Eupatilin alleviates right ventricular fibrosis in rats with pulmonary hypertension induced by monocrotaline

Abstract

Pulmonary arterial hypertension (PAH) is referred to as a tumor of the cardiovascular system and is a major cause of death. It is urgent to develop safe and effective drugs to combat PAH. Eupatilin, a flavonoid extracted from Artemisia, has multiple pharmacological activities. However, the role of Eupatilin in PAH-induced right heart failure is not clear. This study was aimed to investigate the effects of Eupatilin on the PAH via monocrotaline (MCT)-induced rat models and platelet-derived growth factor-BB (PDGF-BB)-induced pulmonary artery smooth muscle cell (PASMC) model. We found Eupatilin inhibits PDGF-BB-induced proliferation of PASMCs in vitro. In addition, Eupatilin affects pulmonary blood flow and right ventricular function induced by MCT in rats. We further revealed Eupatilin alleviates pulmonary artery remodeling induced by MCT. Also, it alleviates myocardial fibrosis induced by MCT. Mechanically, Eupatilin inhibits the JNK/p38 MAPK pathway. Collectively, Eupatilin alleviates MCT-induced pulmonary vascular remodeling as well as right ventricular hypertrophy via JNK/p38 MAPK pathway and could serve as a promising drug to combat PAH.

INTRODUCTION

Pulmonary arterial hypertension (PAH) is referred to as a malignant tumor of the cardiovascular system and is a cause of death globally. In PAH, the increased pressure in the pulmonary artery leads to an increase in the posterior load of the right heart, resulting in right ventricular dysfunction, heart expansion, and ultimately right heart failure as well as death [1,2]. Currently, the treatment of PAH is mainly focused on solving endothelial dysfunction by blocking the endothelin pathway and improving the prostaglandin I2 and nitric oxide pathways pathways [3,4]. Despite significant progress in treatment strategies, PAH has not been fully cured [5]. Moreover, the existing drugs are often expensive and come with significant adverse reactions [5]. Therefore, it is urgent to develop safe and effective drugs to combat PAH.

Pulmonary vascular remodeling is vital in the high-risk stage of PAH [6]. Pulmonary vascular remodeling is mainly mediated by pulmonary artery smooth muscle cells (PASMCs), pulmonary artery endothelial cells, and pulmonary artery fibroblasts [7]. Abnormal proliferation of PASMCs in the media of the pulmonary artery is the main feature of PAH vascular remodeling [7]. The c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) pathway is one of the most critical intracellular pathways, and studies have found that PASMC proliferation depends on the activation of JNK and p38 MAPK phosphorylation [8]. Blocking phosphorylation can inhibit PASMC cell proliferation, and the regulation of this pathway also mediates PH vascular remodeling [9].

Eupatilin, a flavonoid extracted from Artemisia, has pharmacological activities and exhibits anti-inflammatory, anti-cancer, antioxidant, anti-allergic, cardiac protection, and neuroprotective activities [10,11]. Eupatilin alleviates ovalbumin-induced asthma by regulating several pathways [12]. It improves cardiac toxicity caused by doxorubicin by alleviating inflammation, oxidative stress, and cardiomyocyte apoptosis [13]. It also inhibits oxidative stress and cell apoptosis in H9c2 cells after myocardial ischemia/reperfusion injury [14]. Eupatilin regulates cell cycle and induces cell apoptosis to inhibit TGF-β2-induced retinal pigment epithelial cell proliferation [15]. However, the role of Eupatilin in PAH-induced right heart failure is not clear.

Herein, we investigated the effects of Eupatilin on the PAH progression in the monocrotaline (MCT)-induced rat models and platelet-derived growth factor-BB (PDGF-BB)-induced PASMC model. Our data confirmed that Eupatilin alleviates myocardial fibrosis in rats with PAH induced by MCT. Therefore, we thought it could serve as a promising drug to combat PAH.

METHODS

Animals

The use of animals and clinical samples in this study was approved by the Ethics Committee of the Affiliated Hospital of Changchun University of Chinese Medicine (approval No. 2022081), male Sprague–Dawley rats (200–250 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. The animal experiment complies with the ARRIVE guidelines and in accordance with the National Institutes of Health guide for the care and use of Laboratory animals.

MCT-induced PAH model

PAH was induced by a single subcutaneous injection of MCT (60 mg/kg; Beyotime, ST2290) in rats. After MCT injection, the animals were randomly divided into control, MCT, and MCT + Eupatilin treatment groups (5 mg/kg or 10 mg/kg). Eupatilin (MedChemExpress, HY-N0373) was administered via intraperitoneal injection for 21 consecutive days. After euthanasia, the heart was excised, and the right ventricle was carefully separated from the left ventricle and septum. Tissues were blotted dry and weighed individually using a precision balance. This ratio serves as a standard index for right ventricular hypertrophy in PAH models.

Cell culture and PASMC proliferation assay

PASMCs were isolated from rats and cultured in Dulbecco’s Modified Eagle Medium (Beyotime, ST047) supplemented with 10% fetal bovine serum (Gibco, 10099-141) at 37°C in a 5% CO₂ incubator. PASMC proliferation was induced using PDGF-BB (20 ng/mL; Beyotime, ST2558), and Eupatilin was added at various concentrations (2.5, 5, 10 μM). Cell viability was assessed using the CCK-8 assay (Beyotime, ST147) following 24 h of treatment.

Histological analysis

Right ventricular tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 μm thickness. Sections were stained with Masson’s trichrome to visualize collagen deposition (blue) and cardiomyocytes (red). Images were captured using a Zeiss Axio Imager 2 microscope at 20× magnification. Hematoxylin and eosin (H&E) staining was used to evaluate lung tissue morphology. Masson’s trichrome staining was performed to assess fibrosis in right ventricular tissues. Histological images were captured using a Zeiss Axio Imager 2 microscope (Zeiss). Cardiomyocyte cross-sectional area was measured using ImageJ software (National Institutes of Health) by tracing the outline of ≥ 50 randomly selected cardiomyocytes per sample, excluding nuclei and fibrotic areas. Data are presented as mean cross-sectional area (μm²) per group.

Immunoblot analysis

Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, IPVH00010). The membranes were incubated with the following primary antibodies: anti-α-smooth muscle actin (α-SMA) (Abcam, 1:1,000, ab32575), anti-Collagen I (Abcam, 1:1,000, ab138492), anti-proliferating cell nuclear antigen (PCNA) (Abcam, 1:1,000, ab18197), anti-phospho-JNK (Abcam, 1:1,000, ab124956), total JNK (Abcam, 1:1,000, ab179461), anti-phospho-p38 (Abcam, 1:1,000, ab170099), total p38 (Abcam, 1:1,000, ab170099), and anti-β-actin (Beyotime, 1:5,000, AF0003). After incubation with horseradish peroxidase-conjugated secondary antibodies (Beyotime, ST051), the protein bands were visualized using enhanced chemiluminescencereagents (Thermo Fisher, 32106).

Immunofluorescence staining

The sections were blocked with 5% bovine serum albumin (Sigma, A7030) and incubated overnight at 4°C with anti-α-SMA antibody (Abcam, 1:200, ab5694). After washing, the sections were incubated with an Alexa Fluor 594-conjugated secondary antibody (Invitrogen, A11012) for 1 h. DAPI (Thermo Fisher, 62247) was used to stain nuclei. Images were captured using a Zeiss LSM 710 confocal microscope (Zeiss).

Echocardiography

Right ventricular function was assessed by echocardiography using a Vevo 2100 imaging system (VisualSonics) equipped with a 21 MHz transducer. Pulmonary artery acceleration time and right ventricular systolic pressure were measured in anesthetized rats.

Statistical analysis

All quantitative data are presented as mean ± SD. Statistical analysis was performed using GraphPad Prism 8.0 software. All measurements were performed by two blinded investigators to ensure objectivity. Interobserver variability was assessed using the intraclass correlation coefficient (> 0.90). Differences between groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. A p-value < 0.05 was considered statistically significant.

RESULTS

Eupatilin inhibits PDGF-BB-induced proliferation of PASMCs in vitro

To evaluate the effect of Eupatilin on PASMC proliferation, CCK-8 and Edu incorporation assays were performed. Treatment with Eupatilin significantly decreased PASMC viability, as shown by the CCK-8 assay (Fig. 1A). Furthermore, the Edu incorporation assay demonstrated that Eupatilin markedly suppressed PDGF-BB-induced PASMC proliferation (Fig. 1B). These results suggest that Eupatilin effectively inhibits the abnormal proliferation of PASMCs triggered by PDGF-BB stimulation in vitro.

Fig. 1. Eupatilin inhibits PDGF-BB-induced proliferation of PASMCs in vitro.

(A) PASMCs were treated with various concentrations of Eupatilin (0, 2.5, 5, 10, 20, and 40 μM) for 24 h, and cell viability was assessed using the CCK-8 assay. (B) PASMCs were treated with PDGF-BB (20 ng/ml) to induce cell proliferation, followed by treatment with Eupatilin (2.5 and 5 μM). Edu (red) incorporation indicates proliferating cells, and DAPI (blue) stains the nuclei. Scale bar = 50 μm. Values are presented as mean ± SD. Eup, Eupatilin; PDGF-BB, platelet-derived growth factor-BB; PASMCs, pulmonary artery smooth muscle cells; Edu, 5-ethynyl-2’-deoxyuridine; DAPI, 4’,6-diamidino-2-phenylindole. **p < 0.01, ***p < 0.001: compared to the 0 μM (control) group. ^###p < 0.001: compared to the PDGF-BB group.

Eupatilin suppresses pulmonary blood flow and right ventricular function in MCT-induced rats

To assess the impact of Eupatilin on pulmonary function and right ventricular performance in vivo, H&E staining was used to examine the lung tissues of MCT-induced rats. Histological analysis of the lung tissues using H&E staining showed extensive lung tissue damage, including thickened vessel walls, narrowed lumens, and marked alveolar destruction in MCT-treated rats (Fig. 2). However, Eupatilin treatment improved lung structure, with a notable reduction in tissue damage and remodeling compared to the MCT group (Fig. 2). These findings indicate that Eupatilin plays a protective role in preserving pulmonary blood flow and mitigating right ventricular dysfunction in MCT-induced PAH models.

Fig. 2. Effects of Eupatilin on pulmonary blood flow and right ventricular function in MCT-induced rats.

Representative images of H&E staining of lung tissue sections from control, MCT, and Eupatilin-treated groups (5 mg/kg and 10 mg/kg) after 21 days of treatment. Quantitative analysis of pulmonary artery remodeling. Wall thickness and lumen area were measured in pulmonary arteries from control, MCT, and Eupatilin-treated rats. Scale bar = 100 μm. Values are presented as mean ± SD. MCT, monocrotaline; Eup, Eupatilin; H&E, hematoxylin and eosin. p < 0.05 compared to the control group, p < 0.01 compared to the MCT group. ***p < 0.001, ^##p < 0.01, ^###p < 0.001.

Eupatilin alleviates pulmonary artery remodeling induced by MCT

The role of Eupatilin in pulmonary artery remodeling was examined through Western blot and immunofluorescence analyses. MCT increased the expression of α-SMA and PCNA, both markers of vascular remodeling, whereas Eupatilin significantly reduced the expression of these markers, in MCT-induced rats (Fig. 3A). Immunofluorescence staining further confirmed the reduction in α-SMA-positive areas in the pulmonary arteries (Fig. 3B). Together, these data demonstrate that Eupatilin effectively mitigates pulmonary artery remodeling by suppressing the expression of remodeling-related proteins.

Fig. 3. Eupatilin alleviates pulmonary artery remodeling induced by MCT.

(A) Immunoblot analysis of α-SMA and PCNA protein expression in pulmonary artery tissues from control, MCT, and Eupatilin-treated (5 mg/kg and 10 mg/kg) rats. β-actin was used as a loading control. Relative protein expression levels were quantified and shown in the right panel. (B) Immunofluorescence staining of α-SMA (red) and DAPI (blue) in pulmonary artery tissues from control, MCT, and Eupatilin-treated (5 mg/kg and 10 mg/kg) rats. Scale bar = 200 μm. Values are presented as mean ± SD. MCT, monocrotaline; Eup, Eupatilin; α-SMA, alpha-smooth muscle actin; PCNA, proliferating cell nuclear antigen; DAPI, 4’,6-diamidino-2-phenylindole. ***p < 0.001 compared to the control group. ^##p < 0.01, ^###p < 0.001 compared to the MCT group.

Eupatilin attenuates myocardial fibrosis induced by MCT

To investigate whether Eupatilin could alleviate myocardial fibrosis, Masson’s trichrome staining was performed on right ventricular tissue sections. Masson’s trichrome staining of right ventricular tissue from MCT-treated rats revealed extensive collagen deposition, indicating severe fibrosis (Fig. 4A). However, Eupatilin-treated rats exhibited significantly less fibrosis compared to the MCT group, as indicated by the reduced collagen deposition (Fig. 4A). Immunoblot showing the expression of α-SMA and collagen I further confirmed that Eupatilin attenuates ventricular hypertrophy induced by MCT (Fig. 4B). These results suggest that Eupatilin has a protective effect against the development of myocardial fibrosis by inhibiting excessive fibrosis in MCT-induced PAH.

Fig. 4. Eupatilin attenuates myocardial fibrosis induced by MCT.

(A) Representative images of Masson’s trichrome staining of right ventricular tissue sections from control, MCT, and Eupatilin-treated (5 mg/kg and 10 mg/kg) rats, showing the extent of fibrosis. Blue staining indicates collagen deposition, and red staining represents muscle fibers. Scale bar = 200 μm. Quantification of right ventricular hypertrophy. Myocardial fibrosis was assessed by measuring the RV/LV + S weight ratio and cross-sectional area of right ventricular tissue. (B) Immunoblot showed the expression of α-SMA and collagen I in control, MCT, and Eupatilin-treated (5 mg/kg and 10 mg/kg) rats. Values are presented as mean ± SD. MCT, monocrotaline; Eup, Eupatilin; RV, right ventricle; LV + S, left ventricle and septum; α-SMA, α-smooth muscle actin. p < 0.05 compared to the control group, p < 0.01 compared to the MCT group. ***p < 0.001, ^##p < 0.01, and ^###p < 0.001.

Eupatilin inhibits the JNK/p38 MAPK signaling pathway in PAH model

MCT-induced PAH is characterized by the activation of pro-inflammatory and pro-fibrotic signaling pathways, including the JNK/p38 MAPK pathway, which contributes to vascular remodeling and inflammation. Immunoblot analysis of lung tissues from MCT-treated rats showed a significant increase in the phosphorylation of JNK and p38, indicating pathway activation (Fig. 5). Eupatilin treatment, however, significantly suppressed the phosphorylation of both JNK and p38, while total levels of JNK and p38 remained unchanged (Fig. 5). This suggests that Eupatilin inhibits the activation of the JNK/p38 MAPK pathway, thereby mitigating inflammation and vascular remodeling in MCT-induced PAH.

Fig. 5. Eupatilin inhibits the JNK/p38 MAPK signaling pathway.

Immunoblot analysis of p-JNK, total JNK, p-p38, and total p38 in lung tissue from control, MCT, and Eupatilin-treated (5 mg/kg and 10 mg/kg) rats. β-actin was used as a loading control. Relative protein expression levels were quantified and shown in the right panel. Values are presented as mean ± SD. MCT, monocrotaline; Eup, Eupatilin; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; p-JNK, phosphorylated JNK; p-p38, phosphorylated p38; β-actin, beta-actin. ***p < 0.001 compared to the control group. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared to the MCT group.

DISCUSSION

PAH is a severe and progressive disease characterized by elevated pulmonary artery pressure [4,16]. The underlying mechanisms of PAH are multifactorial, involving vascular remodeling, inflammation, and endothelial dysfunction. Inflammatory responses play a significant role in the progression of PAH, with elevated levels of pro-inflammatory cytokines contributing to the disease’s pathophysiology [2,17]. Our study demonstrated that Eupatilin, a flavonoid with known anti-inflammatory properties, significantly alleviates pulmonary hypertension in MCT-induced PAH models by targeting these inflammatory pathways. Unveiling the detailed mechanisms behind inflammation's role in PAH remains critical, as it could lead to more effective treatments. The results of our study underscore the importance of targeting inflammatory processes, which could potentially halt or reverse disease progression.

Pulmonary artery remodeling is a hallmark of PAH, characterized by the proliferation of PASMCs [18,19]. This process contributes to increased vascular resistance and pressure, further exacerbating right ventricular strain [19]. PASMCs play a crucial role in PAH by proliferating abnormally in response to injury or inflammatory stimuli, thereby contributing to vascular obstruction [20,21]. In our research, we observed that Eupatilin inhibits the proliferation of PDGF-BB-induced PASMCs, highlighting its potential to mitigate vascular remodeling. These findings are consistent with previous studies that have linked PASMC proliferation to the pathogenesis of PAH, particularly via the activation of key signaling pathways such as the JNK/p38 MAPK pathway. Furthermore, the animal models used in our study provided robust evidence of Eupatilin’s efficacy in reducing pulmonary artery remodeling, supporting its potential clinical application.

Eupatilin is a multi-functional compound with documented anti-inflammatory, antioxidant, and cardioprotective effects [13,22]. In our study, we demonstrated that Eupatilin alleviated right ventricular fibrosis in MCT-induced PAH models. Previous research has shown that Eupatilin exerts its protective effects by modulating key inflammatory and apoptotic pathways [23]. Our findings extend this knowledge, suggesting that Eupatilin not only reduces inflammation but also plays a crucial role in protecting cardiovascular tissues from fibrosis and hypertrophy. This cardioprotective effect may be attributed to its ability to regulate the JNK/p38 MAPK signaling pathway, as our data showed significant downregulation of this pathway in response to Eupatilin treatment in PAH models.

The role of PASMCs in the progression of PAH cannot be overstated, as their proliferation drives both vascular remodeling and the overall advancement of the disease [7]. Intervening in the proliferation of PASMCs, as we have demonstrated with Eupatilin, offers a promising therapeutic strategy. By targeting PASMCs, we can potentially reverse or prevent the pathological changes associated with PAH, including right ventricular hypertrophy and fibrosis. Our study adds to the growing body of evidence that emphasizes the need for therapies that specifically target PASMCs in PAH.

Eupatilin’s impact on inflammation and related diseases is well-documented in various contexts, including its regulation of the NF-κB and MAPK signaling pathways [13]. In our research, we further confirmed its anti-inflammatory effects in the context of PAH. These effects were evident in both cellular and animal models, where Eupatilin significantly reduced the expression of pro-inflammatory markers and mitigated tissue damage. This suggests that Eupatilin could be a valuable addition to the therapeutic arsenal for PAH, particularly in cases where inflammation plays a prominent role.

The JNK/p38 MAPK pathway is a key intracellular signaling cascade involved in cell proliferation, apoptosis, and inflammatory responses [24,25]. Its activation has been implicated in the progression of PAH, particularly through the regulation of PASMC proliferation and vascular remodeling [24]. Inhibiting this pathway offers a potential therapeutic target for PAH [25]. Our study demonstrated that Eupatilin effectively suppressed the phosphorylation of JNK and p38 MAPK, leading to reduced PASMC proliferation and attenuated pulmonary vascular remodeling. These findings highlight the therapeutic potential of targeting the JNK/p38 MAPK pathway in PAH and other inflammation-driven diseases.

While our study provides compelling evidence of Eupatilin’s efficacy in treating PAH, there are limitations to consider. First, the exact molecular interactions between Eupatilin and the JNK/p38 MAPK pathway require further investigation to fully elucidate the underlying mechanisms. Additionally, while our animal models provide important insights, clinical studies are needed to confirm these findings in human populations. Future research should also explore the long-term effects of Eupatilin on PAH progression and its potential for combination therapy with existing treatments.

The MCT-induced PAH model is commonly used to study PAH, as it effectively mimics key features of the disease, including pulmonary vascular remodeling, right ventricular hypertrophy, and right heart failure. MCT induces endothelial injury and smooth muscle cell proliferation, which are central to PAH pathophysiology. However, the model has limitations, including its acute nature, which may not fully represent the chronic progression of human PAH. Additionally, it primarily focuses on vascular changes and may not capture the complexity of the disease, such as the roles of other cell types and environmental factors.

In conclusion, our research highlights the significant therapeutic potential of Eupatilin in alleviating PAH by targeting key inflammatory and proliferative pathways, particularly the JNK/p38 MAPK signaling cascade. These findings offer a promising avenue for the development of new, more effective treatments for PAH, addressing the current unmet need for safe and affordable therapeutic options.