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Quercetin ameliorates ox-LDL-induced cellular senescence of aortic endothelial cells and macrophages by p16/p21, p53/SERPINE1, and AMPK/mTOR pathways

European Journal of Medical Research volume 30, Article number: 359 (2025) Cite this article

Abstract

Background

Atherosclerosis (AS), a chronic inflammatory disease of the arterial wall, remains a dominant cause of death and disability globally. Quercetin has been evidenced to be effective against AS, but the exact mechanisms are still largely unclear.

Methods

Oxidized low-density lipoprotein (ox-LDL)-induced human aortic endothelial cells (HAECs) and mouse RAW264.7 macrophages were established, with quercetin treatment or p16, p21 or SERPINE1 siRNA transfection. Cellular senescence was assessed by SA-β-gal staining and detection of cellular senescence markers. Cell cycle, apoptosis and intracellular ROS were detected by flow cytometry, with cell proliferation by CCK-8. Lipid accumulation was assessed utilizing oil red O staining. Through transmission electron microscope, autophagosomes and mitochondria were investigated, with detection of autophagy markers. Finally, AS models of ApoE−/− mice were established through feeding high-fat diet, and the effect of quercetin on alleviating AS progression was investigated.

Results

Quercetin protected HAECs from ox-LDL-elicited senescent phenotype, growth arrest and apoptosis and promoted cell viability in a concentration-dependent fashion. Furthermore, quercetin alleviated ox-LDL-elicited cellular senescence, ROS and lipid accumulation in macrophages. In ox-LDL-induced HAECs or/and macrophages, quercetin down-regulated the expression of p16, p21, p53 and SERPINE1, elevated p-AMPK/AMPK levels and decreased p-mTOR/mTOR levels, and these effects of quercetin were ameliorated by SERPINE1 knockdown. In AS mouse models, quercetin treatment alleviated AS progression.

Conclusion

Our findings proposed a novel anti-atherosclerotic mechanism of quercetin by mitigating ox-LDL-elicited senescent phenotype of aortic endothelial cells and macrophages by regulating p16/p21, p53/SERPINE1, and AMPK/mTOR pathways.

Introduction

Atherosclerosis (AS), a chronic inflammatory disease of the arterial wall, remains a dominant cause of death and disability globally [1, 2]. Atherosclerotic lesions are characterized by the lifelong accumulation and conversion of lipid, inflammatory cells, smooth muscle cells, and necrotic cell debris in the inner membrane space beneath the single layer of endothelial cells lining the blood vessels [3,4,5]. As a slowly progressive disease, clinically significant AS occurs mainly in the elderly. In general, the enlargement of the lesion can decrease the blood flow in the lumen by > 50% and may lead to angina, especially during exercise or stress. Lesions become unstable and burst, especially when they contain fatty and inflammatory compositions. When occurring in the coronary arteries, it may lead to local clots that thoroughly block the blood flow, causing myocardial infarction [6, 7]. Or, the clots may escape from the heart to the brain, causing a stroke [8]. With advances in revascularization and prevention approaches, clinical management of AS and its complications has greatly improved [9,10,11]. Nevertheless, the increase in the prevalence of cardiovascular diseases and the incidence of acute events (e.g., myocardial infarction) and the high mortality over the past 30 years require the identification of new treatment strategies to decrease the risk of cardiovascular events [12, 13].

Aging is an independent risk factor for AS, and cellular senescence has been implicated in AS [14,15,16]. Senescent cells are characterized by growth arrest, elevated β-galactosidase activity, and upregulated p16, p21 and p53, and more [17, 18]. In the long run, cellular senescence is harmful, especially because of the secretion of soluble factors [18, 19]. The accumulation of cellular senescence in aged arteries is notably associated with endothelial dysfunction, marking the beginning of AS [20, 21]. Endothelial dysfunction induces monocytes to recruit and adhere to the arterial wall. Once attached, the monocytes differentiate into macrophages and are capable of modified LDL particle ingestion, resulting in foam cell formation and releasing pro-inflammatory factors [22]. The cascade can attract more immune cells and accelerate the progression and instability of atherosclerotic plaques. Therefore, cellular senescence plays a basic and pathogenic role in AS. The p16/p21 and p53/SERPINE1 pathways are key regulators of cellular senescence and have been implicated in the progression of AS through mechanisms involving endothelial dysfunction and macrophage lipid metabolism [17, 23,24,25,26]. Senescence-targeted treatment has been reported to preferentially kill senescent cells and eliminate senescent cells improve AS, and targeted cellular senescence provides an insight into prevention and treatment of AS [27,28,29]. Targeting the p16/p21 and p53/SERPINE1 pathways may represent a potential strategy for the treatment of AS. However, it is needed to further clarify the exact molecular mechanisms of cellular senescence in AS and to develop novel cellular senescence-targeted drugs for the treatment of AS.

Quercetin is a classical natural antioxidant with diverse biological functions (e.g., antioxidant, anti-apoptotic and anti-inflammatory), which plays a critical role in the treatment of aging-related diseases, especially AS [30]. Many studies have suggested that quercetin exerts a protective effect on AS through modulating several biological processes such as inhibition of macrophage pyroptosis [30], macrophage senescence [31] and endothelial dysfunction [32], and endothelial cellular senescence [33]. However, to date, the anti-atherosclerotic mechanisms of quercetin are still largely unclear. In the present study, we found a new anti-atherosclerotic mechanism that quercetin alleviated ox-LDL-elicited senescent phenotype of aortic endothelial cells and macrophages.

Materials and methods

Transcriptome analysis

Microarray expression profiles of human aortic endothelial cells (HAECs) administrated with 50 μg/mL oxidized low-density lipoprotein (ox-LDL) in combination with or without 3 μM quercetin were acquired from the GSE139288 dataset from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE139288) [33]. The microarray samples from ox-LDL group (n = 3) and quercetin group (n = 3) were merged and corrected through principal component analysis. Differential expression analysis between ox-LDL group and ox-LDL + quercetin group was performed with the use of limma package, and differentially expressed genes were identified according to adjusted p < 0.05 by false discovery rate (FDR) correction and fold change > 1.5 [34]. Furthermore, functional enrichment analysis was performed on differentially expressed genes using clusterProfiler package [35, 36].

Cell culture, treatment and transfection

HAECs (HCL-0015; CTCC) and RAW264.7 cells (HCL-0102; CTCC) were cultured in Minimum Essential Medium (SH30008.12; Hyclone) and RPMI-1640 medium (SH30809.01; Hyclone), respectively. The media were supplemented with 10% fetal bovine serum (FBS; SV30087.02; Hyclone) and 1% penicillin–streptomycin solution (BL505 A; Biosharp). All the cells were incubated in a 5% CO2 incubator at 37 °C.

HAECs were administrated with 50 μg/mL ox-LDL in combination with or without 0.5, 1 and 3 μM quercetin for 48 h. RAW264.7 macrophages were exposed to 100 μg/mL ox-LDL in combination with or without 25 μM quercetin, as previously described. The concentrations of ox-LDL and quercetin were selected based on prior literature [24].

For transfection, cells were cultured in a 6-well culture plate (5 × 105 cells/well) for 24 h. Before transfection, each well was replaced with 2 mL fresh culture medium. For each well, 125 μL medium solution without antibiotics and FBS was added, with addition and mixture of 100 pmol of siRNA and 4 μL Lipo8000™ transfection reagent. The prepared mixture was placed at room temperature for 6 h, with addition and mixture of 125 μL Lipo8000™ transfection agent-siRNA mixture. After 48 h of culture, cell samples were collected for subsequent testing.

Senescence-associated β-galactosidase (SA-β-gal) staining

Following the β-galactosidase staining kit (G1580; Solarbio) manufacturer’s instructions, cells were added to 300 μL β-gal fixative and fixed at room temperature for 15 min. Following removal of the fixative, cells were washed with PBS for 3 min × 3 times. The dyeing solution was prepared according to the ratio of B:C:D:E = 5:1:1:93, and cells were treated with 300 μL of dyeing solution at 37 ℃ overnight.

Flow cytometry

Annexin V-FITC/PI kit (CTCC-M009; CTCC) was adopted for assessing cell apoptosis. HAECs were digested with 0.25% trypsin (002PI; CTCC) without EDTA, with 1500 rpm centrifugation for 5 min at room temperature. HAECs were suspended with pre-cooled 1 × PBS (4 ℃) and centrifuged at 1500 rpm for 5 min, with addition of 300 µL 1 × binding buffer to suspend the cells. They were incubated with 5 µL Annexin V-FITC for 15 min and 10 µL PI for 10 min at room temperature avoiding light. For cell cycle, HAECs were centrifuged at 1500 rpm for 5 min, with removal of the supernatant. After rinsing, HAECs were treated with 700 µL pre-cooled 80% ethanol, followed by fixation and centrifugation. After rinsing, 100 µL RNase (50 µg/mL) was added and bathed at 37 ℃ for 30 min. Then, HAECs were stained by 400 µL PI (50 µg/mL) (ST512; Beyotime) at 4 ℃ for 30 min away from light. Cell apoptosis and growth cycle were tested through flow cytometry (FACSVerse; BD), and analyzed by FlowJo 7.6 software.

Cell viability analysis

Cell Counting Kit-8 (CCK-8) kit (M007; CTCC) was employed for evaluating cell viability. HAECs were cultured in a 96-well plate, with addition of 10 μL CCK-8 to each well. After culturing for 1 h at 37 ℃, the optical density 450 of each well was determined utilizing microplate reader (MK3; Thermo Fisher Scientific).

RT-qPCR

RNA was extracted by universal RNA extraction kit (M004; PH Biotechnology), and RNA concentration was measured. cDNA was then synthesized by reverse transcription synthesis kit (Aidlab). The primers were designed and synthesized by Primer Premier 5.0 software (Sangon Biotech), with GAPDH as an internal reference. The primer sequences included: human p21, 5′-CCTGTCACTGTCTTGTACC-3′ (forward), 5′-AATCTGTCATGCTGGTCTG-3′ (reverse); human p16, 5′-GGGTCGGGTAGAGGAGGTG-3′ (forward), 5′-GCTGCCCATCATCATGACCT-3v (reverse); human GAPDH, 5′-GGAGCGAGATCCCTCCAAAAT-3′ (forward), 5′-GGCTGTTGTCATACTTCTCATGG-3′ (reverse); mouse Bcl2, 5′-GAGATCGTGATGAAGTACATAC-3v (forward), 5′-CAGGCTGGAAGGAGAAGA-3′ (reverse); mouse Beclin1, 5′-GCAGAGAACCTGGAGAAG-3′ (forward), 5′-GTGGCATTGAAGACATTGG-3′ (reverse); mouse LC3, 5′-GAGCGAGTTGGTCAAGAT-3′ (forward), 5′-TCATAGATGTCAGCGATGG-3′ (reverse); mouse GAPDH, 5′-GGTGAAGGTCGGTGTGAACG-3′ (forward), 5′-CTCGCTCCTGGAAGATGGTG-3′ (reverse). Reaction solution was prepared according to RT-qPCR reaction system. After PCR amplification, RT-qPCR instrument (7500; ABI) was used for automatic analysis. With the use of 2−ΔΔCT method, relative mRNA expression was calculated.

Western blot

Samples were lysed by RIPA lysis (P0013B; Beyotime) supplemented with PMSF (BP2655; RUIBIO), with subsequent 12000 rpm centrifugation for 10 min. The supernatant was harvested for protein quantification with the use of BCA protein concentration determination kit (BL521 A; Biosharp). Protein extracts were separated via 10–12% SDS-PAGE, with subsequent transference onto PVDF membranes (IPVH00010; Millipore) and closure with 5% BSA at 37 ℃ for 2 h. The membranes were then incubated with primary antibody solution including anti-p16 (1/5000; 10883-1-AP; Proteintech), anti-p21 (1/1000; 10355-1-AP; Proteintech), anti-SERPINE1 (1/1000; 66261-1-Ig; Proteintech), anti-Bcl2 (1/2000; 68103-1-Ig; Proteintech), anti-Beclin1 (1/2000; ab207612; Abcam), anti-GAPDH (1/5000; 60004-1-Ig; Proteintech), anti-AMPK (1/1000; ab32047; Abcam), anti-p-AMPK (1/1000; ab109402; Abcam), anti-mTOR (1/10000; ab134903; Abcam), and anti-p-mTOR (1/10000; ab109268; Abcam) at 4 ℃ overnight. Afterwards, they were washed and incubated with HRP-labeled goat anti-rabbit or anti-mouse secondary antibody solution (1/5000; ZB2301 or ZB2305; ZSGB-BIO) at 37 ℃ for 1 h. After washing, the ECL luminescent solution (ECL-0011; Beijing Dingguo Changsheng Biotechnology Co., LTD) was added to the membranes, and images were taken by chemiluminescence apparatus (ChemiScope 5300 Pro; CLINX).

Reactive oxygen species (ROS) assay

ROS detection kit (S0033S; Beyotime) was utilized for detecting intracellular ROS levels. The final concentration of DCFH-DA was 10 μmol/L by diluting DCFH-DA with serum-free medium at 1:1000. RAW264.7 macrophages were digested and centrifuged at 1500 rpm for 5 min at room temperature. After suspending with pre-cooled 1 × PBS, RAW264.7 macrophages were rinsed and centrifuged at 1500 rpm for 5 min. The cells were incubated with the diluted DCFH-DA at 37 ℃ for 20 min and washed with serum-free culture solution. They were incubated with Hoechst 33342 at 37 ℃ for 10 min, with subsequent addition of complete medium. ROS levels were tested by flow cytometry.

Oil red O staining

Oil red O staining kit (JD003; CTCC) was adopted for Oil red O staining. RAW264.7 macrophages were grown in a 48-well culture plate (6 × 103 cells/well). They were fixed with 10% neutral formaldehyde for 30 min and washed with PBS for 3 times. Next, RAW264.7 macrophages were dyed with 0.5% Oil red work solution for 1 h away from light. After rinsing with isopropyl alcohol, PBS was added, and photographs were acquired.

Immunofluorescence

After rinsing with PBS, RAW264.7 macrophages were fixed with 4% paraformaldehyde (P0099; Beyotime) for 15 min. After washing with PBS for 5 min × 3 times, RAW264.7 macrophages were exposed to 0.1% Triton (P0096; Beyotime) at room temperature for 10 min. RAW264.7 macrophages were incubated with primary antibody of anti-p16 (1/200; 10883-1-AP; Proteintech), anti-p21 (1/200; 10355-1-AP; Proteintech), anti-p53 (1/200; 60283-2-Ig; Proteintech) or anti-SERPINE1 (1/200; 66261-1-Ig; Proteintech) at 4 ℃ overnight, with subsequent goat anti-rabbit or anti-mouse secondary antibody (SA00009-2 or SA00009-1; Proteintech) incubation at 37 ℃ for 1 h. Afterwards, they were incubated with Hoechst 33258 (C1017; Beyotime).

Transmission electron microscopy

RAW264.7 macrophages were immobilized with 2.5% glutaraldehyde (A17876; alfa Aesar) for 4 h. After centrifugation at 1000 rpm, fresh 2.5% glutaraldehyde was added to fix macrophages at 4 °C for 2 h. The macrophages were rinsed with 0.1 M phosphate buffer (PH = 7.4) for 15 min × 3 times. The cells were immobilized with 1% osmic acid ·0.1 M phosphate buffer (pH = 7.4) at 20 ℃ for 2 h. After rinsing with 0.1 M phosphate buffer for 15 min × 3 times, the cells were dehydrated with 50%−70%−80%−90%−95%−100% alcohol for 15 min at a time. Acetone: 812 embedding agent penetrated overnight in a 1:1 mixture, and pure 812 embedding agent penetrated overnight. 48 h after embedding, 60–80 nm ultra-thin sections were prepared. The cells were dyed by 2% uranium acetate saturated solution and lead citrate for 15 min each. The sections were dried overnight at room temperature, which were observed under transmission electron microscopy (HT7700; HITACHI).

Animal experiments

Specific pathogen-free male ApoE−/− mice (age, 6–8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were kept at room temperature of 24 ± 1 °C, relative humidity of 50–70%, and a 12 h light/dark cycle. All mice had AD libitum access to food and water. All animal experiments strictly followed the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) and the ARRIVE guidelines, and were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Harbin Medical University (KY2023-328).

After one week of adaptive feeding, ApoE−/− mice were randomly divided into four groups (n = 6 per group): normal diet (ND) group, high-fat diet (HD) group, HD supplemented with quercetin group, and HD supplemented with atorvastatin group. The mice in the HD group were fed the standard mouse basal diet supplemented with 21% fat and 0.5% cholesterol. The healthy and behavioral status of mice were monitored every 2 weeks. The mice in the HD supplemented with quercetin group were daily administrated with 12.5 mg/kg quercetin by oral gavage for 16 weeks, and those in the HD supplemented with atorvastatin group daily were administrated with 4 mg/kg atorvastatin by oral gavage for 16 weeks, as a positive control. The mice in the ND and HD groups were given an equal volume of distilled water by oral gavage daily for 16 weeks. Body weight, daily food intake and fasting blood glucose were monitored every 2 weeks.

Hematoxylin–eosin (H&E) staining

After the experiment, aorta tissues were gathered and fixed in 10% formalin, followed by paraffinization. The tissues were cut to a thickness of 5 µm. H&E staining was conducted on the aorta sections to evaluate the formation of AS plaque.

Enzyme-linked immunosorbent assay (ELISA)

Serum samples were gathered, and total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low-density lipoprotein (LDL), interleukin (IL)−1β, tumor necrosis factor-α (TNF-α), and IL-6 levels were measured following ELISA kit instructions (Nanjing Jiancheng Bioengineering Institute).

Statistical analysis

All the analyses were implemented through R language (v4.2.0) and GraphPad Prism software (9.0.1). Comparison between two groups was evaluated utilizing unpaired Student’s t test, with one-way ANOVA followed by Tukey’s multiple comparisons test for ≥ 3 groups. P < 0.05 was considered statistically significant.

Results

Quercetin protects aortic endothelial cells from ox-LDL-induced cellular senescence

To present a more in-depth exploration of the anti-atherosclerotic mechanism of quercetin, transcriptome analysis of HAECs with treatment of 50 μg/mL ox-LDL in combination with or without 3 μM quercetin was conducted in our study. There was a distinct transcriptomic difference between quercetin-treated and -untreated ox-LDL-induced HAECs (Supplementary Fig. 1 A, B). In ox-LDL-induced HAECs, 1,365 and 1,331 genes presented significant up-regulation and down-regulation following quercetin treatment, respectively (Fig. 1A, B). Supplementary Table 1 lists the key differentially expressed genes. Next, signaling pathways involved in the differentially expressed genes were studied. All enriched signaling pathways are shown in Supplementary Table 2. We found that several AS-related signaling pathways were significantly enriched by the differentially expressed genes, such as complement and coagulation cascades, autophagy, IL-17 signaling pathway, mTOR signaling pathway, and p53 signaling pathway. Here, we were concerned the p53 and mTOR signaling pathways (Fig. 1C–E), which are closely linked with several pivotal mechanisms, e.g., cell cycle progression, apoptosis, autophagy and cellular senescence. In addition, a few cellular senescence-related genes (IGFBP3, NFATC2, SERPINE1, CXCL8, E2 F3 and TRPM7) occurred expression alterations in ox-LDL-induced HAECs after quercetin administration (Fig. 1F, G), indicating that quercetin might be connected to regulation of cellular senescence. Autophagy was also enriched by the differentially expressed genes (Fig. 1C), and there were protein–protein interactions between p53 and autophagy signaling pathways (Fig. 1H).

Fig. 1
figure 1

Quercetin protects aortic endothelial cells from ox-LDL-induced cellular senescence. A Volcano diagram depicting the differential expression of genes in ox-LDL-induced HAECs with quercetin treatment. Red, upregulated genes; blue, down-regulated genes; grey, no significantly altered genes. Genes with adjusted p < 0.05 by false discovery rate (FDR) correction and fold change > 1.5 were regarded as differential expression. B Heatmap exhibiting the expression of the top 15 up- and down-regulated genes in ox-LDL-induced HAECs with or without quercetin administration. The color from blue to red indicates low to high gene expression. C Enrichment analysis of the differentially expressed genes on KEGG pathways. The top 15 significant pathways were exhibited. D, E Visualization of p53 and mTOR signaling pathways. F Heatmap displaying the differential expression of cellular senescence-related genes in ox-LDL-induced HAECs administrated with quercetin. G Comparison of SERPINE1 expression in ox-LDL-induced HAECs administrated with or without quercetin. H Protein–protein interactions of p53 and autophagy signaling pathways. I Representative SA-β-gal staining of HAECs treated with 50 μg/mL ox-LDL in combination with 0, 0.5, 1 and 3 μM quercetin (Que). Scale bar, 50 μm. J Quantification of SA-β-gal-positive HAECs. ****p < 0.0001

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To confirm that quercetin was involved in the regulation of cellular senescence, SA-β-gal staining was conducted in HAECs administrated with 50 μg/mL ox-LDL in combination with 0, 0.5, 1 and 3 μM quercetin. The data demonstrated that ox-LDL prominently elevated the percentage of SA-β-gal-positive HAECs (Fig. 1I, J), suggesting the senescent phenotype caused by ox-LDL. Quercetin significantly attenuated the percentage of SA-β-gal-positive HAECs with ox-LDL induction in a concentration-dependent fashion (Fig. 1I, J). This revealed that quercetin may protect aortic endothelial cells from ox-LDL-elicited senescent phenotype.

Quercetin protects aortic endothelial cells from ox-LDL-induced cell cycle arrest and apoptosis

Flow cytometry demonstrated that ox-LDL notably elevated the percentage of G0/G1 phase and attenuated the percentage of S phase in HAECs (Fig. 2A–C). 3 μM quercetin significantly attenuated the percentage of G0/G1 phase in HAECs with ox-LDL induction, while 0.5 and 1 μM quercetin had no significant effect on it. Moreover, quercetin significantly elevated the percentage of S phase in ox-LDL-induced HAECs in a concentration-dependent fashion. The data demonstrated that quercetin may ameliorate ox-LDL-induced cell cycle arrest of HAECs. According to the results from flow cytometry, apoptosis of HAECs was significantly increased after exposure to ox-LDL (Fig. 2D, E). Conversely, the ox-LDL-induced apoptosis was notably ameliorated by quercetin in a concentration-dependent fashion. As shown in CCK-8 results, ox-LDL significantly lowered cell viability of HAECs, which was weakened by quercetin in a concentration-dependent fashion (Fig. 2F, G). Hence, our data demonstrated that quercetin may protect HAECs from ox-LDL-induced cell cycle arrest and apoptosis and improve cell viability.

Fig. 2
figure 2

Quercetin protects aortic endothelial cells from ox-LDL-induced cell cycle arrest and apoptosis. A Representative flow cytometry for cell cycle in HAECs with treatment of 50 μg/mL ox-LDL in combination with 0, 0.5, 1 and 3 μM quercetin (Que). B, C Analysis of G0/G1 phase and S phase percentages in the above HAECs. D Representative flow cytometry for apoptosis in HAECs with 50 μg/mL ox-LDL treatment in combination with 0, 0.5, 1 and 3 μM Que. E Quantification of apoptotic HAECs. F Representative white light photographs of HAECs administrated with 50 μg/mL ox-LDL combined with 0, 0.5, 1 and 3 μM Que. Scale bar, 100 μm. G CCK-8 for cell viability of the above HAECs. ns, not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001

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Quercetin ameliorates aortic endothelial cell senescence caused by ox-LDL possibly through down-regulating p16 and p21

In addition to SA-β-gal, p16 and p21 have been recognized as biomarkers of cellular senescence. p16 and p21 expression exhibited significant increase in HAECs in the context of ox-LDL induction (Fig. 3A–E). Quercetin prominently down-regulated the expression of p16 and p21 in ox-LDL-induced HAECs in a concentration-dependent fashion. To knock out the expression of p16 and p21, specific siRNAs against p16 and p21 were transfected into HAECs, respectively. Similar to quercetin, knockdown of p16 and p21 significantly weakened the ox-LDL-induced p16 and p21 up-regulation in HAECs (Fig. 3F–J). In addition, similar to quercetin, the ox-LDL-elicited increase in the percentage of SA-β-gal-positive HAECs was notably weakened by knockdown of p16 or p21 (Fig. 3K, J). Above data suggested that quercetin may alleviate ox-LDL-elicited HAECs senescence possibly through down-regulation of p16 and p21.

Fig. 3
figure 3

Quercetin ameliorates ox-LDL-induced cellular senescence of aortic endothelial cells possibly through down-regulating p16 and p21. A, B RT-qPCR analysis of p16 and p21 mRNA expression in HAECs treated with 50 μg/mL ox-LDL combined with 0, 0.5, 1 and 3 μM quercetin (Que). C Representative western blots of p16 and p21 in HAECs in the context of 50 μg/mL ox-LDL treatment combined with 0, 0.5, 1 and 3 μM Que. D, E Quantification of p16 and p21 protein expression in the above HAECs. F, G RT-qPCR analysis of p16 and p21 mRNA expression in ox-LDL-induced HAECs with treatment of Que or transfection of p16 (si-p16) or p21 (si-p21) siRNA or negative control (si-NC). H Representative western blots of p16 and p21 in HAECs in the context of ox-LDL treatment combined with Que treatment or transfection of si-p16, si-p21 or si-NC. I, J Quantification of p16 and p21 protein expression in the above HAECs. K Representative SA-β-gal staining of HAECs with treatment of ox-LDL in combination with Que treatment or transfection of si-p16, si-p21 or si-NC. Scale bar, 50 μm. L Quantification of SA-β-gal-positive HAECs. ***p < 0.001; ****p < 0.0001

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Quercetin ameliorates ox-LDL-elicited growth arrest and apoptosis in aortic endothelial cells possibly through down-regulating p16 and p21

Knockdown of p16 and p21 significantly attenuated the ox-LDL-induced increase in the percentage of G0/G1 phase and elevated the ox-LDL-induced reduction in the percentage of S phase in HAECs, and the effects were similar to quercetin administration (Fig. 4A–C). Additionally, knockdown of p16 and p21 reduced the ox-LDL-elicited apoptosis of HAECs, which was similar to quercetin administration (Fig. 4D, E). It was also found that knockdown of p16 and p21 significantly improved the ox-LDL-induced inhibition in proliferation of HAECs, and the effects were similar to quercetin administration (Fig. 4F, G). These findings indicated that quercetin may alleviate ox-LDL-elicited cell cycle arrest and apoptosis in HAECs possibly through down-regulating p16 and p21.

Fig. 4
figure 4

Quercetin ameliorates ox-LDL-induced cell cycle arrest and apoptosis of aortic endothelial cells possibly through down-regulating p16 and p21. A Representative flow cytometry for cell cycle of ox-LDL-induced HAECs with treatment of quercetin (Que) or transfection of p16 (si-p16) or p21 (si-p21) siRNA or negative control (si-NC). B, C Quantification of G0/G1 phase and S phase percentages in the above HAECs. D Representative flow cytometry for apoptosis of ox-LDL-induced HAECs treated with Que or transfected with si-p16, si-p21 or si-NC. E Quantification of apoptotic HAECs. F Representative white light photographs of ox-LDL-induced HAECs with Que administration or si-p16, si-p21 or si-NC transfection. Scale bar, 100 μm. G CCK-8 for cell viability of the above HAECs. **p < 0.01; ****p < 0.0001

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Quercetin alleviates cellular senescence, ROS and lipid accumulation and induces autophagy in ox-LDL-treated macrophages

Macrophages are a main source of foam cells, as a hallmark of atherosclerotic plaques. Studies have demonstrated that ox-LDL induces cellular senescence of macrophages [24, 31]. Consistently, this study also observed the senescent phenotype of RAW264.7 macrophages caused by ox-LDL (Fig. 5A, B). Quercetin administration significantly alleviated the macrophage senescence in the context of ox-LDL induction. In addition, ROS production elicited by ox-LDL was weakened by quercetin in RAW264.7 macrophages (Fig. 5C, D). Through Oil red O staining, lipid accumulation was detected. We observed that there was remarkable lipid accumulation in ox-LDL-induced RAW264.7 macrophages (Fig. 5E, F). Conversely, quercetin significantly relieved the ox-LDL-induced lipid accumulation. Altogether, quercetin may mitigate cellular senescence, ROS and lipid accumulation in macrophages in the context of ox-LDL induction. In addition to attenuating SA-β-gal activity, the study observed that quercetin lowered the up-regulation of cellular senescence-related p16, p21, p53 and SERPINE1 caused by ox-LDL in RAW264.7 macrophages (Fig. 5G–M), further revealing the effect of quercetin on inhibiting macrophage senescence driven by ox-LDL. Thus, quercetin alleviates the ox-LDL-induced senescent phenotype of macrophages, which may be linked with the down-regulation of p16, p21, p53 and SERPINE1. In addition, we found that quercetin elevated p-AMPK/AMPK levels and decreased p-mTOR/mTOR levels in ox-LDL-induced RAW264.7 macrophages (Fig. 5N–P), indicating that quercetin may promote autophagy of ox-LDL-treated macrophages through regulating the AMPK/mTOR signaling pathway.

Fig. 5
figure 5

Quercetin alleviates cellular senescence, ROS and lipid accumulation and induces autophagy in ox-LDL-treated macrophages. A Representative SA-β-gal staining of mouse RAW264.7 macrophages treated with ox-LDL with or without quercetin (Que). Scale bar, 50 μm. B Quantification of SA-β-gal-positive mouse RAW264.7 macrophages. C Representative flow cytometry for ROS level in mouse RAW264.7 macrophages with treatment of ox-LDL with or without quercetin. D Quantification of ROS level in the above mouse RAW264.7 macrophages. E Representative Oil red O staining of mouse RAW264.7 macrophages in the context of ox-LDL treatment with or without quercetin treatment. Scale bar, 50 μm. F Quantification of lipid droplet area ratio in the above macrophages. G Representative immunofluorescence staining of p16, p21, p53 and SERPINE1 in RAW264.7 macrophages in the context of ox-LDL treatment with or without quercetin treatment. Scale bar, 20 μm. H–K Quantification of p16, p21, p53 and SERPINE1 expression in RAW264.7 macrophages in the context of ox-LDL with or without quercetin. L Representative western blots of SERPINE1 in RAW264.7 macrophages in the context of ox-LDL with or without quercetin. M Quantification of SERPINE1 expression in the above macrophages. N Representative western blots of AMPK, p-AMPK, mTOR, and p-mTOR expression in RAW264.7 macrophages in the context of ox-LDL with or without quercetin. O, P Quantification of p-AMPK/AMPK and p-mTOR/mTOR levels in the above macrophages. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

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Knockdown of SERPINE1 improves the effects of quercetin on alleviating cellular senescence, ROS and lipid accumulation in ox-LDL-induced macrophages

Further analysis showed that knockdown of SERPINE1 combined with quercetin had a stronger effect on reducing SA-β-gal activity (Fig. 6A, B), ROS accumulation (Fig. 6C, D), and lipid accumulation (Fig. 6E) in ox-LDL-induced macrophages than either of them alone. In addition, knockdown of SERPINE1 in combination with quercetin more effectively attenuated the expression of p16, p21, p53 and SERPINE1 in ox-LDL-induced macrophages than either of them alone (Fig. 6F–J). Therefore, knockdown of SERPINE1 may enhance the effects of quercetin on alleviating cellular senescence, ROS and lipid accumulation in ox-LDL-induced macrophages, demonstrating that SERPINE1 was a key target of quercetin in inhibiting ox-LDL-induced macrophage senescence.

Fig. 6
figure 6

Knockdown of SERPINE1 improves the effects of quercetin on alleviating ox-LDL-elicited cellular senescence, ROS and lipid accumulation in macrophages. A Representative SA-β-gal staining of ox-LDL-induced RAW264.7 macrophages with transfection of SERPINE1 siRNA (si-SERPINE1) or negative control (si-NC) or/and treatment of quercetin (Que). Scale bar, 50 μm. B Quantification of SA-β-gal-positive mouse RAW264.7 macrophages. C Representative flow cytometry for ROS level in ox-LDL-induced RAW264.7 macrophages with transfection of si-SERPINE1 or si-NC or/and treatment of quercetin. D Quantification of ROS level in the above mouse RAW264.7 macrophages. E Representative Oil red O staining of ox-LDL-induced RAW264.7 macrophages in the context of si-SERPINE1 or si-NC transfection or/and quercetin treatment. Scale bar, 50 μm. F Representative immunofluorescence staining of p16, p21, p53 and SERPINE1 in ox-LDL-induced RAW264.7 macrophages in the context of si-SERPINE1 or si-NC transfection or/and quercetin treatment. Scale bar, 20 μm. G–J Quantification of p16, p21, p53 and SERPINE1 expression. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, p > 0.05

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Quercetin protects ox-LDL-induced macrophages against aging-associated detrimental effects through regulating autophagy

The role of autophagy in cellular senescence remains controversial. The transmission electron microscopy showed that quercetin notably increased the autophagosomes in RAW264.7 macrophages with ox-LDL exposure (Fig. 7A), indicating the enhanced autophagy. Moreover, it was observed that RAW264.7 macrophages occurred swollen mitochondria and blurred mitochondrial ridges in the context of ox-LDL, which was mitigated by quercetin. At the mRNA level, quercetin upregulated Bcl2, LC3 and Beclin1 in macrophages in the context of ox-LDL induction (Fig. 7B–D). In addition, the increase in Bcl2, LC3II/I and Beclin1 expression induced by quercetin was also observed at the protein level (Fig. 7E–H). SERPINE1 is a key subset of senescence-associated secretory phenotype (SASP) components. We next investigated whether SERPINE1 was involved in autophagy regulation of quercetin in ox-LDL-induced macrophages. We found that quercetin treatment decreased the expression of Bcl2, LC3II/I and Beclin1 in ox-LDL-induced macrophages with knockdown of SERPINE1 (Fig. 7I–O). Meanwhile, knockdown of SERPINE1 down-regulated the expression of Bcl2, LC3II/I and Beclin1 in ox-LDL-induced macrophages with quercetin treatment. It was also observed that both quercetin treatment and knockdown of SERPINE1 elevated p-AMPK/AMPK levels and decreased p-mTOR/mTOR levels in ox-LDL-induced RAW264.7 macrophages (Fig. 7P–R). This indicated that quercetin may protect ox-LDL-induced macrophages against aging-associated detrimental effects through enhancing autophagy, and when the senescence phenotypic changes (such as SASP) were suppressed, the effects of quercetin on enhancing autophagy of ox-LDL-induced macrophages were alleviated, partly associated with SERPINE1 expression.

Fig. 7
figure 7

Quercetin enhances autophagy of ox-LDL-induced macrophages. A Representative transmission electron microscope photographs of mouse RAW264.7 macrophages in the context of ox-LDL treatment with or without quercetin (Que) treatment. Scale bar, 500 nm. The arrows point to the autophagosomes. B–D RT-qPCR analysis of Bcl2, LC3 and Beclin1 mRNA expression in ox-LDL-induced RAW264.7 macrophages with or without quercetin treatment. E Representative western blots of Bcl2, LC3II/I and Beclin1 in ox-LDL-induced RAW264.7 macrophages with or without quercetin administration. F–H Quantification of Bcl2, LC3II/I and Beclin1 expression in the above macrophages. I–K RT-qPCR analysis of Bcl2, LC3 and Beclin1 mRNA expression in ox-LDL-induced RAW264.7 macrophages with transfection of si-SERPINE1 or si-NC or/and treatment of quercetin. L Representative western blots of Bcl2, LC3II/I and Beclin1 in ox-LDL-induced RAW264.7 macrophages in the context of si-SERPINE1 or si-NC transfection or/and quercetin treatment. M–O Quantification of Bcl2, LC3II/I and Beclin1 expression in the above macrophages. P Representative western blots of AMPK, p-AMPK, mTOR, and p-mTOR in ox-LDL-induced RAW264.7 macrophages in the context of si-SERPINE1 or si-NC transfection or/and quercetin treatment. Q, R Quantification of p-AMPK/AMPK and p-mTOR/mTOR levels in the above macrophages. **p < 0.01; ***p < 0.001; ****p < 0.0001

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Quercetin treatment alleviates AS progression in ApoE−/− mice

Next, we established an AS model using ApoE−/− mice through high-fat diet (HD). ApoE−/− mice that were fed with normal diet (ND) were used as a control. To treat AS progression, ApoE−/− mice were fed with HD supplemented with quercetin or atorvastatin. After the experiment, H&E staining showed that the formation of AS plaque was significantly induced by HD (Fig. 8A). Quercetin treatment effectively suppressed the formation of AS plaque, and its effect was similar to that of atorvastatin. Furthermore, both quercetin and atorvastatin treatment significantly reduced HD-induced increase in body weight and fasting blood glucose, and reduced daily food intake (Fig. 8B–D). In addition, increased serum levels of TC, TG, and LDL and reduced serum levels of HDL were ameliorated by both quercetin and atorvastatin treatment (Fig. 8E–H). This indicated that both quercetin and atorvastatin treatment ameliorated serum lipid levels in ApoE−/− mice with HD. Increased serum levels of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) levels were also attenuated by both quercetin and atorvastatin treatment (Fig. 8I–K), indicating the amelioration in inflammatory response. Collectively, above data suggested that quercetin treatment alleviated AS progression in ApoE−/− mice.

Fig. 8
figure 8

Quercetin treatment alleviates AS progression in ApoE−/− mice. A H&E staining for detecting AS plaque formation in aorta tissues of ApoE−/− mice receiving normal diet (ND), high-fat diet (HD), HD supplemented with quercetin (HD + Que), or HD supplemented with atorvastatin (HD + Ator) (n = 6 per group). Scale bar, 50 μm. B–D Measurement of B body weight, C average daily food intake, and D fasting blood glucose every two weeks. E–H Measurement of serum E TC, F TG, G HDL, and H LDL levels after treatment for 16 weeks. I–K Measurement of serum I IL-1β, J TNF-α, and K IL-6 levels after treatment for 16 weeks. ns, p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001

Full size image

Discussion

AS is a global epidemic of chronic inflammatory disease, which involves the existence of senescent cells that undergo sustaining growth arrest, foster a pro-inflammatory microenvironment and cause the instability of atherosclerotic plaques [37]. Owing to the epidemiological features and significance of AS, much effort has been devoted to developing effective treatment choices. Quercetin has been demonstrated to be effective in the treatment of AS, but the exact mechanisms remain to be further explored. This study found that quercetin exerted an anti-atherosclerotic effect by alleviating ox-LDL-induced senescent phenotype both in HAECs and macrophages, establishing a theoretical basis for its clinical application.

The lysosomal β-galactosidase in normal cells operates in the acidic range of pH 4.0–4.5, whereas senescent cells exhibit SA-β-gal activity at pH 6.0 [38]. The capacity in detecting cellular senescence utilizing SA-β-gal activity has been well characterized [39,40,41]. Cellular senescence has the features of persistent growth arrest and up-regulation of cell cycle inhibitors p16 and p21 [17, 42]. Dietary quercetin reduces oxidant-induced endothelial dysfunction and AS progression in high-fat feeding ApoE−/− mice [43]. Our data demonstrated that quercetin may protect HAECs from ox-LDL-induced cellular senescence, cell cycle arrest and apoptosis, and promoted cell viability in a concentration-dependent fashion. Consistently, a prior study reported that quercetin prevents AS through suppressing endothelial cell senescence driven by ox-LDL [33]. Moreover, quercetin attenuated the increase in p16 and p21 expression driven by ox-LDL in HAECs, and knockdown of p16 and p21 had the similar effects to that of quercetin, indicating that quercetin might mitigate senescent phenotype of HAECs driven by ox-LDL possibly through down-regulating p16 and p21.

Macrophages determine the development of atherosclerotic lesions [5, 44, 45]. As reported by Luo et al.’s findings, quercetin inhibits senescence of plaque macrophages driven by ox-LDL and alleviates AS through suppressing p38 MAPK/p16 signaling pathway [31]. p53 is a key co-activator of SERPINE1 that encodes PAI-1 (a serine protease inhibitor) transcription both in the genomic profibrotic and cellular senescence programs [46]. A recent study reported that SERPINE1 facilitates alveolar epithelial type II cell senescence by inducing p53 and activating p53–p21–pRb pathway [47]. Our data showed that quercetin mitigated macrophage senescence driven by ox-LDL, with the down-regulation of senescence-associated biomarkers p16, p21, p53 and SERPINE1, indicating the inhibition effect of quercetin on p16/p21 and p53/SERPINE1 pathways. We also found that ox-LDL-induced ROS production was alleviated by quercetin in macrophages. Quercetin attenuates atherosclerotic plaque development in high fructose feeding mouse models through anti-inflammation and anti-apoptosis, which is linked with regulation of ROS-mediated PI3 K/AKT signaling pathway [48]. Macrophages in plaques display high heterogeneity and plasticity, which influence the evolution of the plaque microenvironment, especially resulting in excessive lipid accumulation [49]. Quercetin was evidenced to ameliorate macrophage lipid accumulation induced by ox-LDL, thus preventing AS progression. Furthermore, knockdown of SERPINE1 improved the effects of quercetin on alleviating cellular senescence, ROS and lipid accumulation in ox-LDL-induced macrophages. However, knockdown of SERPINE1 did not influence the senescent phenotype of HAECs (data not shown), indicating the cell type-specificity of SERPINE1 in regulating cellular senescence.

Autophagy is a cellular self-catabolic process that is essential for protecting cellular homeostasis from harmful conditions [50,51,52]. Autophagy highly participates in the process of cellular senescence and senescent effect. It is an important mechanism to maintain the stability of the intracellular microenvironment, which is intertwined with cellular senescence. In the tissues of ATG5i mice chronically treated with doxycycline, there were upregulated senescence markers, positive SA-β-gal staining, and increased telomere-associated γ-H2 AX foci, indicating that the loss of basal autophagy contributes to cellular senescence [53]. Oppositely, basal autophagy maintains homeostasis through suppressing cellular senescence sand maintaining stem cells [54,55,56]. LC3-II shows up-regulation in oncogene-induced senescent cells, but not in quiescent cells, suggesting that autophagosome accumulation is associated with cellular senescence rather than cell cycle arrest [57]. Additionally, senescent cells can further activate autophagy through inducing negative feedback modulation of the PI3 K-mTOR pathway and SASP production to promote cellular senescence in a paracrine or autocrine manner [58]. In the present study, we found that quercetin enhanced autophagy of ox-LDL-induced macrophages, as demonstrated by increased autophagosomes, upregulated Bcl2, LC3II/I and Beclin1, elevated p-AMPK/AMPK levels and decreased p-mTOR/mTOR levels. Quercetin may mitigate ox-LDL-induced cellular senescence of macrophages through improving autophagy. However, the effects of quercetin on enhancing autophagy of ox-LDL-induced macrophages seemed to be attenuated when SERPINE1 expression was suppressed. Also, knockdown of SERPINE1 attenuated the autophagy of ox-LDL-induced macrophages induced by quercetin treatment. We inferred that when the senescence of macrophages induced by ox-LDL was ameliorated, the effects of quercetin on enhancing autophagy were attenuated, partly associated with SERPINE1 expression. Therefore, how quercetin prevents cellular senescence by regulating autophagy still needs further investigation.

The limitations of this study should be pointed out. Firstly, although we investigated the anti-atherosclerotic effects of quercetin both in vitro and in vivo, the exact mechanisms need to be further explored. Secondly, potential off-target effects of quercetin should be evaluated. Thirdly, clinical trials will be conducted to investigate the clinical treatment effects of quercetin in AS progression in future studies. The optimal dose, treatment duration time, and potential adverse effects should be evaluated in humans.

Conclusion

Collectively, we found that quercetin exerts an anti-atherosclerotic mechanism by mitigating cellular senescence driven by ox-LDL both in HAECs and macrophages, which may be linked with p16/p21, p53/SERPINE1, and AMPK/mTOR pathways. Moreover, quercetin alleviates cellular senescence of macrophages through regulating autophagy, partly associated with SERPINE1 expression. Our findings provide more experimental evidence that quercetin holds promise as a treatment drug for AS. However, the molecular mechanisms of quercetin in preventing cellular senescence in the treatment of AS require in-depth exploration.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

AS:

Atherosclerosis

HAECs:

Human aortic endothelial cells

ox-LDL:

Oxidized low-density lipoprotein

FBS:

Fetal bovine serum

SA-β-gal:

Senescence-associated-β-galactosidase

CCK-8:

Cell counting kit-8

ROS:

Reactive oxygen species

ND:

Normal diet

HD:

High-fat diet

H&E:

Hematoxylin–eosin

ELISA:

Enzyme-linked immunosorbent assay

TC:

Total cholesterol

TG:

Triglyceride

HDL:

High density lipoprotein

LDL:

Low-density lipoprotein

IL:

Interleukin

TNF-α:

Tumor necrosis factor-α

SASP:

Senescence-associated secretory phenotype

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Acknowledgements

Not applicable.

Funding

This study was supported by Natural Science Foundation of Heilongjiang Province of China (LH2020H032), National Natural Science Foundation of China (81670296), National Natural Science Foundation of China(82100364).

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Authors and Affiliations

  1. Department of Cardiology, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, Heilongjiang, China

    Xiao Liang, Jiangbo Yu, Jiyi Zhao & Shusen Yang

  2. Department of Cardiology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310009, Zhejiang, China

    Xiao Liang & Jingyuan Zhang

  3. First Department of Medicine, Medical Faculty Mannheim, University Medical Centre Mannheim (UMM), University of Heidelberg, 68167, Mannheim, Germany

    Xiao Liang & Jingyuan Zhang

  4. DZHK (German Center for Cardiovascular Research), Partner Site, Heidelberg-Mannheim, 68167, Mannheim, Germany

    Xiao Liang & Jingyuan Zhang

Authors
  1. Xiao Liang
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  2. Jingyuan Zhang
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  3. Jiangbo Yu
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Contributions

Shusen Yang conceived and designed the study. Xiao Liang, Jingyuan Zhang and Jiangbo Yu conducted most of the experiments and data analysis, and wrote the manuscript. Jiyi Zhao participated in collecting data and helped to draft the manuscript. All authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Shusen Yang.

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Ethics approval and consent to participate

The animal experiments were approved by the Animal Ethics Committee of The First Affiliated Hospital of Harbin Medical University (KY2023-328).

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The authors declare no competing interests.

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Supplementary Information

40001_2025_2562_MOESM1_ESM.pdf

Supplementary Material: Fig. 1. Transcriptome analysis of HAECs with treatment of 50 μg/mL ox-LDL in combination with or without 3 μM quercetin.Correlation analysis between different samples.Principal component analysis

40001_2025_2562_MOESM2_ESM.xlsx

Supplementary Material 2: Table 1.The key differentially expressed genes in ox-LDL-induced HAECs with or without quercetin treatment

Supplementary Material 3: Table 2. Signaling pathways enriched by the differentially expressed genes

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Liang, X., Zhang, J., Yu, J. et al. Quercetin ameliorates ox-LDL-induced cellular senescence of aortic endothelial cells and macrophages by p16/p21, p53/SERPINE1, and AMPK/mTOR pathways. Eur J Med Res 30, 359 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40001-025-02562-y

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40001-025-02562-y

Keywords

European Journal of Medical Research

ISSN: 2047-783X

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