a natural flavonoid, exhibits various biological activities, including antioxidant, anti-inflammatory, anticancer, antiviral, and immunomodulatory effects. However, the role of CAPE in EMT-related diseases such as pulmonary fibrosis remains unclear. This study aimed to investigate whether CAPE can target the Sirtuin 1 (Sirt1)/Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) pathway to regulate mitochondrial function, thereby inhibiting EMT and pulmonary fibrosis. Our in vitro and in vivo findings demonstrate that CAPE significantly improves the quantity and function of mitochondria including mitochondrial DNA (mtDNA) content, ATP level, or mitochondrial membrane potential in altered alveolar epithelial cells, increases the ratio of alveolar epithelial to mesenchymal cell markers, and reduces ROS level or collagen expression, ultimately alleviating the degree of fibrosis. These findings establish a robust preclinical foundation for the translational application of CAPE in EMT-related diseases. In conclusion, CAPE may serve as a potential therapeutic agent for pulmonary fibrosis by modulating mitochondrial function through the Sirt1/PGC-1α pathway, underscoring its potential for clinical translation and merit for further investigative efforts in other EMT-associated conditions.","manuscriptTitle":"Caffeic Acid Phenethyl Ester Ameliorates Pulmonary Fibrosis by Inhibiting Epithelial-Mesenchymal Transition via the Sirt1/PGC-1α/Mitochondrial Axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-30 09:19:38","doi":"10.21203/rs.3.rs-7559730/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c0df3f16-c8eb-4bba-a185-ca990f88402a","owner":[],"postedDate":"September 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-10T15:07:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-30 09:19:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7559730","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7559730","identity":"rs-7559730","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}