Human aortic smooth muscle cells (HASMCs; Catalog No. HTX2073P, Otwo Biotech, Shenzhen, China) were cultured in Smooth Muscle Cell Medium (SMCM; Catalog No. 1101, ScienCell, San Diego, CA, USA). Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. HASMCs between passages three and eight were used for all experiments. Cell line identity was validated by short tandem repeat (STR) profiling, and all cultures tested negative for mycoplasma.

Cellular senescence was induced by treating HASMCs with 2 µM Ang II (Catalog No. MB1677, Meilunbio, Dalian, China) for 72 h; this group was designated as the Ang II group . To evaluate the protective effects of $\beta$-Ecd, cells were pretreated with 50, 100, or 200 µM $\beta$-Ecd (Catalog No. S25531, Yuanye, Shanghai, China) prior to Ang II exposure, designated as the low-, medium-, and high-dose $\beta$-Ecd groups, respectively. For mechanistic analyses, cells in the high-dose $\beta$-Ecd group were additionally treated with 50 nM bafilomycin A1 (BafA1; Catalog No. HY-100558, MCE, Shanghai, China), 10 µM SC79 (Catalog No. HY-18749, MCE, Shanghai, China), or 2.5 mM N-acetyl-L-cysteine (NAC; Catalog No. A9165, Sigma, Shanghai, China), referred to as the BafA1, SC79, and NAC groups, respectively. In parallel, cells in the Ang II group were treated with 10 µM MK-2206 (Catalog No. GC16304, Glpbio, Shanghai, China) and designated as the MK-2206 group. Untreated HASMCs served as the control group.

HASMCs were seeded into 96-well plates and treated with different concentrations of $\beta$-Ecd for 72 h. Subsequently, 10 µL of CCK-8 reagent (Catalog No. MA0218-5, MeilunBio, Dalian, China) was added to each well, followed by incubation at 37 °C for 4 h. Absorbance was measured at 450 nm using a microplate reader (SpectraMax iD3; Molecular Devices, USA). Cell viability was analyzed and plotted using GraphPad Prism version 10.1.2 (GraphPad Software LLC, San Diego, CA, USA).

HASMCs (2.5 $\times$ 10⁴ cells/well) were seeded in 24-well plates, pretreated with $\beta$-Ecd or BafA1 as described above, and subsequently exposed to Ang II for 72 h. Cells were fixed and stained using a commercial SA-$\beta$-gal staining kit (Catalog No. GC16304, Beyotime, Shanghai, China), followed by incubation overnight at 37 °C in a CO₂-free incubator. Senescent cells exhibiting blue staining were observed and quantified using an IX73 inverted microscope (Olympus, Tokyo, Japan).

Cell culture supernatants were collected after treatment with varying concentrations of $\beta$-Ecd or Ang II for 72 h to measure IL-6 and MCP-1 levels. ELISA assays were performed according to the manufacturer’s instructions (Catalog No. E-EL-H6156, Elabscience, Wuhan, China; Catalog No. E-EL-H6005, Elabscience, Wuhan, China). Absorbance was measured at 450 nm using a SpectraMax iD3 microplate reader (Molecular Devices, USA).

Total RNA was extracted from HASMCs in the Ang II group and the 200 µM $\beta$-Ecd group (n = 3 per group) after 72 h of treatment. RNA library preparation and sequencing were conducted on the Illumina NovaSeq X Plus platform (Illumina, San Diego, CA, USA) by Hangzhou Kaitai Biotechnology (Hangzhou, China). Differentially expressed genes (DEGs) were identified using edgeR version 4.0.0 (Bioconductor, http://www.bioconductor.org) with thresholds of $|$log₂ fold change$|$ $>$1 and adjusted p 0.05. Volcano plots were generated using ggplot2. Functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, were performed using clusterProfiler (p 0.05). Gene set enrichment analysis (GSEA) was performed using the fgsea package, with $|$normalized enrichment score$|$ $>$1 and p 0.05. Data visualization was performed using ggplot2, TBtools-II, and the Bioinformatics platform (https://www.bioinformatics.com.cn).

HASMCs were treated with 200 µM $\beta$-Ecd or 2.5 mM NAC, followed by exposure to 2 µM Ang II for 72 h. Cells were fixed in 70% ethanol overnight at 4 °C, stained with propidium iodide (Catalog No. C1052, Beyotime, Shanghai, China), and analyzed by flow cytometry using a LongCyte C2060 system (Beijing Challen Biotechnology, China).

Cells were lysed using RIPA buffer (Catalog No. P0013B, Beyotime, Shanghai, China), and protein concentrations were determined using a BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes (Catalog No. ISEQ00010, Sigma-Aldrich, USA). After blocking, membranes were incubated overnight at 4 °C with primary antibodies against p53 (Catalog No. AP062-1, Beyotime, Shanghai, China), p21 (Catalog No. AP021-1, Beyotime, Shanghai, China), LC3 (Catalog No. 14600-1-AP, Proteintech, Wuhan, China), p62 (Catalog No. 18420-1-AP, Proteintech, Wuhan, China), mTOR (Catalog No. 66888-1-Ig, Proteintech, Wuhan, China), phosphorylated mTOR (p-mTOR; Catalog No. 67778-1-Ig, Proteintech, Wuhan, China), AKT (Catalog No. BM4400, Boster, Wuhan, China), phosphorylated AKT (p-AKT; Catalog No. BM4744, Boster, Wuhan, China), and GAPDH (Catalog No. BM3874, Boster, Wuhan, China), followed by incubation with HRP-conjugated secondary antibodies (Catalog No. AS014, ABclonal, Wuhan, China; Catalog No. AS003, ABclonal, Wuhan, China). Protein bands were visualized using enhanced chemiluminescence (MeilunBio, Dalian, China) and quantified with ImageJ software (NIH, Bethesda, MD, USA).

Cells were incubated with 10 µM DCFH-DA (Catalog No. S0033S, Beyotime, Shanghai, China) for 20 min at 37 °C, washed with PBS. Samples were acquired on a BD FACSAria II flow cytometer (BD Biosciences, USA) and analyzed using FlowJo software version 10.8.1 (BD Biosciences, Ashland, OR, USA) to quantify intracellular ROS levels.

HASMCs cultured on coverslips were fixed with ethanol, permeabilized with Triton X-100, and blocked with goat serum. Cells were incubated overnight at 4 °C with a primary antibody against LC3 (Catalog No. 14600-1-AP, Proteintech, Wuhan, China), followed by incubation with a fluorophore-conjugated an AF647-labeled Goat Anti-Rabbit IgG (H+L) secondary antibody (Catalog No. A0468, Beyotime, Shanghai, China). Nuclei were counterstained, and LC3-positive puncta were visualized using a Revolve fluorescence microscope (Aperbio, Suzhou, China).

Cells were incubated with DAL Green dye (Catalog No. D675, Dojindo, Japan) for 30 min, followed by treatment with Ang II, $\beta$-Ecd, or BafA1 for 6 h. Fluorescence signals were observed using a Revolve confocal imaging system (Aperbio, Suzhou, China).

The three-dimensional structure of $\beta$-Ecd was obtained from PubChem and optimized using Chem3D version 22.0 (PerkinEImer Inc., Waltham, MA, USA). Crystal structures of AKT1, AKT2, AKT3, and mTOR were downloaded from the RCSB Protein Data Bank. Molecular docking simulations were performed using AutoDock Vina, and docking poses were visualized with PyMOL. Binding energies lower than –5 kcal/mol were considered indicative of stable interactions. Additional docking validation was conducted using the CB-Dock2 platform (https://cadd.labshare.cn/cb-dock2).

All experiments were independently repeated at least three times. Data are presented as mean $\pm$ standard deviation (SD). Statistical analyses were performed using GraphPad Prism version 10.1.2. Data normality was assessed using the Shapiro–Wilk test. Comparisons among multiple groups were conducted using one-way analysis of variance followed by Tukey’s post hoc test. A value of p 0.05 was considered statistically significant.

Cell viability assays showed that exposure to $\beta$-Ecd at concentrations up to 200 µM for 72 h did not significantly affect HASMCs’ viability compared with controls, indicating that $\beta$-Ecd within this concentration range was not cytotoxic (Fig. 1A).

Fig. 1.

$\beta$-Ecd attenuates Ang II–induced senescence in HASMCs. (A) Effects of $\beta$-Ecd on HASMCs viability after 72 h of treatment, as determined by CCK-8 assay. (B) Representative SA-$\beta$-gal staining images showing SA-$\beta$-gal–positive HASMCs under the indicated treatment conditions. Blue staining indicates SA-$\beta$-gal-positive cells. Scale bar = 100 µm. (C) Quantification of SA-$\beta$-gal–positive cells (n = 3). (D) Western blot analysis of p53 and p21 protein expression in HASMCs treated with Ang II in the presence or absence of $\beta$-Ecd. (E,F) Quantitative analysis of p53 and p21 protein expression normalized to GAPDH (n = 3). (G) Flow cytometric analysis of cell cycle distribution in HASMCs treated with Ang II and/or $\beta$-Ecd. (H) Statistical analysis of the proportions of cells in the G0/G1, S, and G2/M phases (n = 3). (I,J) ELISA-based quantification of IL-6 and MCP-1 secretion in HASMCs culture supernatants following Ang II stimulation with or without $\beta$-Ecd treatment (n = 3). Values are expressed as mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001. HASMCs, Human aortic smooth muscle cells; CCK-8, Cell Counting Kit-8; $\beta$-Ecd, Ecdysterone; Ang-II, Angiotensin II; SA-$\beta$-gal, Senescence-Associated $\beta$-Galactosidase; p53, Tumor protein p53; p21, Cyclin-dependent kinase inhibitor 1A; ELISA, Enzyme-linked immunosorbent assay.

To evaluate the effects of $\beta$-Ecd on Ang II–induced senescence, HASMCs were pretreated with increasing concentrations of $\beta$-Ecd and subsequently exposed to Ang II. SA-$\beta$-gal staining demonstrated a marked increase in senescent cells following Ang II treatment. Pretreatment with 200 µM $\beta$-Ecd significantly reduced the proportion of SA-$\beta$-gal-positive HASMCs (Fig. 1B,C). Consistent with these results, Ang II–induced upregulation of p53 and p21 was significantly attenuated by $\beta$-Ecd pretreatment, as determined by Western blot analysis (Fig. 1D–F).

Cell cycle analysis further revealed that Ang II induced pronounced G0/G1 phase arrest, accompanied by a reduction in G2/M phase cells. $\beta$-Ecd treatment partially reversed Ang II–induced cell cycle arrest, promoting progression into the G2/M phases (Fig. 1G,H). Because cellular senescence is often accompanied by activation of the senescence-associated secretory phenotype (SASP), we next assessed inflammatory cytokine secretion. ELISA analysis showed that Ang II increased IL-6 and MCP-1 secretion in HASMCs, whereas 200 µM $\beta$-Ecd significantly inhibited this response (Fig. 1I,J). Overall, these results indicate that $\beta$-Ecd mitigates Ang II–induced HASMC senescence, as evidenced by normalization of the cell cycle distribution and reduced SASP factor production.

RNA sequencing analysis revealed 137 DEGs between the $\beta$-Ecd–treated group and the Ang II groups, of which 130 were upregulated and 7 downregulated (Fig. 2A; Supplementary Table 1). KEGG pathway enrichment analysis revealed 18 significantly enriched pathways, with the PI3K–AKT signaling pathway among the most prominently represented (Fig. 2B). GO enrichment analysis indicated that the DEGs were significantly enriched in terms related to cytoplasmic microtubule organization, TOR signaling, protein kinase A binding, and small GTPase binding (Fig. 2C).

Fig. 2.

Transcriptomic changes induced by $\beta$-Ecd in senescent HASMCs. (A) Volcano plot of DEGs identified in HASMCs treated with $\beta$-Ecd compared with the Ang II group. Red dots indicate upregulated genes, green dots indicate downregulated genes, and gray dots represent genes that did not exhibit significant changes in expression. (B) KEGG pathway enrichment analysis of DEGs. Bar height represents the number of enriched genes, and color intensity reflects the level of statistical significance. *p 0.05, **p 0.01, ***p 0.001. (C) GO enrichment analysis of DEGs displayed as a bubble plot. Bubble size corresponds to the number of enriched genes, and bubble color denotes enrichment significance. (D) GSEA of Reactome pathways associated with DEGs. (E) Bubble plot summarizing significantly enriched Reactome pathways identified by GSEA. (F) Heatmap showing enriched Reactome pathways and their associated DEGs. DEGs, Differentially Expressed Genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis.

GSEA identified 99 enriched Reactome pathways (Supplementary Table 2), among which 12 were positively enriched. These pathways were mainly involved in cell cycle regulation, PLK1-mediated signaling, G2/M phase transition, DNA repair, centrosome maturation, and mitotic progression (Fig. 2D,E). Heatmap visualization further highlighted 47 DEGs with relatively high normalized expression, including CDK1, NEK2, HSP90AA1, and CENPF, which are involved in mitotic control and cell proliferation (Fig. 2F). Taken together, these results indicate that $\beta$-Ecd treatment is associated with changes in gene expression related to cell cycle progression and proliferation, potentially relevant to Ang II–induced senescence.

Immunofluorescence analysis showed that LC3 puncta were significantly reduced in Ang II–treated HASMCs compared with control cells. This reduction was partially inhibited following $\beta$-Ecd treatment (Fig. 3A,B). Consistent with these observations, Western blot analysis demonstrated that Ang II treatment decreased the LC3II/LC3I ratio and increased p62 protein levels, whereas $\beta$-Ecd treatment attenuated these changes. The addition of the autophagy inhibitor BafA1 abolished the effects of $\beta$-Ecd, resulting in LC3II accumulation and increased p62 levels (Fig. 3C–E). Autophagic flux analysis showed that Ang II reduced autolysosome formation, while $\beta$-Ecd significantly enhanced it. In contrast, BafA1 treatment markedly reduced autolysosome formation in Ang II + $\beta$-Ecd–treated cells (Fig. 3F).

Fig. 3.

$\beta$-Ecd enhances autophagy activity in HASMCs. (A) Representative immunofluorescence images showing LC3 distribution in HASMCs under the indicated treatment conditions (scale bar = 50 µm). (B) Quantitative analysis of LC3 fluorescence intensity (n = 3). (C) Western blot analysis of the autophagy-related proteins LC3 and p62 in HASMCs. (D,E) Densitometric analysis of LC3 and p62 protein expression levels normalized to GAPDH (n = 3). (F) Autolysosome staining was used to assess autophagic flux in HASMCs under the indicated conditions (scale bar = 50 µm). Values are expressed as mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001. LC3, Microtubule-associated protein 1A/1B light chain 3; p62, sequestosome 1; Baf A1, Bafilomycin A1.

ROS measurements revealed that Ang II significantly increased intracellular ROS levels. Treatment with NAC inhibited ROS generation, while $\beta$-Ecd produced a similar suppressive effect. In contrast, BafA1 attenuated the antioxidant effect of $\beta$-Ecd (Fig. 4A–D). Consistent with these findings, SA-$\beta$-gal staining demonstrated that $\beta$-Ecd treatment reduced Ang II-induced cellular senescence, whereas BafA1 restored senescence levels (Fig. 4E,F). Similarly, $\beta$-Ecd decreased Ang II–induced upregulation of p53 and p21, while BafA1 partially counteracted this effect (Fig. 4G–I). Overall, these findings indicate that the anti-senescent effects of $\beta$-Ecd in HASMCs are closely associated with enhanced autophagic activity and reduced ROS accumulation.

Fig. 4.

Autophagy-associated regulation of oxidative stress and senescence by $\beta$-Ecd in HASMCs. (A) Representative flow cytometry plots showing intracellular ROS levels in HASMCs following the indicated treatments. (B) Quantification of intracellular ROS levels in each group (n = 3). (C) Flow cytometric analysis of ROS levels following combined $\beta$-Ecd and BafA1 treatment. (D) Quantitative comparison of ROS levels in HASMCs (n = 3). (E) Representative SA-$\beta$-gal staining images used to evaluate cellular senescence in HASMCs (scale bar = 100 µm). (F) Quantification of SA-$\beta$-gal–positive cells (n = 3). (G) Western blot analysis of the senescence-associated proteins p53 and p21 in HASMCs. (H,I) Quantitative analysis of p53 and p21 protein expression levels normalized to GAPDH (n = 3). Values are expressed as mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001. NAC, N-acetyl-L-cysteine.

Western blot analysis showed that Ang II treatment significantly increased the ratios of p-AKT/AKT and p-mTOR/mTOR compared with control cells. Pretreatment with $\beta$-Ecd markedly reduced both p-AKT/AKT and p-mTOR/mTOR ratios, suggesting reduced activation of AKT/mTOR signaling (Fig. 5A–C). In addition, compared with the Ang II group, treatment with the AKT inhibitor MK-2206 significantly decreased the levels of p-AKT/AKT, p-mTOR/mTOR, as well as the senescence-associated proteins p53 and p21 (Fig. 5D–I). Conversely, treatment with the AKT activator SC79 largely inhibited the effects of $\beta$-Ecd, restoring AKT and mTOR phosphorylation and the expression of p53 and p21 (Fig. 5D–I). These results further support the involvement of the AKT/mTOR pathway in the anti-senescence effect of $\beta$-Ecd on HASMCs.

Fig. 5.

$\beta$-Ecd attenuates Ang II–induced activation of the AKT/mTOR signaling pathway in HASMCs. (A) Representative Western blot images showing total and phosphorylated AKT and mTOR protein levels in HASMCs under the indicated treatment conditions. (B,C) Quantitative analysis of the p-AKT/AKT and p-mTOR/mTOR ratios in each group (n = 3). (D) Representative Western blot images showing p-AKT, AKT, p-mTOR, mTOR, p53, and p21 expression in HASMCs following modulation of AKT signaling with MK-2206 or SC79. (E,F) Quantitative analysis of p-AKT/AKT and p-mTOR/mTOR ratios (n = 3). (G–I) Quantification of p53 and p21 protein expression levels normalized to GAPDH (n = 3). Data are presented as mean $\pm$ SD. *p 0.05, **p 0.01, ***p 0.001. AKT, Protein Kinase B; p-AKT, Phosphorylated AKT; mTOR, Mechanistic Target of Rapamycin; p-mTOR, Phosphorylated mTOR.

Molecular docking analysis suggested potential interactions between $\beta$-Ecd and components of the AKT/mTOR pathway. $\beta$-Ecd showed favorable predicted binding energies with AKT1, AKT2, AKT3, and mTOR (–8.0, –8.5, –8.7, and –8.6 kcal/mol, respectively) and formed multiple hydrogen bonds. Specifically, $\beta$-Ecd formed hydrogen bonds with ARG328, TYR38, and LYS30 on AKT1; VAL272, TYR327, ASP324, and LEU52 on AKT2; GLU154, GLN44, GLN38, and GLU109 on AKT3; and GLU95, GLN140, ASP322, and ARG227 on mTOR (Fig. 6A–D). These interactions were further supported by CB-Dock2 analysis, which yielded comparable or slightly stronger predicted binding energies (–8.2 to –9.4 kcal/mol; Fig. 6E–H). Taken together, these results indicate that $\beta$-Ecd attenuates Ang II–induced HASMC senescence, associated with reduced AKT/mTOR signaling activity, which may involve interactions with components of this pathway.

Fig. 6.

Molecular docking analysis of $\beta$-Ecd with components of the AKT/mTOR signaling pathway. (A–D) Predicted binding modes of $\beta$-Ecd with AKT1, AKT2, AKT3, and mTOR, as determined by AutoDock Vina and visualized using PyMOL. (E–H) Independent docking analysis of $\beta$-Ecd with AKT1, AKT2, AKT3, and mTOR was performed using the CB-Dock2 platform.