A Bifidobacterium adolescentis LA20k9 strain was isolated from the feces of an elderly dog (accession no: CCTCC-M-2025638). For LA20k9 fermentation and freeze-drying, the seed culture of Bifidobacterium adolescentis LA20k9, preserved in 15% glycerol (Oxoid; Thermo Fisher Scientific, Inc.), was inoculated into De Man-Rogosa-Sharpe medium (Oxoid; Thermo Fisher Scientific, Inc.) containing 2% (v/v) brown algae extract (derived from Laminaria japonica; Shanxi Benhe Biotechnology Engineering Co., Ltd.) and anaerobic culture was carried out at 38˚C for 48 h.

Furthermore, 15% glycerol without Bifidobacterium adolescentis LA20k9 was fermented under the same conditions, with blank substrate fermentation metabolite (BMFM) excluding LA20-k9. The supernatant of the fermentation broth was collected after room temperature centrifugation for 10 min at 2,000 x g and freeze-dried. CVECs [Global Pet Cell Resource Center (PETCC)-199 cell line; cat. no. PETCC#199] were obtained from the PETCC (Tianjin, China). Doxorubicin (DOX) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Hanks' balanced salt solution (HBSS), the senescence-associated β-galactosidase (SA-β-gal) staining kit and SA-β-gal activity assay kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. Trypsin, FBS, TRIzol^® and M-3 medium were obtained from Gibco (Thermo Fisher Scientific, Inc.). Canine ELISA kits for MMP-1 (cat. no. ml031816), MMP-3 (cat. no. ml024650), VEGF (cat. no. ml031966), basic fibroblast growth factor (bFGF; cat. no. ml031890), granulocyte-macrophage colony stimulating factor (GM-CSF; cat. no. ml002650) and inflammatory factors IL-1β (cat. no. ml024767), TNF-α (cat. no. ml031931), IL-6 (cat. no. ml024780), IL-8 (cat. no. ml024786) and monocyte chemotactic protein-1 (MCP-1; cat. no. ml002710) were obtained from Shanghai Enzyme-linked Biotechnology Co., Ltd. Oxidative stress-associated kits for superoxide dismutase (SOD; cat. no. S0101S), malondialdehyde (MDA; cat. no. S0131S) and glutathione (GSH; cat. no. S0053).

Disaccharide and monosaccharide contents of LA20k9 and BMFM were determined using precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) (cat. no. A501894-0250; Sangon Biotech Co., Ltd.) and high-performance liquid chromatography (HPLC). LA20k9 and BMFM were hydrolyzed in 10 ml 7.2% sulfuric acid (cat. no. S962529; Shanghai Macklin Biochemical Co., Ltd.) under nitrogen at 110˚C for 2 h. After hydrolysis, 0.5 ml the sample was neutralized and diluted with water to 1 ml. Monosaccharide standards [glucose (cat. no. D823520) and fucose (cat. no. L809666) were obtained from Shanghai Macklin Biochemical Co., Ltd.] or hydrolyzed LA20k9 and BMFM were derivatized with 0.2 ml 0.3 M sodium hydroxide (cat. no. R885161; Shanghai Macklin Biochemical Co., Ltd.) and 0.4 ml PMP solution. The mixture was incubated at 70˚C for 60 min under nitrogen. Afterward, 0.2 ml 0.3 M hydrochloric acid (cat. no. C0680110271; Nanjing Chemical Reagent Co., Ltd.) was added and the volume was adjusted to 2 ml with water. Chloroform (1.5 ml; cat. no. C1481519267; Nanjing Chemical Reagent Co., Ltd.) was added, shaken and the organic layer discarded. The aqueous phase was filtered through a 0.45 µm aqueous membrane and analyzed on an Agilent 1200 HPLC system (Agilent Technologies, Inc.) with a C18 (250.0x4.6 mm; 5 µm) column and UV detection. The method of monosaccharide concentration detection followed the same procedure as described for polysaccharide content analysis, excluding the hydrolysis step.

Protein content. LA20k9 and BMFM protein content was determined using the Pierce^™ BCA protein assay kit (cat. no. A65453; Thermo Fisher Scientific, Inc.). A 20 mg sample was accurately weighed and dissolved in 1 ml ultrapure water, followed by a 200-fold dilution to prepare the sample solution. Then, 20 µl solution was mixed with 200 µl BCA working reagent. The reaction mixture was incubated at 37˚C for 30 min and the absorbance at 562 nm was measured at the end of the incubation.

Detection of amino acid content. LA20k9 and BMFM amino acid content was determined by HPLC coupled with pre-column derivatization using o-phthaldialdehyde (OPA) (cat. no. 26025; Thermo Fisher Scientific, Inc.). LA20k9 and BMFM were hydrolyzed in 10 ml 6 M hydrochloric acid under nitrogen at 110˚C for 24 h to prevent oxidation. After hydrolysis, 100 µl sample solution or amino acid standard [aspartic acid (cat. no. L708685), threonine (cat. no. H818497), serine (cat. no. D794545), glutamic acid (cat. no. D665473), glycine (cat. no. G800880), alanine (cat. no. D688836), cystine (cat. no. D804429), valine (cat. no. V828207), methionine (cat. no. L675564), isoleucine (cat. no. H811900), leucine (cat. no. D688815), tyrosine (cat. no. D818619), phenylalanine (cat. no. D793640), histidine (cat. no. D810956), lysine (cat. no. D856989), arginine (cat. no. H677986) and proline (cat. no. D794397) obtained from Shanghai Macklin Biochemical Co., Ltd.] was mixed with 200 µl OPA reagent. The mixture was vortexed vigorously for 30 sec and allowed to react at room temperature in the dark for 15-20 min. After reaction, the mixture was filtered through a 0.22 µm aqueous membrane to remove particulates prior to HPLC analysis. HPLC separation was performed on an Agilent 1200 HPLC system (Agilent Technologies, Inc.) equipped with a C18 reversed-phase column. The mobile phase consisted of 0.1 M PBS (pH 8.0; solvent A) and a mixture of methanol (cat. no. C0690110124; Nanjing Chemical Reagent Co., Ltd.) and acetonitrile [cat. no. C0560116005; Nanjing Chemical Reagent Co., Ltd.; 1:1 (v/v) solvent B]. A linear gradient elution was employed as follows: 0-10 min, 10-30% B; 10-25 min, 30-50% B; 25-30 min, 50-10% B; and 30-35 min, 10% B for equilibration. The flow rate was set at 1.0 ml/min and the column temperature was maintained at 30˚C. Amino acids were emission wavelengths set at 340 and 450 nm, respectively.

Detection of organic acid content. Organic acid content was determined by HPLC. The analysis was performed on a reversed-phase C18 column with 0.01 mol/l potassium dihydrogen phosphate solution (pH 2.5-3.0; cat. no. P837011; Shanghai Macklin Biochemical Co., Ltd.) as the mobile phase at a flow rate of 1.0 ml/min. The column temperature was maintained at 30˚C and the detection wavelength was set at 210 nm. The injection volume was 20 µl. Samples or organic acid standard [tartaric acid (cat. no. D666102), malic acid (cat. no. D689340), lactic acid (cat. no. D742481), pyruvic acid (cat. no. P796878), oxalic acid (cat. no. S818087), acetic acid (cat. no. A801299), citric acid (cat. no. C805019), succinic acid (cat. no. S874398), propionic acid (cat. no. P757623) and benzenolactic acid (cat. no. L795422) obtained from Shanghai Macklin Biochemical Co., Ltd.] were dissolved in water, vortexed to mix, prepared as standard working solutions at room temperature and filtered through a 0.22 µm aqueous membrane before injection. Quantification was carried out using the external standard method (23,24). Calibration curves were constructed by plotting peak areas against corresponding standard concentrations. The contents of individual organic acids in samples were calculated based on the calibration equations.

CVECs were authenticated by the supplier and tested negative for Mycoplasma contamination. The cells were cultured in modified M3-DMEM medium (PETCC) supplemented with 12% FBS. Cultures were maintained in an atmosphere humidified with 5% CO₂ until 90% confluence before use.

Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) assay (Beyotime Biotechnology). CVECs were stained with trypan blue at room temperature for 5 min, counted and adjusted to 1x10⁴ cells/ml. The cells were seeded into 96-well plates and incubated for 24 h at 37˚C with numerous concentrations of LA20k9 (0, 20, 40, 80, 160, 320, 640 and 1,280 µg/ml), BMFM (0, 20, 40, 80, 160, 320, 640 and 1,280 µg/ml) and DOX (0, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 µmol/l). After removing the drug solutions, 110 µl HBSS containing 10% CCK-8 was added, mixed for 30 sec and incubated for 2 h. Absorbance was then measured at 450 nm. HBSS without drugs served as the negative control (NC). DOX at 60-70% viability was used to induce senescence.

The following groups were established to assess the capacity of LA20k9 and BMFM to restore the viability of DOX-induced vascular ECs: HBSS with only DOX (senescence model group); DOX with varying concentrations of LA20k9 (LA20k9 treatment groups); DOX with varying concentrations of BMFM (BMFM treatment groups); and HBSS alone (NC group). The procedures were the same as those used to determine the drug concentrations.

CVECs were seeded in 6-well plates at a density of 1x10⁶ cells/well and cultured at 37˚C and 5% CO₂ for 24 h until the cells adhered. Drug treatment groups were the same as those used for the cell viability assay. After 24 h, quantitative and qualitative analyses were conducted using the SA-β-gal activity detection kit and the SA-β-gal staining kit, respectively, following the manufacturer's protocols.

CVECs were seeded in 6-well plates at a density of 1x10⁶ cells/well and cultured at 37˚C and 5% CO₂ for 24 h until the cells adhered. Drug treatment groups were the same as those used in the cell viability assay. After 24 h, the levels of MMP-1, MMP-3, VEGF, bFGF, IL-1β, TNF-α, IL-6, IL-8, GM-CSF and MCP-1 were measured using ELISA kits. Canine ELISA kits for MMP-1 (cat. no. ml031816), MMP-3 (cat. no. ml024650), VEGF (cat. no. ml031966), basic fibroblast growth factor (bFGF; cat. no. ml031890), granulocyte-macrophage colony stimulating factor (GM-CSF; cat. no. ml002650) and inflammatory factors IL-1β (cat. no. ml024767), TNF-α (cat. no. ml031931), IL-6 (cat. no. ml024780), IL-8 (cat. no. ml024786) and monocyte chemotactic protein-1 (MCP-1; cat. no. ml002710) were obtained from Shanghai Enzyme-linked Biotechnology Co., Ltd. Specifically, 50 µl supernatant from each treatment group was added to wells pre-coated with primary antibodies specific to either canine protein. HRP-conjugated secondary antibodies were then added and the plates were incubated at 37˚C for 60 min. After incubation, the wells were washed five times with PBS with Tween 20, followed by the addition of 100 µl 3,3',5,5'-tetramethylbenzidine substrate for color development. Absorbance was measured at 450 nm.

CVECs were seeded in 6-well plates at a density of 1x10⁶ cells/well and cultured at 37˚C and 5% CO₂ for 24 h until the cells adhered. The treatment groups were the same as used for the cell viability assay. After 24 h of action, the SOD content was detected using a total SOD activity detection kit, following the manufacturer's protocols and absorbance was measured at 450 nm. MDA content was used to assess lipid oxidation levels. MDA was measured in cell lysates using a colorimetric lipid oxidation detection kit, following the manufacturer's protocols. Absorbance was measured at 532 nm. GSH content was measured using a GSH detection kit, following the manufacturer's protocols. GSH levels were measured by mixing 20 µl sample with 5,5'-dithiobis (2-nitrobenzoic acid) solution, allowing the reaction to proceed for 5 min and reading the absorbance at 405 nm. An enhanced NAD⁺ detection kit was used to measure the NAD⁺ content through sequential incubation with ethanol dehydrogenase, followed by the addition of a colorimetric reagent in strict accordance with the manufacturer's instructions. Protein concentrations were quantified using a BCA kit (Thermo Fisher Scientific, Inc.).

Cells from different treatment groups were lysed using TRIzol reagent. Phase separation was achieved by adding chloroform, followed by RNA precipitation with isopropanol and washing with ethanol. The isolated RNA was dissolved in RNase-free water (cat. no. R0022; Beyotime Biotechnology). RNA concentration and integrity were verified using an Agilent 5400 Bioanalyzer (Agilent Technologies, Inc.). mRNA was enriched using Oligo(dT) beads and fragmented in a buffer with divalent cations [NEBNext^® Poly(A) mRNA Magnetic Isolation Module; cat. no. E7490; New England Biolabs, Inc.]. First-strand cDNA was synthesized using Moloney Murine Leukemia Virus Reverse Transcriptase (New England Biolabs, Inc.), followed by second-strand synthesis with DNA polymerase I and RNase H (NEBNext^® Ultra^™ II RNA Library Prep; cat. no. E7775L; New England Biolabs, Inc.). The double-stranded cDNA underwent end repair, A-tailing, adaptor ligation and AMPure XP bead purification, then PCR amplification to construct the cDNA library (NEBNext^® Ultra^™ II RNA Library Prep; cat. no. E7775L; New England Biolabs, Inc.). The sequencing was carried out using the NovaSeq 6000 platform (Illumina, Inc.) employing a paired-end 150 bp approach. The final loading concentration of the library was 1.5 µg/µl.

To generate clean data, sequencing reads were filtered to remove adapters, ambiguous bases and low-quality sequences. The filtered reads were aligned to the reference genome with Hierarchical Indexing for Spliced Alignment of Transcripts (version 2.2.1; https://daehwankimlab.github.io/hisat2/download/). Gene expression quantification was performed using featureCounts (version 2.0.6; https://subread.sourceforge.net/featureCounts.html) to tally reads mapped to each gene, with results expressed as fragments per kilobase of transcript per million (FPKM) mapped reads. Principal component analysis was applied to the FPKM values. Differential expression was analyzed using the ‘DESeq2’ package (version 1.42.0; R software; Posit Software, PBC; https://bioconductor.org/packages//release/bioc/html/DESeq2.html). Raw read count data were normalized to correct for sequencing depth differences. Statistical models calculated P-values for each gene, which were adjusted for multiple testing using the Benjamini-Hochberg method to obtain adjusted P-values. Differentially expressed genes (DEGs) were identified based on a fold change of ≥1.2 and adjusted P≤0.05. Normalization and hierarchical clustering of DEGs' FPKM expression matrices were performed using the ‘ggplot2’ package in R software (version 3.0.3; Posit Software, PBC; https://ggplot2.tidyverse.org/), with heatmap clustering conducted using the ‘pheatmap’ package (https://cran.r-project.org/web/packages/pheatmap/index.html).

To determine impacted signaling pathways and biological processes, Gene Set Enrichment Analysis (GSEA) was conducted using Gene Ontology (GO; https://www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Genes were ranked by differential expression between groups using ‘Signal2Noise’ as the ranking metric. Enrichment scores for each gene set were calculated using permutation tests and normalized enrichment scores were obtained to account for gene set size. Significance was assessed by computing P-values and false discovery rates (FDR), with FDR <0.25 indicating significant enrichment. The EnrichmentMap tool (version 3.3.1; https://apps.cytoscape.org/apps/enrichmentmap) was used to visualize GSEA results.

Untargeted metabolomic profiling was performed to investigate treatment-related metabolic alterations in CVECs. After treatment, CVECs were digested with trypsin for 2 min at 37˚C and then neutralized with PBS. Cell pellets were obtained by centrifuging at 1,000 x g for 5 min (at 4˚C; 5702 R centrifuge; Eppendorf SE). Samples were sent to Novogene Co., Ltd. for analysis using a liquid chromatography-tandem mass spectroscopy platform (Thermo Fisher Scientific, Inc.).

All data are expressed as the mean ± SD. Each group contained ≥4 replicates and CCK-8 assays were performed with a minimum of 12 replicates per condition. Statistical analyses were conducted using SPSS software (version 26.0; IBM Corp.). One-way ANOVA followed by Least Significant Difference or Games-Howell post-hoc tests was used for the datasets. The linear correlation between the transcriptome analysis samples was assessed by Pearson's correlation analysis and the correlation coefficient (r) was calculated. P-values were calculated using unpaired t-test. P<0.05 was considered to indicate a statistically significant difference.

Components of LA20k9 and BMFM were analyzed (Table SI). The most significant difference between the two was in the organic acid component. LA20k9 contained 26.63 mg/g of malic acid and 59.17 mg/g of succinic acid, while these two organic acids were absent in BMFM. These two metabolites are the main biologically active metabolites produced by LA20k9. Malic acid and succinic acid are used in anti-aging studies due to their excellent performance in repairing oxidative damage and anti-inflammatory activity (25,26). Therefore, the results suggest that LA20k9 has anti-aging effects.

Fig. 1A shows the cell viability after LA20k9 treatment. The three concentrations with the highest cell survival rates (320, 640 and 1,280 µg/ml) were selected for subsequent experiments. As shown in Fig. S1A, for BMFM, concentrations of 160, 320 and 640 µg/ml were utilized for comparative analysis.

The effects of different DOX concentrations (0-1.0 µmol/l) on cell viability were evaluated to determine the optimal concentration for inducing aging. With increases in DOX concentration, the viability of CVECs gradually decreased, demonstrating DOX dose-dependency (Fig. 1B). A DOX concentration of 0.2 µmol/l significantly reduced cell viability to 70.2% (P<0.05) and was selected as the concentration for DOX-induced CVEC senescence.

After adding 320, 640 and 1,280 µg/ml LA20k9 to 0.2 µmol/l DOX, the viability of CVECs increased to varying degrees (Fig. 1C). Among them, 1280 µg/ml LA20k9 most significantly improved cell viability (P<0.05), which was be restored to 184.8%, markedly exceeding the level of the NC group. As shown in Fig. S1B, after adding 320 µg/ml BMFM to 0.2 µmol/l DOX, the cell viability of CVEC showed the most significant improvement, reaching 90.7% (P<0.05). Notably, the regenerative capacity of LA20k9 markedly surpassed that of the BMFM. This performance gap suggests that the bioactive potency was not inherent to the raw algal substrate but was specifically generated through B. adolescentis fermentation. Therefore, subsequent experiments were conducted at 0.2 µmol/l DOX + 1,280 µg/ml LA20k9 and 0.2 µmol/l DOX + 640 µg/ml BMFM.

SA-β-gal accumulates in lysosomes and has become a commonly used biomarker for identifying senescent cells (27,28). After treating CVECs with DOX for 24 h, the SA-β-gal activity significantly increased by 16% compared with the control group (P<0.05) and the number of blue-infiltrated senescent cells also increased (Fig. 2A and B). After treatment with 1280 µg/ml LA20k9, SA-β-gal activity was significantly reduced by 16% (P<0.05) and the number of blue-infiltrated senescent cells was also decreased.

By contrast, BMFM failed to mitigate this response, exhibiting activity levels statistically indistinguishable from the DOX model group. This quantitative profile was visually corroborated by microscopic imaging (Fig. 2B). These results indicated that the anti-aging effect was not derived from the non-fermented algae extract itself but was specifically produced through the fermentation process of Bifidobacterium adolescentis.

Effect of LA20k9 on the levels of senescence-associated secretory phenotype (SASP) markers in CVECs. SASP markers represent the hallmark features of cellular senescence, influencing intercellular communication through paracrine signaling within the tissue microenvironment and thereby contributing to diverse physiological and pathological outcomes (29-32). The expression levels of SASP markers, including MMP-1, MMP-3, VEGF and bFGF, were evaluated in CVECs to assess the impact of LA20k9 on DOX-induced senescence.

DOX treatment significantly increased the levels of MMP-1 and MMP-3 and significantly reduced VEGF and bFGF content (P<0.05; Fig. 3). However, MMP-1 and MMP-3 levels significantly decreased after LA20k9 treatment, while VEGF and bFGF content significantly increased (P<0.001). Specifically, MMP-1 levels increased from 53.4 ng/ml (NC) to 80.5 ng/ml (DOX) and decreased to 52.8 ng/ml with DOX + LA20k9 treatment. MMP-3 levels increased from 21.0 ng/ml (NC) to 26.6 ng/ml (DOX) and decreased to 24.8 ng/ml with DOX + LA20k9 treatment. VEGF levels decreased from 132.9 pg/ml (NC) to 102.6 pg/ml (DOX) and increased to 129.6 pg/ml with DOX + LA20k9 treatment. bFGF levels decreased from 23.7 pg/ml (NC) to 20.8 pg/ml (DOX) and increased to 31.4 pg/ml with DOX + LA20k9 treatment. These results indicated that LA20k9 protected the structure and function of vascular ECs under senescence conditions by inhibiting the expression of MMP-1 and MMP-3 and upregulating the expression of VEGF and bFGF in CVECs, thereby preventing DOX-induced ECM degradation and the renewal of senescent cells.

NAD⁺ is a central coenzyme in cells that participates in energy metabolism, redox reactions and signal transduction. During cellular senescence, a decline in NAD⁺ levels leads to mitochondrial dysfunction, reduced DNA repair capacity and increased inflammation. Supplementation with NAD⁺ precursors, such as nicotinamide mononucleotide, has been shown to delay the aging process (33). Meanwhile, oxidative stress and inflammatory responses are recognized as the most significant exogenous factors triggering the senescence response (34).

In CVECs, after DOX treatment, the levels of inflammatory factors IL-1β, TNF-α, IL-6, IL-8, GM-CSF and MCP-1, as well as the oxidative stress-associated index MDA, significantly increased (P<0.05), while the NAD⁺ content, SOD activity and GSH concentration significantly decreased (P<0.05; Figs. 4 and 5). After co-treatment with LA20k9, the levels of inflammatory factors and MDA decreased significantly (P<0.05), while the levels of NAD⁺, SOD and GSH increased significantly (P<0.05). Specifically, IL-1β levels increased from 50.4 ng/ml (NC) to 174.5 ng/ml (DOX) and decreased to 86.8 ng/ml with DOX + LA20k9 treatment. TNF-α levels increased from 17.5 ng/ml (NC) to 38.3 ng/ml (DOX) and decreased to 22.9 ng/ml with DOX + LA20k9 treatment. IL-6 levels increased from 86.0 ng/ml (NC) to 116.2 ng/ml (DOX) and decreased to 92.9 ng/ml with DOX + LA20k9 treatment. IL-8 levels increased from 45.5 ng/ml (NC) to 55.1 ng/ml (DOX) and decreased to 45.6 ng/ml with DOX + LA20k9 treatment. GM-CSF levels increased from 174.0 ng/ml (NC) to 254.5 ng/ml (DOX) and decreased to 174.8 ng/ml with DOX + LA20k9 treatment. MCP-1 levels increased from 84.4 ng/ml (NC) to 95.7 ng/ml (DOX) and decreased to 83.6 ng/ml with DOX + LA20k9 treatment. MDA content increased from 0.151 µmol/mg prot (NC) to 0.162 µmol/mg prot (DOX) and decreased to 0.152 µmol/mg prot with DOX + LA20k9 treatment. NAD⁺ content decreased from 0.978 µM (NC) to 0.426 µM (DOX) and increased to 0.719 µM with DOX + LA20k9 treatment. SOD activity decreased from 27.2 U/mg prot (NC) to 12.8 U/mg prot (DOX) and increased to 32.1 U/mg prot with DOX + LA20k9 treatment. GSH content decreased from 8.1 µM (NC) to 5.3 µM (DOX) and increased to 7.4 µM with DOX + LA20k9 treatment. These findings indicated that LA20k9 not only increased cell survival rates but also restored oxidative balance and reduced inflammatory responses.

Following transcriptome sequencing, 12 cDNA libraries were constructed, yielding a total of 35.68 Gb of raw data. The proportion of Q30 bases exceeded 95.25% across all samples, indicating high base-calling accuracy. Clean reads from each sample were aligned to the reference genome, with mapping efficiencies ranging from 85.03-92.94%. The results demonstrated that the sequencing quality for all samples was high and suitable for downstream analysis.

The mechanism of LA20k9 effect on aging was investigated by conducting a statistical analysis of the DEGs between the control group and the DOX-induced aging group, as well as between the DOX-induced aging group and the LA20k9 intervention group. Reliability was evaluated using Pearson's correlation coefficient (r). The results showed that the sample r² within each treatment group exceeded 0.80, indicating a strong correlation (Fig. 6A). Venn diagram analysis was used to present the number of DEGs, visually reflecting the overlapping associations among different DEG sets and the distribution of genes within each set. This provided clear and visual data support for subsequent research. Venn analysis showed that 454 genes were co-expressed in the NC group and the LA20k9 group (Fig. 6B). The expression profiles of the NC group and the LA20k9 group were more similar (Fig. 6C).

DEGs were screened based on changes in gene expression levels between the two sample groups and were further classified into upregulated and downregulated genes according to their expression trends. The statistical summary of DEGs between NC and DOX (Fig. 6D and E) showed that a total of 2,519 genes were upregulated including TOB1 (an anti-proliferative protein that negatively regulates cell cycle progression), UBR4, TM7SF2, TUBB4B, PIDD (a p53-induced death domain protein mediating apoptosis and cell cycle arrest), FOSL1, ESYT3, FAAH, CD70 and CHRD. Conversely, 3,888 genes were downregulated, including WDR75, CFI (a trypsin-like serine protease involved in complement system regulation), HSPA13, MSH2, NOP58, CCDC112, SNX2, ZNF25, ETAA1 (an ATR kinase activator mediating replication stress response and genome stability maintenance) and NUDT12. The functional annotations of the DEGs between NC and DOX are described in Table SII.

The comparison of DOX and LA20k9 DEGs identified 4,120 upregulated genes, including EIF3E, CFI, SULT1C4, IMPACT, PPP4R2 (a regulatory subunit of protein phosphatase 4 mediating NF-κB signaling and DNA repair), SERINC1, SLC38A2, YME1L1, SCFD1 and ZC3H15 (a zinc-finger protein involved in mRNA processing and cellular homeostasis). By contrast, 2,915 genes were downregulated, including MKI67, INPP5J (an inositol 5-phosphatase regulating phosphoinositide signaling), PLA2G3 (a secretory phospholipase A2 associated with lipid metabolism and inflammation), CYR61 (a cellular communication network factor involved in cell adhesion, migration and stress responses), UBR4 (an E3 ligase associated with protein ubiquitination and stress signaling), TM7SF2, MVD, DHCR7, FADS2 and FASN. The functional annotations of the DEGs between DOX and LA20k9 are described in Table SIII. These results indicate that gene expression changed significantly under different treatment conditions and the data quality was reliable, making it suitable for subsequent in-depth analysis.

GO and KEGG pathway enrichment analyses of DEGs in the NC vs. the DOX group and the DOX vs. the LA20k9 group were performed to explore the effect of LA20k9 treatment on the senescence response of CVECs.

The GO annotations of DEGs included three functional categories: Biological processes, cellular composition and molecular function (Fig. 6F and G). The top terms in the DOX group were ‘chromosome’, ‘protein-DNA complex’, ‘nucleosome’ and ‘chromatin’. DOX exhibited an upregulatory effect on these biological functions. DOX induces DNA damage-dependent cellular senescence by intercalating into double-stranded DNA, triggering DNA damage and oxidative stress (35,36). During this process, transcriptomic analysis revealed significant associations with cellular composition elements, including chromosomes, chromatin, nucleosomes and DNA-protein complexes. These changes are closely associated with chromatin remodeling and structural reinforcement that occur in senescent cells to maintain genomic stability. The most abundant terms in the LA20k9 group were ‘peptidyl-prolyl cis-trans isomerase activity’, ‘cis-trans isomerase activity’, ‘protein peptidyl-prolyl isomerization’ and ‘peptidyl-proline modification’. Transcriptome analysis demonstrated that LA20k9 exhibited a downregulatory effect on these biological functions. This suggested that LA20k9 may exert the function associated with peptidyl-prolyl cis-trans isomerase, reduce the levels of protein peptidyl-prolyl isomerization and modification, thereby inhabiting the excessive activation of the p53-centered senescence pathway, which may prevent the sustained amplification of DNA damage downstream signaling. Ultimately, this effects may help alleviate cellular senescence and restore the normal cellular state. Therefore, the downregulation of these functions is highly consistent with the anti-senescence effect of LA20k9 against DOX-induced senescence, suggesting that inhibiting peptidyl-prolyl cis-trans isomerase-associated activities may represent an important molecular mechanism underlying the protective effect of LA20k9.

Further KEGG pathway enrichment analysis was performed to systematically characterize the functional distribution of DEGs at the signaling pathway level. The top 20 most significantly enriched pathways are displayed in Fig. 6H and I. DOX treatment was associated with the enrichment of genes involved in the ‘p53 signaling pathway’, a core regulatory cascade governing the DNA damage response, cell cycle arrest, apoptosis and cellular senescence. Mechanistically, DOX upregulated a series of pivotal genes in this pathway, including ATR, SESN2, GADD45G, PIDD, CCNG1/2, TP73, ZNF385A and GADD45B. Meanwhile, DOX downregulated CCNE1 and CCNE2. Collectively, these transcriptomic changes suggest that DOX may trigger the robust activation of the p53-dependent DNA damage surveillance network, which, in turn, promotes growth arrest, DNA repair responses, apoptosis and stress-associated signaling, ultimately leading to permanent cell cycle exit and establishing cellular senescence in CVECs.

Further mechanistic insight was obtained by examining the regulatory effects of LA20k9 on key signaling and functional pathways. LA20k9 was associated with the enrichment of genes involved in homologous recombination and cell cycle regulation, while concurrently suppressing pathways associated with chemical carcinogenesis and reactive oxygen species (ROS)-driven genotoxicity. At the molecular level, these protective effects were associated with the upregulation of a core set of genes key in DNA double-strand break repair, DNA replication and cell cycle progression, including RPA1, UIMC1, BRIP1, MRE11A, POLD3, MCM2, MCM3, RAD21, ORC4, ORC5 and CCNE2. In addition, LA20k9 enhanced the expression of genes involved in anti-inflammatory and cytoprotective signaling, such as TANK, ATG5 and CHUK. Concomitantly, LA20k9 downregulated genes associated with mitochondrial dysfunction, oxidative stress and ROS-mediated cellular damage, including UQCRC1, CYP1A1 and SLC25A5. Collectively, these findings indicate that LA20k9 exerts its senescence-attenuating effects by restoring homologous recombination-mediated DNA repair, sustaining normal cell cycle progression and alleviating ROS-associated oxidative injury. The present findings suggest that LA20k9 could effectively counteract the deleterious senescence program triggered by DOX, possibly through reinforcing genomic stability, reducing oxidative stress and normalizing cell cycle dynamics, thus potentially protecting cells against DNA damage-induced growth arrest and functional decline.

LA20k9 anti-aging mechanism was analyzed using non-targeted metabolomics technology. Based on the aforementioned data, systematic pathway annotations were made for all identified metabolites and differential analysis and KEGG analysis were conducted for all differentially expressed metabolites. Metabolites were classified according to their chemical structure, among which, lipids and lipid-like molecules (29.22%) accounted for a relatively high proportion (Fig. 7A). Venn diagrams visually present the overlap and unique metabolites between different groups, and illustrate the relationships between multiple groups of differentially expressed metabolites (Fig. 7B). Hierarchical cluster analysis showed that the metabolic patterns of the NC and LA20k9 groups were similar (Fig. 7C).

Relative to the NC group, the levels of terezine A, parinol, metomidate, methylaphyllate, pyro-L-glutaminyl-L-glutamine, 9(11)-dehydroaxinystero, pentylone and arginyltryptophan were significantly upregulated in the DOX group (Tables I and II). The majority of these metabolites are closely associated with oxidative stress, inflammatory activation and endothelial dysfunction, which are key pathogenic characteristics of vascular ECs senescence. By contrast, monolaurin, (6E,10E)-2,6,10-trimethyldodeca-2,6,10-triene, clitocybulol B, 3-oxotetradecanoic acid, prosopinine and (E,E)-12-hydroxy-6,10-dimethyl-6,10-dodecadien-2-one were significantly upregulated in the LA20k9 group compared with the DOX group. These metabolites are primarily involved in anti-inflammatory responses, maintaining membrane stability, improving lipid metabolism and enhancing endothelial protection, thereby contributing to the alleviation of vascular endothelial senescence (37-40).

KEGG pathway analysis indicated that ‘global and overview maps’ and ‘lipid metabolism’ contained the highest number of differentially expressed metabolites in both the DOX and LA20k9 groups, suggesting that metabolic reprogramming and lipid homeostasis are key in regulating vascular ECs senescence (Fig. 7D and E). Enrichment analysis revealed that pathways including ‘caffeine metabolism’, ‘thyroid hormone synthesis’, ‘ether lipid metabolism’ and ‘asthma’ were significantly enriched in the DOX group compared with the NC group, with the largest number of differential metabolites involved in ‘glycerophospholipid metabolism’, ‘choline metabolism in cancer’, ‘efferocytosis’ and ‘steroid hormone biosynthesis’ (Fig. 7F and G). These pathways are closely associated with oxidative stress injury, mitochondrial dysfunction, inflammatory responses and endothelial barrier damage, all of which promote the occurrence and progression of vascular endothelial senescence induced by DOX (41-44). Conversely, compared with the DOX group, the LA20k9 group showed significant enrichment in the ‘cAMP signaling pathway’, ‘amino sugar and nucleotide sugar metabolism’, ‘parathyroid hormone synthesis, secretion and action’ and ‘retinol metabolism’ pathways. The most highly represented pathways included ‘ABC transporters’, ‘arginine and proline metabolism’, ‘biosynthesis of unsaturated fatty acids’, ‘β-alanine metabolism’ and ‘vitamin digestion and absorption’. The cAMP pathway protects endothelial function by mitigating inflammation, oxidative stress and SASP. Retinol metabolism sustains endothelial integrity and delays senescence. Arginine/proline metabolism optimizes vascular homeostasis, while unsaturated fatty acid biosynthesis preserves membrane and mitochondrial function. ABC transporters alleviate intracellular metabolic damage (45-49). This result therefore suggests that LA20k9 improves vascular endothelial function and resists cellular senescence mainly by regulating metabolic homeostasis, activating the cAMP signaling pathway to inhibit inflammation and oxidative stress, enhancing retinol metabolism and endothelial repair capacity and maintaining lipid balance and mitochondrial function, thereby exerting protective effects against DOX-induced vascular ECs senescence.