The antioxidant effects of decursin inhibit EndMT progression through PI3K/AKT/NF-κB and Smad signaling pathways

Abstract

Endothelial cells (ECs) undergo endothelial-to-mesenchymal transition (EndMT) during the pathophysiology of cardiovascular diseases, a complex cellular transdifferentiation process closely associated with increased oxidative stress under adverse conditions such as myocardial infarction (MI). Decursin, a major constituent of Angelica gigas Nakai, displays diverse pharmacological properties. This study aimed to examine the antioxidant impact of decursin on EndMT regulation in both in vitro and in vivo models as a potential therapeutic strategy for MI. In vitro the inhibitory effects of decursin treatment were analyzed by measuring the expression of EndMT-associated genes, assessing endothelial function, intracellular ROS levels, and mitochondrial membrane potential. Furthermore, the study elucidated antioxidation-related signaling mechanisms within EndMT-induced ECs. In vivo, the therapeutic potential of decursin was investigated using a mouse model of MI. Decursin administration attenuated the EndMT process by upregulating CD31 and VE-Cadherin while decreasing fibronectin and α-SMA expression in EndMT-induced ECs. It also lowered ROS levels, preserved mitochondrial membrane potential, and modulated functional properties, resulting in enhanced LDL uptake and diminished endothelial permeability. Endothelial integrity was sustained via regulation of the PI3K/AKT/NF-κB and Smad-dependent signaling pathways, both responsive to oxidative stress during EndMT. In the MI mouse model, decursin reversed EndMT, lessened myocardial fibrosis and apoptosis, and promoted recovery of infarcted regions. The treated hearts demonstrated improved cardiovascular performance. Decursin represents a novel therapeutic strategy targeting intracellular oxidative stress induced by EndMT. By exerting antioxidant activity through the PI3K/AKT/NF-κB and Smad-dependent pathways, decursin maintains endothelial function, suppresses myocardial fibrosis, and supports cardiac recovery following MI therapy.

INTRODUCTION

Endothelial cells (ECs) reside on the inner surfaces of blood vessel walls within the cardiovascular system and are integral to the pathophysiology of cardiovascular diseases (CVDs) through multiple biological processes (1, 2). EC dysfunction serves as a defining feature of various CVDs, including myocardial infarction (MI) (3). Upon exposure to diverse stressors, ECs undergo a dynamic cellular transdifferentiation process called the endothelial-to-mesenchymal transition (EndMT), characterized by the loss of endothelial properties and acquisition of mesenchymal phenotypes (4, 5). Under pathological states, this cellular transition elevates oxidative stress, disrupts homeostatic balance, induces endothelial fibrosis, and aggravates myocardial dysfunction (6, 7). Recent investigations have concentrated on the molecular mechanisms and therapeutic potential of targeting MI-induced EndMT. Furthermore, patients who have limited responsiveness to current therapies necessitate innovative approaches to enhance the pharmacological outcomes of MI treatment.

Decursin is a pyranocoumarin compound derived from the roots of Angelica gigas Nakai and has been traditionally applied in the management of gynecological disorders (8). In the past decade, research on decursin has expanded, investigating its therapeutic potential across a broad spectrum of clinical applications. This compound displays diverse pharmacological properties, including anti-inflammatory, anticancer, and neuroprotective activities (9). Decursin mediates its effects by modulating immune responses to inhibit inflammatory conditions and by providing neuroprotection in neuronal injury models (9, 10). As a result, the molecular mechanisms and related signaling pathways have been examined in emerging contexts. Recent evidence demonstrates that decursin mitigates oxidative stress in a range of disease models. Specifically, it suppresses osteoarthritis progression by acting on the phosphatidylinositol 3-kinase (PI3K)/AKT and nuclear factor kappa B (NF-κB) signaling pathways in in vitro and in vivo systems (11). Furthermore, decursin inhibits hepatocellular carcinoma progression by modulating Hippo/YAP signaling pathway-related proteins (12). Despite these established activities, its antioxidant capacity in the context of EndMT has not been extensively investigated. Notably, research on the cardioprotective actions of decursin remains in the preliminary stages, with ongoing efforts to clarify the relevant signaling pathways and mechanisms. Therefore, this study aims to elucidate the role of decursin in modulating EndMT and reducing oxidative stress via distinct molecular pathways.

In the present study, we investigated the antioxidative capacity of decursin utilizing both in vitro EndMT progression and in vivo MI models. Additionally, we sought to delineate the underlying mechanisms through which decursin modulates signaling pathways, including PI3K/AKT/NF-κB and Smad-dependent pathways, involved in antioxidant responses in EndMT-induced ECs. Although reperfusion therapy aims to restore blood flow and address symptoms in infarcted hearts, it does not adequately target the molecular mechanisms driving disease progression. Therefore, we propose decursin as a novel drug candidate and therapeutic approach for treating MI and other CVDs, based on evidence that decursin alleviates EndMT by regulating pathways associated with oxidative stress.

RESULTS

Decursin suppresses transforming growth factor (TGF)-β2/ interleukin (IL)-1β-induced EndMT progression in human umbilical vein endothelial cells (HUVECs)

Previous reports have shown that mRNA levels of TGF-β2 and IL-1β are elevated in MI mouse models (13). To reproduce EndMT progression in HUVECs, we employed TGF-β2 and IL-1β as inducers. HUVECs exposed to TGF-β2/IL-1β exhibited morphological changes, transitioning from a typical cobblestone-like appearance to an elongated and spindle-shaped morphology (Fig. 1A). Prior to examining the effect of decursin on EndMT-induced ECs, we evaluated its cytotoxicity to confirm suitability for further investigation. Our data showed that decursin did not elicit significant cytotoxicity at any concentration tested (10-50 μM) (Fig. 1C). EndMT-induced HUVECs were then treated with escalating concentrations of decursin to assess its molecular effects on EndMT. Administration of decursin led to dose-dependent upregulation of the endothelial markers CD31 and VE-cadherin, while reducing the mesenchymal markers α-smooth muscle actin (SMA) and fibronectin in a similar dose-dependent manner. These results demonstrate that decursin suppressed cellular transition in ECs (Fig. 1D, E). To further evaluate endothelial function, TGF-β2/IL-1β-treated EndMT-induced HUVECs were labeled with low-density lipoprotein (LDL) in the presence or absence of decursin. EndMT induction resulted in loss of LDL uptake capacity in HUVECs, signifying diminished endothelial identity and enhanced mesenchymal phenotype. Decursin exposure restored LDL uptake, indicating its role in preventing EndMT-induced phenotypic changes (Fig. 1F, G). Moreover, increased permeability typically associated with EndMT was reduced in a dose-dependent manner upon decursin treatment (Fig. 1H). Collectively, these findings indicate that decursin has strong potential as a therapeutic agent for modulating EndMT progression by inhibiting the mesenchymal transition and maintaining endothelial function.

Fig. 1.

DE suppresses TGF-β2/IL-1β-induced EndMT progression in HUVECs. (A) Representative images showing HUVEC morphological alterations. (B) DE chemical structure. (C) Results of DE cytotoxicity assays. (D, E) Levels of endothelial and mesenchymal marker expression. (F, G) Immunofluorescence detection of LDL uptake. (H) Alterations in endothelial monolayer permeability in EndMT-induced HUVECs following increasing concentrations of DE. N.S., not significant; ^###P < 0.001 vs. HUVECs not exposed to TGF-β2/IL-1β and DE; *P < 0.05, ***P < 0.001, vs. EndMT-induced HUVECs without DE treatment.

Decursin suppresses HUVEC migration by modulating Rho GTPase pathways and influencing cofilin activity

Cell migration and invasion play key roles in the progression of numerous human diseases (14). During EndMT (4), the acquisition of mesenchymal morphology is associated with enhanced migratory and invasive cellular behaviors. To examine the effects of decursin on EndMT-related migration and invasion, we conducted wound healing and transwell assays. The wound healing assay revealed that decursin treatment of TGF-β2/IL-1β-induced HUVECs significantly delayed scratch wound closure, reflecting decreased migration compared to the heightened migratory activity seen in EndMT-induced HUVECs (Fig. 2A, B). In concordance, the transwell assay demonstrated that increasing concentrations of decursin resulted in a progressive decline in the number of cells traversing the membrane (Fig. 2C, D). RhoA and cell division cycle (Cdc) 42, critical Rho GTPase family members, are known to regulate actin dynamics and promote cellular motility (14, 15). Cofilin, an actin-binding protein, facilitates cell migration by remodeling the actin cytoskeleton and modulating actin-driven protrusions (16). To clarify whether decursin alters molecular regulators of cellular migration, we analyzed phosphorylation levels of RhoA, cdc42, and cofilin in EndMT-induced HUVECs. TGF-β2/IL-1β-treated HUVECs exhibited marked suppression of the intracellular pathways associated with enhanced phosphorylation of these key molecules (Fig. 2E, F). Collectively, these results indicate that decursin influences actin dynamics-related pathways and interferes with cytoskeletal reorganization, thus impairing EndMT-mediated migration and restricting EndMT progression.

Fig. 2.

DE restricts HUVEC migratory ability by influencing Rho GTPase signaling and cofilin activation. (A, B) Analysis of wound closure. (C, D) Quantification of cell migration. (E, F) Assessment of RhoA, cdc42, and cofilin phosphorylation in EndMT-induced HUVECs following DE exposure. ^###P < 0.001 vs. HUVECs not treated with TGF-β2/IL-1β and DE; *P < 0.05, **P < 0.01, ***P < 0.001 vs. EndMT-induced HUVECs not treated with DE.

Decursin mitigates intracellular reactive oxygen species (ROS) generation and alleviates mitochondrial dysfunction in EndMT-induced ECs

Mitochondria serve as primary generators of cellular ROS when exposed to a variety of stressors (17). Increased ROS production induces persistent mitochondrial membrane impairment, contributing to progressive cellular dysfunction and adverse pathological outcomes (18). We investigated the mitochondrial protective effects of decursin under conditions of intracellular oxidative stress in EndMT-induced ECs (Fig. 3A, B). Fluorescence microscopy revealed that decursin-treated groups displayed dose-dependent reductions in green fluorescence specific to the DCF-DA probe relative to the untreated EndMT-induced group. We evaluated the capacity of decursin to protect mitochondria from intracellular oxidative stress in EndMT-induced ECs (Fig. 3A, B). The impact of decursin on preserving mitochondrial membrane potential was assessed by measuring tetramethylrhodamine (TMRE) intensity, which showed a dose-dependent prevention of mitochondrial depolarization in EndMT-induced ECs (Fig. 3C, D). These findings establish that decursin effectively suppresses EndMT-associated intracellular ROS accumulation and prevents mitochondrial depolarization, thereby preserving mitochondrial function.

Fig. 3.

DE exerts protective effects by reducing intracellular ROS levels and preserving mitochondrial function. (A) Microscopy images depicting intracellular ROS generation (B) and quantification of green fluorescence intensity for ROS. (C) Evaluation of mitochondrial membrane potential by TMRE staining (D) and quantification of red fluorescence intensity of active mitochondria. (E, F) Influence of DE on PI3K/AKT/NF-κB and Smad-mediated signaling pathways. ^###P < 0.001 vs. HUVECs not treated with TGF-β2/IL-1β and DE; *P < 0.05, **P < 0.01, ***P < 0.001 vs. EndMT-induced HUVECs not treated with DE.

Decursin inhibits EndMT progression via PI3K/AKT/NF-κB and Smad-dependent signaling pathways in EndMT-induced ECs

EndMT progression is governed by the modulation of cellular signaling pathways, specifically the PI3K/AKT/NF-κB and Smad-dependent pathways, which are crucial in regulating intracellular ROS and contributing to cardiac dysfunction (19, 20). Furthermore, decursin modulates several cellular signaling pathways as part of its therapeutic effects in various diseases, with particular evidence supporting its activity in the PI3K/Akt, NF-κB, and Smad pathways (11, 21). To determine the impact of decursin on these pathways in EndMT-induced HUVECs, we measured the expression levels of major proteins involved. We observed that treatment with TGF-β2/IL-1β increased the phosphorylation of PI3K, AKT, NF-κB, and Smad2/3, indicative of EndMT advancement and resulting endothelial dysfunction. In contrast, decursin intervention reduced the phosphorylation of these proteins in EndMT-induced HUVECs (Fig. 3E, F). This demonstrates that decursin inhibits activation of the implicated signaling pathways, thereby blocking EndMT progression and diminishing ROS-associated endothelial injury. Collectively, these data support the potential of decursin as a therapeutic agent to suppress EndMT by modulating key signaling pathways and thus safeguarding against ROS-mediated endothelial dysfunction and cardiac pathology.

Decursin alleviates MI-induced EndMT and subsequent fibrosis in the post-ischemic myocardium

To assess the therapeutic efficacy of decursin in modulating EndMT progression following MI, animals received decursin (25 mg/kg) at predetermined intervals after MI (Fig. 4A). The expression of cell transition markers α-SMA and VE-cadherin was analyzed using immunofluorescence staining. In the ischemic myocardium, α-SMA expression was elevated, while VE-cadherin expression declined, indicating EndMT activation within the infarcted region. Administration of decursin resulted in decreased α-SMA levels alongside enhancement of VE-cadherin expression. These findings indicate that decursin may suppress EndMT and contribute to the preservation of infarcted myocardium (Fig. 4B). To further characterize the effects of decursin on MI-induced fibrosis and EndMT, we examined the mRNA levels of key EndMT-associated genes, specifically, Snail, ferroptosis suppressor protein 1 (Fsp1), collagen type I alpha 1 chain (Col1a1), TGF-b1 and vimentin. The transcriptional upregulation of these genes post-MI indicated the induction of EndMT. Treatment with decursin significantly downregulated the expression of all aforementioned markers, supporting its role in inhibiting EndMT processes following MI (Fig. 4C). Furthermore, we evaluated cardiac fibrosis and EndMT progression markers, including Col1a1, Col3a1, matrix metalloproteinase 2 (MMP-2), and MMP-9 after MI. Col1a1 and Col3a1 are structural components of the extracellular matrix, while MMP-2 and MMP-9 are responsible for matrix breakdown and remodeling. Expression of these markers was significantly enhanced in MI hearts compared to the Normal group. Decursin administration led to marked suppression of their protein expression, thereby mitigating excess fibrosis in the affected myocardium (Fig. 4D). Collectively, these results demonstrate that decursin exerts protective effects against EndMT progression and reduces fibrotic remodeling in cardiac tissue after MI.

Fig. 4.

DE attenuates EndMT process in infarcted myocardium. (A) Schematic diagram of the experimental design of MI-induced mice with DE. (B) Immunofluorescence staining of α-SMA (green) and VE-cadherin (red). (C) Quantitative analysis of EndMT-associated genes. *P < 0.05 vs Normal. (D, E) Western blotting analysis of cardiac fibrosis and EndMT-related proteins. *P < 0.05 vs. MI.

Therapeutic impact of decursin on apoptosis and cardiac function in post-ischemic hearts

Finally, we investigated the therapeutic efficacy of decursin utilizing an animal model of MI. The triphenyltetrazolium chloride (TTC) staining analysis revealed that the MI group had a substantial infarcted myocardium (yellow-lined white region), representing significant tissue damage. Conversely, decursin-treated animals showed a reduction in infarct size, indicating a protective effect of decursin against myocardial injury (Fig. 5A). Apoptotic cell death in the damaged heart was assessed using 5-bromo-2’-deoxyuridine 5’-triphosphate (Br-dUTP) and 7-aminoactinomycin D (7-AAD) staining. Strong Br-dUTP and 7-AAD fluorescence in the MI group indicated increased apoptotic cell presence. However, decursin-treated myocardium displayed a decreased occurrence of apoptotic cell death, supporting a protective role of decursin against MI-induced cellular apoptosis (Fig. 5B). Building on previous data, we validated alterations in myocardial fibrosis-associated protein expression. Administration of decursin significantly reduced myocardial fibrosis relative to the non-treated MI group, which exhibited marked fibrotic regions. We subsequently quantified microvessel density in decursin-treated ischemic hearts by analyzing CD31 expression. Immunofluorescence studies demonstrated that MI led to increased EndMT, while decursin treatment enhanced CD31-positive fluorescence in the injured myocardium. These results suggest that decursin interfered with EndMT progression following MI (Fig. 5D). Finally, the therapeutic benefit of decursin on cardiac performance was assessed by examining left ventricular (LV) pressure–volume (PV)-loops, as well as ejection fraction (EF) and end-systolic pressure-volume relationship (ESPVR). The MI group showed deficits in EF, LV pressure, and volume regulation compared with the normal group. All cardiac function parameters improved upon decursin treatment in comparison to the MI group lacking decursin intervention (Fig. 5E-G). Collectively, these findings demonstrate that decursin attenuates myocardial structural remodeling and promotes restoration of cardiac function in MI therapy.

Fig. 5.

DE presents cardioprotective effects post-MI. (A) TTC staining for assessing infarcted area. *P = 0.0010 MI vs. MI ＋ Decursin. (B) and TUNEL assay for measuring apoptotic cell death. *P = 0.0006. (C) Detection of the myocardial fibrotic region using Masson’s trichrome staining. *P = 0.0043 MI vs. MI ＋ Decursin. (D) Evaluation of CD-31-positive fluorescence density. *P < 0.005 vs. Normal **P = 0.0017 MI vs. MI ＋ Decursin. (E) EF, ***P < 0.05 vs. normal ^###P < 0.001 vs. MI. (F) ESPVR, (G) and LV PV-loops.

DISCUSSION

Ischemic heart disease arises mainly from restricted blood supply, resulting in a critical imbalance between oxygen availability and the metabolic requirements of cardiac tissue. This process ultimately culminates in cell death within the infarcted region (22, 23). While restoring blood flow is essential, reperfusion paradoxically intensifies tissue injury by generating ROS, thereby triggering an oxidative stress cascade (24). Oxidative stress serves as a central mediator of ischemic damage. In addition to amplifying cellular injury and apoptosis, it also precipitates cardiac dysfunction (25). In this study, we aimed to elucidate the significance of therapeutic approaches that reduce ROS production and restore cardiac function in ischemic hearts. Specifically, we investigated ECs, which play a pivotal role in maintaining vascular homeostasis; dysregulation in these cells is a defining feature of several CVDs (26). After MI, ECs become particularly susceptible to oxidative stress, which leads to profound endothelial dysfunction and subsequent cardiac dysfunction, presented through increased endothelial permeability and elevated expression of adhesion molecules (27, 28). These alterations facilitate inflammatory cell infiltration and aggravate myocardial injury. Additionally, oxidative stress has been identified as a contributor to EndMT, a phenomenon in which ECs lose their specific markers and acquire mesenchymal traits (7). The transition of ECs diminishes the regenerative potential of the endothelium and drives fibrotic responses in the infarcted myocardium, resulting in increased extracellular matrix accumulation and fibrosis. This study examined the interrelation among EndMT, oxidative stress, and endothelial dysfunction, which collectively play crucial roles in the intricate pathophysiological responses following MI (29, 30). Targeting signaling pathways involved in EndMT-induced oxidative stress may provide innovative therapeutic options to preserve endothelial integrity, attenuate fibrosis, and enhance cardiac outcomes following MI.

Decursin is a key constituent of Angelica gigas Nakai. Multiple recent in vitro and in vivo studies have reported its beneficial pharmacological activities in the treatment of various diseases. Decursin and decursinol angelate inhibit adipogenesis by activating the β-catenin signaling pathway, thereby suppressing major adipogenic markers such as C/EBPα and PPARγ (31). This study further advances current understanding of decursin by providing evidence for its antioxidant capacity, which helps to alleviate oxidative stress. The findings also elucidate the significant role of decursin in modulating signaling pathways that govern EndMT progression and offer new perspectives on the involved molecular mechanisms. Both chemical and physical factors can stimulate EC transition through EndMT (32). TGF-β1 is pivotal in the development of tissue fibrosis. In our previous experiments, we observed increased TGF-β2 and IL-1β mRNA expression levels in MI mouse models. Consequently, we chose TGF-β2 and IL-1β to induce in vitro EndMT progression. Our results demonstrate that decursin impedes EndMT and concurrently inhibits EndMT-associated cellular activities by promoting LDL uptake and attenuating EndMT-induced cellular migration in a concentration-dependent fashion (Fig. 1 and 2). EndMT results in the impairment of cell–cell adhesion and polarity, consequently enhancing migratory and invasive properties (33). The reduction in migration observed with decursin treatment likely stems from its effect on suppressing the activation of key signaling molecules—including RhoA, cdc42, and cofilin—that govern cytoskeletal dynamics and cell movement (34, 35). Decursin achieves this by inhibiting phosphorylation of these targets, which disrupts the actin cytoskeleton rearrangement essential for migration (Fig. 2). Additionally, decursin mitigates EndMT-induced intracellular ROS levels and protects against mitochondrial dysfunction in a dose-dependent fashion. In addition, our data revealed that decursin modulates EndMT-related signaling cascades including the PI3K/AKT/NF-κB and Smad-dependent pathways, underscoring crosstalk between signaling and intracellular ROS regulation. The PI3K/AKT pathway represents an intracellular ROS-dependent non-canonical signaling axis (11, 21). Smad-related events are associated with canonical signaling, and activation of Smad2/3 is regulated by the elevated intracellular ROS that emerges during EndMT progression (Fig. 6) (36). These results support the notion that decursin mitigates ROS-mediated mitochondrial membrane injury and depolarization. The main objective of this study was to characterize not only the pathological changes in EndMT-driven ECs but also the regulatory signaling pathways involved in these alterations. The progression of EndMT and its associated ROS-antioxidant mechanisms are intricately connected to the modulation of intracellular signaling pathways, with a particular emphasis on the PI3K/AKT/NF-κB and Smad-dependent cascades (37, 38). These signaling pathways play a substantial role in the development of cardiac dysfunction by facilitating EndMT, which results in enhanced fibrosis and deleterious cardiac remodeling (37). To explore these mechanisms, we examined the influence of decursin on these pathways through western blot analysis. In EndMT-induced HUVECs, decursin administration markedly suppressed the phosphorylation of proteins involved in the PI3K/AKT/NF-κB pathway. Furthermore, decursin inhibited the activation of Smad2/3, thereby impeding EndMT progression via the Smad-dependent signaling route (Fig. 3). Through inhibition of these pathways, decursin maintains endothelial cell integrity and lessens both fibrosis and pathological remodeling within infarcted myocardial tissue. The broad inhibitory effects of decursin on critical EndMT-related signaling networks, combined with its antioxidant properties, underscore its promise as a therapeutic option for ischemic heart disease. After establishing the antioxidant role of decursin in in vitro EndMT-induced ECs, we further assessed its therapeutic potential using in vivo mouse models. The in vivo findings present compelling evidence supporting decursin’s efficacy in reducing EndMT, fibrosis, and cardiac dysfunction in infarcted myocardium. In the MI mouse model, decursin therapy reduced α-SMA levels and upregulated VE-cadherin, indicating reversal of EndMT pathology. Moreover, decursin downregulated transcription factors associated with EndMT and fibrosis after treatment. The in vivo results align with the in vitro data, showing that decursin considerably inhibits EndMT progression through modulation of oxidative stress, mitochondrial dysfunction, and the PI3K/AKT/NF-κB and Smad signaling pathways. This consistency between in vitro and in vivo results demonstrates that decursin’s cardioprotective effects in infarcted hearts are, in part, attributed to its antioxidative activity influencing EndMT regulation, thereby enhancing the translational value of our study.

Fig. 6.

The schematic diagram depicts the proposed mechanism underlying the antioxidant effects of DE in EndMT-induced models. DE exerts its cardioprotective action via modulation of the PI3K/AKT/NF-κB and Smad signaling pathways, which inhibit ROS-mediated endothelial dysfunction and attenuate cardiac complications.

In conclusion, decursin demonstrates significant therapeutic potential by modulating EndMT progression, alleviating endothelial dysfunction, reducing intracellular oxidative stress, and inhibiting cellular fibrosis in both in vitro and in vivo models. Decursin exerts its regulatory effects on EndMT and oxidative stress responses through the PI3K/AKT/NF-κB and Smad-dependent signaling pathways in EndMT-induced in vitro ECs. Furthermore, decursin confers cardioprotective effects and promotes functional recovery of the heart in an EndMT-induced in vivo mouse model. Collectively, these findings support the clinical potential of decursin as a novel therapeutic approach for CVDs and other diseases by targeting key molecular mechanisms.

MATERIALS AND METHODS

Cell culture

HUVECs (ATCC, VA, USA) were maintained in endothelial growth medium-2 (Lonza, NJ, USA) supplemented with 1% penicillin–streptomycin (Welgene, Daegu, Korea) at 37°C in a 5% CO₂ humidified incubator.

Cell viability assay

HUVECs were plated onto a 96-well plate and exposed to increasing concentrations of decursin (0, 10, 25, and 50 μM) for 24 h. Subsequently, the cells were treated with cell counting kit 8 solution (Dojindo, Kumamoto, Japan) at 37°C for 3 h. Absorbance at 450 nm was measured using a microplate reader. The assay was carried out in triplicate for each group.

Wound healing assay

HUVECs were seeded at 1 × 10⁵ cells/well in 12-well plates and grown to 90% confluence, followed by treatment with 0, 1, 3, 5, or 10 μM decursin for 24 h and serum starvation for 12 h. After starvation, scratches were introduced using 200 μl pipette tips, and the culture medium was replaced with medium containing TGF-β2 (10 ng/ml) and IL-1β (1 ng/ml). Images were obtained by microscopy. The extent of migration was documented at 0 and 24 h post TGF-β2 induction, and the recovered area percentage was determined. Triplicate experiments were performed per group.

Transwell assay

HUVECs were placed in the upper chamber of a 24-well transwell insert (Corning, NY, USA) with TGF-β2/IL-1β and decursin. Complete medium was added to the lower chamber for 24 h. Following incubation, non-migrated cells were removed from the upper membrane surface, and migrated cells on the lower surface were fixed with 4% paraformaldehyde for 10 min, then stained using 0.5% crystal violet solution for 15 min. Stained cells were quantified in five randomly selected fields per insert with a light microscope at 200× magnification. Each group was analyzed in triplicate.

LDL uptake and permeability assays

The HUVECs were incubated with different concentrations of decursin for 24 h, after which they were exposed to 10 μg/ml fluorescently labeled LDL to evaluate LDL uptake. For the permeability assay, HUVECs were plated onto a 24-well plate pre-coated with 1% gelatin. The lower chamber contained endothelial cell growth medium (EBM)-2, while the upper chamber medium was supplemented with TGF-β2/IL-1β and decursin for 24 h. The concentration of fluorescein isothiocyanate–dextran that permeated to the lower chamber was quantified using a microplate reader. Each group was assayed in triplicate.

ROS and mitochondrial membrane potential detection

The HUVECs were treated with varying concentrations of decursin for 24 h, consistent with previous protocols. After a 1 h incubation with TGF-β2/IL-1β, cells were stained using 5 μM H2DCFDA to visualize ROS. Fluorescent signals were captured using excitation wavelengths of 492-495 nm and emission at 517-527 nm. For analysis of mitochondrial membrane potential, cells were stained with 200 nM TMRE and fluorescence was recorded at excitation and emission wavelengths of 549 nm and 575 nm, respectively. Each group underwent triple independent experiments.

Western blotting assay

Following decursin exposure, total cellular proteins were isolated using a cell lysis buffer. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking, membranes were incubated overnight at 4°C with primary antibodies targeting β-actin, CD31, VE-cadherin, α-SMA, fibronectin, Ras homolog family member A, Cdc42, p-Cdc42, cofilin, p-cofilin, PI3K, p-PI3K, AKT, p-AKT, NF-κB p65, p-NF-κB p65, Smad2/3, and p-Smad2/3. Membranes were then washed with tris buffered saline containing Tween 20 and incubated with the correct horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced ECL detection system. Experiments were performed in triplicate for each group.

MI model and decursin treatment

All animal experimental procedures were conducted in strict accordance with the approved guidelines of the Institutional Animal Care and Use Committee of the Catholic Kwandong University College of Medicine (No. CKU 01-2020-009) and the standards established by the Association for Assessment and Accreditation of Laboratory Animal Care. Male C57BL/6 mice (10 weeks old) were randomly assigned to one of three experimental groups: (1) Normal, healthy mice not subjected to MI (n = 15); (2) MI, mice undergoing MI surgery (n = 15); and (3) MI ＋ Decursin (n = 15), MI-operated mice that received decursin (25 mg/kg) intraperitoneally 6 h prior to MI surgery and again at two 2-day intervals after MI. Mice were euthanized at 1 and 4 weeks post-treatment for pathological and functional assessment of decursin-treated hearts. To minimize variability in myocardial infarction induction due to differences in coronary artery bifurcation, ligation was performed at a location of 3 mm or less from the atrium.

Histological and immunofluorescence analyses of the MI model

In order to assess the ischemic area, isolated hearts were perfused with 2% 2,3,5-TTC (Sigma-Aldrich, MO, USA) at 37°C for 1 h and fixed overnight in 4% formaldehyde at 4°C. To identify fibrotic regions, collagen fibers in the infarcted myocardium were visualized with Masson’s trichrome according to established protocols. Apoptotic and necrotic cells in the MI area were detected by performing a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay following the manufacturer’s instructions. For immunohistochemical analysis, tissue sections prepared from mouse hearts were incubated with primary antibodies targeting α-SMA and VE-cadherin. Sections were then incubated with Alexa Fluor 488 and 647 secondary antibodies (Abcam, Cambridge, UK) for detection. Fluorescent images were obtained using a confocal microscope (Carl Zeiss, Germany). Three mice per group were utilized for quantitative analyses.

Real-time polymerase chain reaction (PCR) analysis

Total RNA was isolated from heart tissue samples using TRIzol reagent, and cDNA was synthesized employing the RevertAid First Strand cDNA Synthesis Kit in accordance with the manufacturer’s protocols. Real-time PCR was conducted with the following cycling parameters: initial denaturation at 95°C for 5 min, followed by 40 cycles consisting of denaturation at 94°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 15 s. The relative mRNA expression of target genes was determined using the 2^−∆∆CT method, with normalization to β-actin in each sample. Analyses were performed using three mice per group.

Cardiac catheterization

LV catheterization was conducted 4 weeks after MI to collect invasive hemodynamic data. A PV loop catheter (Millar Instruments, TX, USA) was used to record LV pressure and volume as well as real-time volume loops. The resulting measurements were analyzed with LabChart v8.1.5 software (Millar). For this analysis, we used a cohort of three mice per group.

Statistical analysis

Statistical analyses were performed with GraphPad Prism version 9.4.1 (GraphPad Software, Inc., San Diego, CA, USA). All outcome data are presented as mean ± standard error of the mean. Student’s t-test was utilized for comparisons between two groups. For analyses involving more than two groups, a one-way ANOVA followed by Bonferroni’s post-hoc test was applied to determine statistical significance. Results with P < 0.05 were considered statistically significant.