
==== Front
Animal Model Exp Med
Animal Model Exp Med
10.1002/(ISSN)2576-2095
AME2
Animal Models and Experimental Medicine
2096-5451
2576-2095
John Wiley and Sons Inc. Hoboken

38970173
10.1002/ame2.12457
AME212457
AMEM-2024-0006.R3
Original Article
Themed Section: Study on Cardiovascular and Cerebrovascular Diseases
Themed Section: Original Article
Cucurbitacins mitigate vascular neointimal hyperplasia by suppressing cyclin A2 expression and inhibiting VSMC proliferation
Yuan et al.
Yuan Ruqiang 1
Qian Lei 1
Xu Hu 2 xuhu1024@126.com

Yun Weijing https://orcid.org/0000-0002-8711-0231
1 yunweijing@163.com

1 Advanced Institute for Medical Sciences Dalian Medical University Dalian China
2 Health Science Center East China Normal University Shanghai China
* Correspondence
Weijing Yun, Advanced Institute for Medical Sciences, Dalian Medical University, Dalian 116044, China.
Email: yunweijing@163.com
Hu Xu, Health Science Center, East China Normal University, Shanghai 200241, China.
Email: xuhu1024@126.com

05 7 2024
8 2024
7 4 10.1002/ame2.v7.4 Themed Issue: Study on Cardiovascular and Cerebrovascular Diseases 397407
08 1 2024
30 5 2024
© 2024 The Author(s). Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

Background

Restenosis frequently occurs after percutaneous angioplasty in patients with vascular occlusion and seriously threatens their health. Substantial evidence has revealed that preventing vascular smooth muscle cell proliferation using a drug‐eluting stent is an effective approach to improve restenosis. Cucurbitacins have been demonstrated to exert an anti‐proliferation effect in various tumors and a hypotensive effect. This study aims to investigate the role of cucurbitacins extracted from Cucumis melo L. (CuECs) and cucurbitacin B (CuB) on restenosis.

Methods

C57BL/6 mice were subjected to left carotid artery ligation and subcutaneously injected with CuECs or CuB for 4 weeks. Hematoxylin–Eosin, immunofluorescence and immunohistochemistry staining were used to evaluate the effect of CuECs and CuB on neointimal hyperplasia. Western blot, real‐time PCR, flow cytometry analysis, EdU staining and cellular immunofluorescence assay were employed to measure the effects of CuECs and CuB on cell proliferation and the cell cycle in vitro. The potential interactions of CuECs with cyclin A2 were performed by molecular docking.

Results

The results demonstrated that both CuECs and CuB exhibited significant inhibitory effects on neointimal hyperplasia and proliferation of vascular smooth muscle cells. Furthermore, CuECs and CuB mediated cell cycle arrest at the S phase. Autodocking analysis demonstrated that CuB, CuD, CuE and CuI had high binding energy for cyclin A2. Our study also showed that CuECs and CuB dramatically inhibited FBS‐induced cyclin A2 expression. Moreover, the expression of cyclin A2 in CuEC‐ and CuB‐treated neointima was downregulated.

Conclusions

CuECs, especially CuB, exert an anti‐proliferation effect in VSMCs and may be potential drugs to prevent restenosis.

Cucurbitacins suppress the vascular smooth muscle cell (VSMC) proliferation. Cucurbitacins improve vascular neointimal hyperplasia induced by left carotid artery ligation in mice. Cucurbitacins mediate VSMC cell cycle arrest at the S phase by targeting cyclin A2

CuB
Cucurbitacin
cyclin A2
restenosis
vascular smooth muscle cell
Scientific Research Fund Project of Liaoning Provincial Department of EducationLJKMZ20221267 LJKZ0840 LJKZ0847 National Natural Science Foundation of China Grants81900267 source-schema-version-number2.0
cover-dateAugust 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:03.09.2024
Yuan R , Qian L , Xu H , Yun W . Cucurbitacins mitigate vascular neointimal hyperplasia by suppressing cyclin A2 expression and inhibiting VSMC proliferation. Anim Models Exp Med. 2024;7 :397‐407. doi:10.1002/ame2.12457

Ruqiang Yuan and Lei Qian contributed equally to this work.
==== Body
pmc1 INTRODUCTION

Coronary artery disease poses a serious threat to people's health. 1 , 2 Currently, percutaneous coronary intervention (PCI) is widely used for the treatment of severe arterial stenosis. However, postangioplasty restenosis remains a major obstacle, occurring in 20%–30% of the patients who undergo angioplasty. 3 It is widely recognized that neointimal hyperplasia constitutes a critical pathological feature underlying restenosis, 4 with vascular smooth muscle cells (VSMCs) being a key component of the neointima. 5 The proliferation and migration of VSMCs and the deposition of extracellular matrix are critically involved in the development of neointimal hyperplasia following angioplasty. 6 Therefore, the suppression of VSMC proliferation has long been considered a primary cellular target in preventing neointimal hyperplasia. Consequently, there is an urgent need to develop effective drugs for anti‐neointimal hyperplasia.

Cucurbitacins have various pharmacological activities, such as anti‐inflammatory, anti‐tumor, and antiviral activities, as well as liver protection and immune regulation. 7 , 8 , 9 , 10 , 11 , 12 There are a total of 40 known types of cucurbitacin and their derivatives including cucurbitacin A, B, C, D, E, I, H, Q, R, and dihydrocucurbitacin B. Among these compounds, cucurbitacin B (CuB) is the most abundant and significant constituent extracted from the stalk of Cucumis melon L. (CuEC). 13 Our previous study proved that CuEC exerts a hypotensive effect by regulating vascular tone. 14 On this basis, we speculate that CuEC or CuB may improve restenosis by suppressing VSMC proliferation, especially in patients with hypertension.

Cyclin proteins are a class of proteins that play important regulatory roles in the cell cycle. Cyclin proteins form complexes with kinase protein activators called cyclin‐dependent kinases (CDKs), which regulate transitions between different stages of the cell cycle. 15 Common cyclin proteins include cyclin A, cyclin B, cyclin D, and cyclin E. Each type of cyclin protein plays a distinct role at different stages of the cell cycle. 16 Cyclins and cell cycle progression are involved in the regulation of cell self‐renewal and apoptosis. 17 Overexpression or abnormal degradation of cyclins proteins may lead to an uncontrolled cell cycle and abnormal proliferation. Cyclin A2 protein is a highly conserved cell cycle protein that binds to CDK1 and CDK2, controls cell cycle progression, and promotes cell mitosis. 18 VSMCs are highly plastic and can be transformed and activated from a contractile phenotype to a secretory phenotype under pathological conditions. 19 , 20 Cyclin‐dependent kinases (CDKs) and cyclins are pivotal regulators of cell proliferation, playing a critical role in the development of vascular restenosis. 21 , 22 , 23 In general, during the transition from a contractile to secretory phenotype in vascular smooth muscle cells (VSMCs), there is an upregulation in the expression of CDKs and cyclins. This upregulation facilitates VSMC proliferation and contributes to vascular remodeling. 24 In our current study, we have demonstrated that CuEC and CuB can effectively inhibit restenosis in a mouse model with left carotid artery ligation. The underlying mechanism involves the suppression of excessive VSMC proliferation by CuEC and CuB through targeted inhibition of cyclin A2.

2 METHODS

2.1 Reagents and antibodies

CuECs were extracted from the stalk of Cucumis melon L. as previously. 14 In brief, dried melon stems were crushed and the powder was soaked in 6 times the volume of 70% ethanol for 30 min. The extracts were refluxed at 80°C three times for 1 hour each. The extracts were combined, filtered, and dried to obtain the extract. The dried extracts were suspended in 100 mL of water and separated using D101 macroporous resin. Samples were eluted with 200 mL of distilled water, 400 mL of 30% ethanol, 400 mL of 50% ethanol, and 600 mL of 70% ethanol. The fractions eluted with 70% ethanol were collected, concentrated, and dried to constant weight. Finally, the fractions were eluted through silica gel (300–400 mesh). The elution solvents were petroleum ether‐ethyl acetate (10:1–1:1) and chloroform‐methanol (5:1), and the chloroform‐methanol elution fraction was collected. Cucurbitacin B (PHL82226) was purchased from Sigma‐Aldrich. Phospho‐Akt (4060), phospho‐mTOR‐C (5536), P53 (2524), cyclin A2 (18678), and α‐SMA (19254) antibody were purchased from Cell Signaling Technology. eIF5 (sc‐135 894) and GAPDH (sc‐32 233) antibody were purchased from Santa Cruz.

2.2 Mouse carotid artery ligation model

Male C57BL/6 mice (8–10 weeks old and 25–30 g weight) were used in this study. The surgery was performed as previously described. 25 After the mice were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg), the left common carotid artery (LCA) was exposed and ligated. Then, the mice were treated with CuEC (0.5 mg/kg/48 h), CuB (0.5 mg/kg/48 h) or vehicle. After 4 weeks, the mice were killed by inhalation of excess carbon dioxide, then perfused with PBS and 4% paraformaldehyde. The left and right carotid arteries were resected and embedded in paraffin for histological analysis. All procedures were by the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). All animal experiments in this study were approved by the Animal Care and Use Review Committee of Dalian Medical University.

2.3 Cell culture

Male Sprague–Dawley rats (6–8 weeks) were used to isolate aortic VSMCs by enzymatic digestion, as described previously. 26 For all experiments, VSMCs in passages 3–6 were utilized. The VSMCs were cultured on plates and when the cells reached 70% confluence, the medium was changed to serum‐free DEME and incubated overnight for 24 h. Subsequently, cells were used for follow‐up tests (western blot, real‐time PCR, flow cytometry analysis and cellular immunofluorescence assay).

2.4 Hematoxylin–Eosin (H&E) staining and immunofluorescence staining

The ligated LCA and the non‐ligated right carotid artery (RCA) were cut into 5 μm sections with a freezing microtome. The resulting slides were used for H&E staining and immunofluorescence. 27 , 28 DAPI (Beyotime, C1002) was used to counterstain the nuclei. H&E staining and immunofluorescence were performed according to standard procedures. For cellular immunofluorescence analysis, VSMCs cultured on coverslips were fixed, permeabilized, incubated with primary antibodies, then incubated with secondary antibodies before being counterstained with DAPI. Immunofluorescence was imaged under a microscope (Leica, Germany). 29

2.5 Immunohistochemistry (IHC)

A microtome (Leica, Germany) was used to cut the paraffin‐containing carotid arteries into 4 μm sections.

Briefly, sections were heated to 100°C, incubated with 5% donkey serum, and then incubated with primary antibodies. After rinsing, the secondary antibody was incubated. Visualization was accomplished using DAB (Yeasen, China). 30

2.6 Cell proliferation assays

VSMCs were implanted on 96‐well plates with 2 × 103 cells per well in 100 μL medium. When cells reached 70% confluence, different concentrations of CuEC and CuB were added to each well along with 5% FBS for 24 h. MTT reagent (10 μL) was then added to each well and incubated at 37°C for 4 h, after which 150 μL DMSO was added to each well. A microplate reader (Bio‐Rad, Hercules, CA, USA) was used measured the optical density (OD) at 490 nm. Cell viability was calculated as (experimental group absorbance value/control group absorbance value) × 100%. 30

For EdU assay of cell proliferation experiments, cells were first digested and embedded into 6‐well plates at a density of approximately 40%. After cell attachment, the cells were treated with FBS‐free medium for 6 h and then treated with the corresponding drugs for 24 h. The EdU working solution was prepared at a concentration of 10 μmol/L. Next, the EdU working solution was added to the well plates and incubated for 2 h. Cells were subsequently fixed, washed, and permeabilized. EdU detection was performed using the Click reaction. Click reaction solution was added to each well and incubated for 30 min before washing. After that, Hoechst 33342 was added for nuclear staining. Finally, fluorescence detection was performed to evaluate cell proliferation. 31

2.7 Western blot

Western blot was performed using standard procedures. 32 Briefly, VSMCs were lysed in RIPA lysis buffer after washed with PBS twice. A BCA assay kit was employed to quantified the protein concentrations. Proteins were then subjected to SDS‐PAGE and transferred onto a nitrocellulose filter membrane. The membranes were blocked with 5% BSA, followed by incubation with primary antibodies overnight at 4°C. They were then incubated with HRP‐labeled secondary antibodies for 1 h at room temperature. The bands on the membranes were detected in a Chemiluminescent Imaging System (Tanon 5200, China) using enhanced chemiluminescence (ECL) incubation. Band intensities were quantified using Image J software (NIH).

2.8 Real‐time PCR

Total RNA extracted from the VSMCs was reverse transcribed to cDNA. The primer pairs for real‐time PCR are listed in Table S1. 18S RNA was used as a marker. A mix of NTP and SYBR Green was used and real‐time PCR was performed on the LightCyler96 Sequence Detection System (Roche). 32

2.9 Flow cytometry analysis

Cell cycle progression was assessed using a previously established protocol. 33 Cells were trypsinized and centrifuged at 2000 r/min for 5 min, then fixed with 70% ethanol for 24 h at 4°C and then stained with propidium iodide (PI) (50 μg/mL in sample buffer containing 100 μg/mL RNase A) for 30 minutes at 37°C. PI‐stained cells were filtered using a 5 mL polystyrene round‐bottom tube and analyzed by flow cytometry (BD FACS Verse) and Cell Quest Research Software (FlowJo_V10). The proportions of cells in the G0/G1, S, and G2/M phases of the cell cycle were analyzed using the ModFit LT software (Verity Software House, Topsham, ME, USA).

2.10 Molecular docking

Molecular docking analysis was performed using Auto Dock 4.2 with MGL tools 1.5.6 (The Scripps Research Institutes, San Diego, CA, USA). Chem3D Pro software was used to get the PDB files of CuB. The crystal structure of cyclin A2 (PDB ID: 1ol2) was obtained from the RCSB Protein Data Bank. According to standard procedures for docking studies, ions, water molecules and non‐standard amino acid residues from the proteins were removed. Then, the potential interactions of CuB with cyclin A2 were performed by AutoDock Vina software. 34

2.11 Statistics

GraphPad Prism 8.3.0 software was used to analyze experimental data and plot the results. Data are presented as means ± SEM. Analysis of variance (ANOVA) was employed to compare multiple groups, while Student's t test was used for statistical analysis of differences between two groups. A p value <0.05 was considered to indicate a significant difference.

3 RESULTS

3.1 Effects of CuEC and CuB on neointimal hyperplasia after left carotid ligation

The effect of CuEC on the inhibition of ligation‐induced neointimal hyperplasia was assessed by quantifying the ratio of intima to media (I/M ratio) after ligating a mouse left carotid artery for 28 days. As shown in Figure 1A, CuEC reduced the I/M ratio compared with the vehicle. This result demonstrated that CuEC exerted an efficaciously protective effect on neointimal hyperplasia in mice. We analyzed the composition of CuEC using LC–MS. As shown in Figure 2, CuEC primarily consisted of 4 compounds: namely cucurbitacin B (CuB), cucurbitacin D (CuD), cucurbitacin E (CuE), and cucurbitacin I (CuI). The proportions of CuB, CuD, CuE, and CuI in CuEC are presented in Tables S2–S5, with CuB accounting for the largest proportion (48.03%) in CuEC. Therefore, we propose that CuB is the key substance responsible for inhibiting neointimal hyperplasia mediated by CuEC. Consistent with our hypothesis, mice treated with CuB after ligation exhibited a decrease in the I/M ratio as shown in Figure 1B. Furthermore, no significant changes were observed in body weights throughout the experimental period, suggesting that both CuEC and CuB had no toxic or side effects on animals at this dosage level, as demonstrated by Figure S1.

FIGURE 1 Effect of CuEC and CuB on vascular neointimal hyperplasia induced by ligation in mice. The ligations were performed in mice LCA, after which the mice were treated with CuEC (A) or CuB (B) for 28 days. The whole LCA was then harvested for H&E staining and quantitative analyses. Bar = 200 μm. Data are presented as mean ± SEM; n = 6–7. ***p < 0.001 vs. vehicle.

FIGURE 2 LC–MS analysis result of CuEC. Panels show the extracted ion chromatograms (XIC) for CuEC (A), CuB (B), CuD (C), CuE (D), and CuI (E).

3.2 CuEC and CuB inhibited VSMC proliferation

In the model group, the thickening of vessels primarily resulted from an increase in vascular smooth muscle cells (VSMCs) (Figure S2). To verify the inhibitory effects of CuEC and CuB on VSMC proliferation, MTT assay, EdU staining and microscopy were used to detected the cell density. The MTT results showed that CuEC or CuB at different concentrations gradually inhibited the proliferation of VSMCs induced by FBS (Figure 3A). In addition, the results of EdU staining and microscopic observation showed that CuEC and CuB significantly inhibited the increase of VSMCs density caused by FBS (Figure 3B,C). Additionally, we evaluated the migratory capacity of VSMCs using a wound‐healing assay to investigate the inhibitory action of CuB. Our findings indicated that CuB effectively inhibited VSMC proliferation in vitro (Figure S3).

FIGURE 3 Effect of CuEC and CuB on the cell proliferation of VSMCs. VSMCs were treated with different concentrations of CuEC or CuB for 24 h. (A) After treatment, the cell density of the VSMCs was detected by MTT assay. (B) Representative microscope images. Bar = 100 μm. (C) Representative images of EdU staining are shown. Data are presented as mean ± SEM; n = 6, ### p < 0.001 vs. control, ***p < 0.001 vs. 5% FBS.

3.3 CuEC and CuB mediated cell cycle progression in VSMCs

Flow cytometry was used to analyze the proportion of cells at different stages (Figure 4A). The analysis revealed that the majority of cells were in the G0/G1 phase, constituting 86.27% under 0% FBS conditions. Upon supplementation with 5% FBS, the distribution of cell phases shifted, comprising 72.69%, 21.11%, and 6.2% in the G0/G1, S, and G2/M phases, respectively (Figure 4B). When VSMCs were treated with CuEC or CuB, there was a decrease in the proportion of cells in the G0/G1 phase and an increase in the proportion of cells in the S phase compared to the 5% FBS group, especially when the drug concentration reached 0.5 μg/mL. This difference was statistically significant (Figure 4B,C). The results indicated that CuEC and CuB inhibit the transition from the S phase to the G2/M phase but have no effect on the transition from the G0/G1 phase to the S phase, thus arresting the cell cycle in the S phase.

FIGURE 4 Effects of CuEC and CuB on cell cycle progression. (A) Cell cycle distribution of different treatment groups. (B, C) Quantitative analysis of the distribution of cell populations at different phases of the cell cycle. Data are presented as mean ± SEM; n = 3–5. **p < 0.01 vs. vehicle; *p < 0.05 vs. vehicle.

3.4 Cyclin A2 is a potential target of CuEC and CuB in the inhibition of VSMCs proliferation

Previous studies revealed that CuB suppressed the proliferation of cancer cells by activating the PI3K/Akt/mTOR signal pathway. 35 We also determined the expression of proteins phospho‐Akt, phospho‐mTOR‐C1, phospho‐mTOR‐C2, and P53 using western blotting. To our surprise, neither administration of CuEC nor CuB reversed the increases of these markers induced by FBS (5%) (Figure S4). This suggested that CuEC and CuB inhibit proliferation in VSMCs independently of the PI3K/Akt/mTOR signal pathway. Considering that CuEC and CuB arrest the cell cycle progression in VSMCs (increasing the S phase and decreasing the G0/G1 phase), we investigated the expression of cell cycle‐related markers (CDK2, CDK4, CDK6, cyclin A2, and cyclin D1), as well as markers related to phenotypic transformation and cell proliferation (SM22α, α‐SMA, and Mki‐67), using Q‐PCR. As shown in Figure 5A–H, treatment with 0.5 μg/mL of CuEC or CuB reversed the increased mRNA expressions of Mki‐67, CDK2, and cyclin A2 induced by FBS. Overall, these results suggest that cyclin A2 may be a key target of CuEC and CuB in VSMC proliferation.

FIGURE 5 Discovery of targets for inhibition of VSMC proliferation by CuEC and CuB. (A–H) Real‐time PCR analysis of SM22α, α‐SMA, Mki‐67, cyclin A2, CDK2, cyclin D1, CDK4, and CDK6 mRNA levels in VSMCs. Data are presented as mean ± SEM; n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

3.5 CuEC and CuB suppressed the expression of cyclin A2 in vitro

The ability of CuB, CuD, CuE and CuI to bind to cyclin A2 was calculated by molecular docking. As shown in Figure 6A, CuB interacted with the active amino acid residues (Trp217, Arg250, Gln254) in cyclin A2 to form three hydrogen bonds and the binding energy was −8.03 kcal/mol. Asp216, Arg250, Gln254, and Gln406 in cyclin A2 interacted with CuD, with a binding energy of −7.65 kcal/mol., CuE interacted with Trp216, Ile250, Gln254 in cyclin A2, with a binding energy of −7.65 kcal/mol. CuI interacted with Met210, Gln254, Ile281, Thr282 in cyclin A2 with binding energies of 7.41 kcal/mol. Collectively, these results suggested that CuEC inhibits cell proliferation by targeting on cyclin A2 and CuB had a stronger hydrogen bonding and hydrophobic effects compared to other cucurbitins. The results of western blotting showed that the expression of cyclin A2 was significantly increased after FBS treatment, and pretreatment with CuEC or CuB reversed this phenomenon (Figure 6B,C). In addition, immunofluorescence showed that CuEC and CuB substantially abolished the FBS‐induced increased expression of cyclin A2 in VSMCs (Figure 6D,E). These results demonstrated that CuEC and CuB inhibit VSMC proliferation via inhibition of cyclin A2 activation.

FIGURE 6 CuEC and CuB regulated the expression of cyclin A2 in vitro. (A) The molecular docking results of CuB, CuD, CuE, CuI and cyclin A2. (B) Inhibition by CuEC of 5% FBS‐stimulated protein expression of cyclin A2. (C) Inhibition by CuB of 5% FBS‐stimulated protein expression of cyclin A2. (D) Cyclin A2 expression measured by confocal microscopy. Bar = 25 μm. Data are presented as mean ± SEM; n = 3, ### p < 0.001 vs. control, *p < 0.05, **p < 0.01, ***p < 0.001 vs. 5% FBS.

3.6 CuEC and CuB attenuated the expression of cyclin A2 in neointima

The levels of cyclin A2 were determined by immunofluorescence to further evaluate the inhibition of CuEC and CuB on vascular restenosis after carotid ligation. The results revealed the abundant presence of cyclin A2 positive cells after carotid ligation in the control group (Figure 7). As expected, treatment with both CuEC (Figure 7A) and CuB (Figure 7B) significantly decreased the cyclin A2‐positive cells. These results further demonstrated that CuEC and CuB suppressed vascular restenosis by inhibiting cyclin A2 activity.

FIGURE 7 Cyclin A2 expressions were upregulated by CuEC and CuB in vivo. The mice were treated with CuEC (A) or CuB (B) for 28 days after ligation, then whole LCAs were harvested for staining with cyclin A2, α‐SMA and DNA (DAPI). Bar = 50 μm. Data are presented as mean ± SEM; n = 6–7, ***p < 0.001 vs. vehicle.

4 DISCUSSION

Restenosis presents a significant challenge for numerous patients following PCI. The development of restenosis primarily arises from neointimal hyperplasia and vascular remodeling. Neointimal hyperplasia refers to the process of intimal thickening caused by endothelial injury, while vascular remodeling leads to changes in vessel size. 36 Carotid artery ligation is a commonly used an animal models of neointimal hyperplasia and induces alterations in shear stress and inflammation, thereby increasing the secretion of cytokines and growth factors, subsequently stimulating the activation of VSMCs. Activated VSMCs enter the cell cycle and synthesize a massive extracellular matrix, resulting in increased medial thickness and neointima formation. 25 , 28 , 37 In this study, we demonstrated that CuEC, especially CuB, exerted a remarkable anti‐restenosis effect in mice.

According to reports, cucurbitacins A, B, D, E, I, Q, IIa and their derivatives have attractive anticancer activity against lung cancer. 38 Cucurbitacin I and B have the surprising effect of inducing cell death. 39 Eight cucurbitacins exhibit antiproliferative activity, including cucurbitacins B, D, E, I, IIa, L, Q, and R. 40 In our experiments, the results showed that CuEC and CuB exerted comparable effects on restenosis in vivo and on VSMC proliferation in vitro at the same dose. Additionally, the binding energy of CuB to cyclin A2 was −8.03 kcal/mol, which is higher than that of CuD, CuE and CuI (−7.65 kcal/mol, −7.65 kcal/mol, and − 7.41 kcal/mol, respectively). Further studies are needed to clarify the effect of CuD, CuE, and CuI on restenosis. However, in our study, CuB was found to be the most efficacious compound in CuEC.

The cell cycle is crucial for cellular proliferation as it consists of four phases: G1 (Gap 1 and presynthetic growth), the S‐phase (DNA synthesis), G2 (Gap 2 or premitotic growth), and the M phase. 41 The progression of the cell cycle is regulated by cyclin subunits and cyclin‐dependent kinase (CDK). 33 The phases in which cucurbitacins induce cell cycle arrest vary among different cell types. CuB induces S and G2/M cell cycle arrest in various cancers. 42 , 43 CuD and IIa induce cell cycle arrest in the G2/M phase in breast cancer. 44 CuI mediates S–G2 transition in colon cancer cells. 45 In this study, CuEC and CuB significantly inhibited the expression of CDK2 and cyclin A2, which normally mediate progression through the S phase. DNA is synthesized during the S phase, resulting in cell proliferation. 46 These results suggest that CuEC and CuB blocked the cell cycle in the S phase, causing VSMCs to fail to proliferate, thereby preventing restenosis.

5 CONCLUSIONS

In conclusion, our study has demonstrated that CuEC and CuB inhibited the cyclin A2 pathway, thereby suppressing VSMC proliferation and preventing vascular restenosis. Our study provides firsthand evidence for the potential of CuEC and particularly CuB in regulating cyclin A2 to suppress VSMC proliferation. Therefore, both CuEC and CuB show promise as candidates for treating vascular restenosis.

AUTHOR CONTRIBUTIONS

Ruqiang Yuan and Hu Xu: Conceived and designed the experiments. Ruqiang Yuan: performed the biological experiments and phytochemical analysis. Lei Qian: calculated and supervised the study. Hu Xu and Weijing Yun: drafted the manuscript. All authors read and approved the final manuscript.

FUNDING INFORMATION

This work was supported by the National Natural Science Foundation of China Grants (81900267), Scientific Research Fund Project of Liaoning Provincial Department of Education (LJKMZ20221267, LJKZ0840, LJKZ0847).

CONFLICT OF INTEREST STATEMENT

None.

ETHICS STATEMENT

All procedures were by the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). All animal experiments in this study were approved by the Animal Care and Use Review Committee of Dalian Medical University.

Supporting information

Appendix S1.
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