The fruits of F. hispida used in this study were collected in Wenchang city of Hainan Province, People’s Republic of China, in September 2022 (latitude 19.5614° N, longitude 110.8023° E). A voucher specimen (20220901) was deposited in the Herbarium of Materia Medica, School of Pharmaceutical Sciences, Hunan University of Medicine. Isowighteone, 3′-(3-methylbut 2-enyl)biochanin A, and myrsininone A was isolated from the collected fruits. The purity of these isolated compounds was assessed using HPLC and was found to be greater than 98%.

This study was approved by the Research Ethics Committee of Guangdong Provincial People’s Hospital and was performed in accordance with the Declaration of Helsinki. The enrolled patients included 8 males and 3 females, with a mean age of 62.5 years (range: 53–72 years). The etiology of aortic dissection among participants primarily included hypertension-induced degeneration, and atherosclerosis. HASMCs were isolated from the aorta of a patient with aortic dissection. Briefly, the median membrane of the aorta was removed and the cells were incubated with 1 mg/mL trypsin (Gibco, 12605-010) for 10 min. The cells were washed with HBSS buffer (Hyclone, SC30588.01). Aortic tissue was digested in 250 U/mL collagenase type II solution (Worthington, 47D17411A) at 37 ° C for 7 h. The resulting cells were resuspended in α-MEM containing 10% fetal bovine serum (Gibco, 16000-044), 100 U/mL penicillin, and 100 mg/mL streptomycin (HyClone, SH40003.01.) SV30010) in growth medium and plated on a 25 cm² flask coated with 0.25 μg/cm² type I collagen (Gibco, A1048301). Two to four generations of cells were used in this study.

HASMCs were inoculated into 6-well plates at a density of 1.0 × 105 cells/well and cultured with growth medium. As mentioned above, calcification was simply induced by culturing HASMCs to fusion and treating with a control (1.0 mM Pi/1.8 mM Ca) or a calcifying medium (50 μg/mL ascorbate/2.5 mM Pi/2.7 mM Ca) for 7 days. Pi was prepared using NaH2PO4/Na2HPO4 as solvent and pH = 7.4. To evaluate the effect of isowighteone on calcifying HASMCs in vitro, a certain concentration (50 μM) of isowighteone was added to the medium. The medium was changed every 2/3 days.

Male C57BL/6 mice, 6–8 weeks old, purchased from Rueger Biology Co., LTD. The 45 mice were randomly divided into three groups with 15 mice in each group. To establish a mouse calcification model, high doses of vitamin D (5*105 IU/kg/day) were injected subcutaneously into the abdominal cavity of each mouse for 3 consecutive days and continued to be fed for 4 days. After the vitamin Dinjection, mice in the model group were given normal saline by gavage, and mice in the treatment group were given 50 mg/kg isowighteone by gavage daily for 4 consecutive days. On day 7, at the end of the study, the mice were euthanized under 2% pentobarbital anesthesia and the mouse aorta was excised. The control group received no treatment, and the aorta was collected on day 7. The research plan of this animal experiment was approved by the Animal Ethics Committee of Guangdong Provincial People’s Hospital, Guangzhou, China. All animal procedures were performed in accordance with the recommendations of the National Science and Technology Commission of the People’s Republic of China in the Guide to the Care and Use of Laboratory Animals.

Cell viability was assessed by CCK-8 assay (Biosharp, BS350A). Specifically, HASMCs was plated in 96-well plates at a density of 2 × 10^4 cells per well and then incubated overnight in a 5% CO² atmosphere at 37 °C to allow cell attachment and stabilization. After incubation, the effects of the compound on cell viability at different doses (50 μM, 100 μM and 150 μM) were measured at 24 and 48 h time points. Before detection, 10 μL CCK-8 solution was added to each well and incubated for 2 h. The optical density of each well was then quantitatively measured at a wavelength of 450 nm, providing a direct reading of cell viability for the treatment condition.

To assess RUNX2 expression, HASMCs were cultured with control or calcified media in the presence or absence of 50 μM isowightone for up to 7 days. Cells were fixed, infiltrated with 0.5% Triton X-100(Beyotime Biotechnology, P0013B) and treated with anti-RUNX2 (1:500, proteintech, 20700-1-AP) at 4 °C overnight. After frozen sectioning of isolated mouse aorta, antigen repair was performed and antiRUNX2 (1:500, Proteintech, 20700-1-AP) was treated overnight at 4 °C. After washing with PBS, Alexa Fluor^®488 anti-rabbit antibody (1:1,000, Invitrogen, A11008)were incubated in blocking buffer at 37 °C in the dark for 2 h. Fluorescence signals were detected under a Leica DMRB fluorescence microscope (Leica SP8) with a DAPI-stained glass mask.

Total RNA was extracted from HASMCs using Trizol (Invitrogen, 1596026) according to the manufacturer’s instructions. Quantification and reverse transcription of RNA was performed using HiScript III RT SuperMix for qPCR(+gDNA wiper) (Vazyme, R323-01). qRT-PCR was performed in a QuantStudio 5 real-time system (Life technologies) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-01). Each PCR was repeated in triplicate. All gene expression data were calculated using 2^−ΔΔCT and normalized to β-actin. β-actin was used as the housekeeping gene after confirming its stable expression under both control and calcifying conditions. The control value is expressed as 1 to represent the exact fold change value for each gene of interest. Primer sequences of the target genes are summarized in Supplementary Table S1.

HASMCs was harvested using a radioimmunoprecipitation (RIPA) lysis buffer (Beyotime Biotechnology, P0013B) containing protease and phosphatase inhibitor (Thermo, A32961). Western blot was performed. An equal amount of protein lysate was separated on an SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with primary antibodies overnight at 4 °C: anti-HSP90AA1 (1:2,000, Proteintech, 13171-1-AP), anti-RUNX 2 (1:2,000, Proteintech, 82636-2-RR), anti-BMP2 2 (1:1,000, Proteintech, 66383-1-Ig), anti-p-Akt (1:2,000, Proteintech, 66444-1-Ag), anti-Akt (1:2,000, Proteintech, 10176-2-AP), anti-MSX2 (1:2,000, Proteintech, 68550-1-Ig), anti-PI3K(1:1,000, Cell Signaling Technology, 4292), anti-p-PI3K(1: 1,000, Cell Signaling Technology, 3011S), anti-beta-actin (1:2,000, Proteintech, 66009-1-Ig), followed by anti-mouse (1:400, Cell Signaling Technology, 7076S) or anti-rabbit (1:400, Cell Signaling Technology, 7074S) secondary antibodies coupled to the membrane with horseradish peroxidase (HRP) were incubated at room temperature for 1 h. The immune complex was visualized using a chemiluminescent Western blot Substrate (Millipore, WBKLS0500). Semi-quantitative assessment of band intensity was performed using ImageJ software (National Institutes of Health).

The Search Tool for Interacting Chemicals (STITCH) and the SwissTargetPrediction database were used to predict interacting molecular targets associated with isowightone. The collection of targets associated with VC was obtained from Genebank.

PPI data of SO-related molecular targets were collected using STRING online database, Cytoscape 3.2.1 software was run to visualize the PPI relationship network.

Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the DAVID database (https://david.ncifcrf.gov/). This comprehensive analysis included the three major categories of GO: biological process (BP), cellular component (CC), and molecular function (MF). Pathways and GO terms with p < 0.05 were considered significantly enriched, and false discovery rate (FDR) correction was applied to adjust for multiple comparisons. The genes most significantly associated with enriched GO terms and KEGG pathways were prioritized based on their logP values.

Structural data for the two primary target proteins were obtained from the Universal Protein Resource (UniProt: https://www.uniprot.org/) and the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB: https://www.rcsb.org/).Simultaneously, the three-dimensional structures of two key small-molecule ligands were retrieved from PubChem (https://pubchem.ncbi.nlm.nih.gov/). All receptor proteins were processed and visualized using PyMOL version 2.5.2. Prior to molecular docking with AutoDock Vina version 1.1.2, a grid box was defined around the active sites of the receptors to ensure accurate interaction analysis. The docking results were evaluated based on binding affinity, and the conformation with the lowest binding energy was selected as the most favorable ligand–receptor binding mode.

Isowighteone (10 mg) was dissolved in anhydrous dimethyl sulfoxide (DMSO, 1 mL).Separately, Cy5-NHS ester (Lumiprobe, United States) was dissolved in DMSO (10 mM stock). The two solutions were mixed at a 1:1.5 M ratio (isowighteone:Cy5-NHS), and 3 equivalents of DIPEA (N,N-diisopropylethylamine) were added to catalyze the reaction. The mixture was stirred under nitrogen at room temperature, protected from light, for 12 h. After the reaction, the product was purified using C18 reverse-phase HPLC, and the conjugate was identified by UV–Vis spectroscopy (showing peaks at ∼290 nm and ∼650 nm) and LC-MS to confirm the expected molecular weight. The final compound was lyophilized and stored at −20 °C in the dark.

HASMCs were seeded in 24-well plates containing coverslips and cultured to approximately 70% confluence. Cells were incubated with either free Cy5(500 nM) or an equimolar concentration of Isowighteone-Cy5 conjugate in serum-free medium for 12 h at 37 °C. After incubation, cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with DAPI. Coverslips were mounted on slides, and fluorescence images were acquired using a confocal laser scanning microscope (excitation/emission for Cy5: 650/670 nm).

Male C57BL/6J mice (8–10 weeks old) were randomly divided into two groups and injected via tail vein with either free Cy5 or Isowighteone-Cy5(1 nmol in 100 µL PBS). Whole-body fluorescence imaging was performed using an in vivo imaging system (IVIS Spectrum, PerkinElmer) at 0, 1, 2, and 4 h post-injection.

Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons using GraphPad Prism (GraphPad software version 7.0, San Diego, CA, United States). P < 0.05 was considered statistically significant.

Three isoflavonoids, isowighteone, 3′-(3-methylbut 2-enyl)biochanin A (Biochanin A) (hereafter referred to as Biochanin A), and myrsininone A (Figure 1A), were isolated from the fruit of F. hispida. CCK-8 assays demonstrated that all three compounds exerted minimal cytotoxicity on HASMCs at a concentration of 50 μM over 24 and 48 h (Figures 1B–D), establishing 50 μM as the optimal concentration for subsequent experiment.

Exposure of HASMCs to 50 μM isowighteone significantly reduced calcium deposition after 7 days, as evidenced by alizarin red staining and calcium quantification (Figures 2A,B). In contrast, 3′-(3-methylbut-2-enyl)biochanin A and myrsininone A did not notably affect calcification levels. Furthermore, isowighteone attenuated the upregulation of osteogenic markers RUNX2, BMP2, and MSX2 at both mRNA and protein levels (Figures 2C–G). Immunofluorescence imaging corroborated the reduction in nuclear RUNX2 expression upon isowighteone treatment (Figure 2H). Collectively, isowighteone exhibits superior efficacy in inhibiting calcification and osteogenic gene expression compared to the other tested compounds.

A comprehensive target analysis identified 5,604 VC-related targets from the GeneCards database and 17 from the OMIM database, totaling 5,613 unique targets after deduplication. Intersection with 100 isowighteone-related targets revealed 64 overlapping targets (Figure 3A; Supplementary Table S2). Based on the candidate protein targets of isowighteone,we constructed the PPI network, which contained 64 nodes and 337 edges (Figure 3B; Supplementary Table S3). The top 4 proteins were considered as the critical molecular targets mediating the anti-VC effects of isowighteone. It was found that EGFR, ESR1, BCL2 and HSP90AA1 were separately linked to other 39,34,32 and 31 targets, respectively (Figure 3C).

To further verify the biological characteristics of 64 isowighteone targets on VC, we performed enrichment analysis of KEGG pathways to explore potential pathways. The 20 most enriched pathways are shown in Figure 3D. The KEGG pathways are detailed in Supplementary Table S4. These enriched targets are associated with a variety of pathways, including primarily those associated with inflammation and angiogenesis. Among the 64 overlapping targets, 26 closely interacted with pathways associated with the cancer pathway, the PI3K−Akt signaling pathway, MicroRNAs in cancer pathway and the RAS pathway (Figure 3D). Details of KEGG analysis are provided in Supplementary Table S4. Next, we performed GO enrichment analysis to clarify their biological processes. The top 20 entries based on the value of p. The GO enrichment analysis provided insights into the roles of intersecting targets across the domains of BP, CC, and MF. In the BP domain, the genes were primarily involved phosphorylation, protein phosphorylation, signal transduction, and positive regulation of transcription by RNA polymerase II (Figure 3E). Within the CC domain, the predominant genes were associated with plasma membrane, cytoplasm, and nucleus (Figure 3F). For the MF domain, the key genes were involved in protein binding, ATP binding and identical protein binding, among other molecular functions (Figure 3G). Details of GO analysis are provided in Supplementary Tables S5–S7. These findings suggest that isowighteone may exert its anti-VC effects through modulation of these key signaling pathways.

Molecular docking studies were performed to evaluate the binding affinity of isowighteone to its core target genes. The main targets identified due to their centrality in the network include BCL2 (PDB ID: 2XA0), EGFR (PDB ID: 1YY9), ESR1 (PDB ID: 1A52), and HSP90AA1, (PDB ID: 1BYQ) as protein receptors. Isowighteone, as a natural compound, acts as a ligand in the validation of molecular docking procedures (Figure 4A). The docking results showed that the binding energies of these compounds and the target were all below −6.90 kcal/mol, indicating that there was a strong binding effect between them (Figure 4B). It is noteworthy that isowighteone interacts particularly strongly with HSP90AA1, showing the most favorable binding energy. The binding pattern between isowighteone and the central gene is shown in the figure, which shows multiple interaction types at the active site, including van der Waals forces, hydrogen bonds, hydrophobic interactions, and electrostatic forces.Subsequently, qRT-PCR and Western blot analyses confirmed that isowighteone significantly downregulated HSP90AA1 expression in calcifying cells (Figures 4C–E). These findings suggest that isowighteone may exert its anti-VC effects through modulation of HSP90AA1. Given the pivotal role of the PI3K/Akt pathway in VC, Western blot analyses revealed that isowighteone reduced the phosphorylation levels of PI3K and Akt without altering their total protein expression (Figures 4F,G). These results implicate isowighteone as an inhibitor of the PI3K/Akt pathway, thereby attenuating VC.

To investigate the anti-calcification effects of isowighteone in vivo, we established a mouse model of VC through high-dose vitamin D administration (Figure 5A). Oral administration of isowighteone did not significantly affect body weight, suggesting a favorable safety profile (Figure 5B). Alizarin red staining and quantitative calcium analysis revealed that isowighteone markedly attenuated aortic calcification in the VC model (Figures 5C,D). Furthermore, fluorescence staining demonstrated a significant reduction in RUNX2 expression in the aortic tissues of treated mice (Figure 5E). Given the critical role of osteogenic genes in calcification, we evaluated the expression of RUNX2, BMP2, and MSX2, and found that isowighteone effectively suppressed their levels in vivo (Figure 5G). These findings collectively indicate that isowighteone significantly reduces calcification in the VC model.

To elucidate the underlying mechanisms, we examined the expression of HSP90AA1 in mouse aortic smooth muscle cells. qRT-PCR confirmed that isowighteone downregulated HSP90AA1 expression (Figure 5F). Additionally, considering the pivotal role of the PI3K/Akt signaling pathway as a downstream effector, we assessed its activity via Western blot (Figures 5G–I). The results demonstrated that isowighteone suppresses PI3K/Akt signaling transduction. Together, these in vivo results underscore the therapeutic potential of isowighteone in mitigating VC.

To investigate the biodistribution of isowighteone in vivo, we synthesized a Cy5-labeled isowighteone (Isowighteone-Cy5) conjugate. Following incubation with HASMCs for 12 h, immunofluorescence analysis revealed a markedly higher intracellular fluorescence signal in the Isowighteone-Cy5 group compared to cells treated with free Cy5, indicating enhanced cellular uptake of isowighteone (Figure 6A).

For in vivo evaluation, Isowighteone-Cy5 or free Cy5 was intravenously administered via tail vein injection in mice. Fluorescence imaging at 0, 1, 2, and 4 h post-injection showed that Isowighteone-Cy5 exhibited slower clearance compared to free Cy5. Notably, the conjugate exhibited evident accumulation around vascular structures, demonstrating that isowighteone is capable of accumulating in perivascular tissues in vivo (Figure 6B).