Introduction: Senescent cells play a key role in the initiation and development of various age-related diseases. Human umbilical vein endothelial cells (HUVECs) senescence is closely associated with age-related cardiovascular diseases. Accumulating evidence has demonstrated that senolytics, the combination of dasatinib and quercetin (D+Q), could selectively eliminate senescent cells. N6-methyladenosine (m6A), the most abundant internal transcript modification, greatly influences RNA metabolism and modulates gene expression. We aimed to investigate whether RNA m6A functions in lipopolysaccharide (LPS)-induced HUVECs senescence and D+Q suppress HUVECs senescence by regulating RNA m6A. Methods: Senescence-associated β-galactosidase activity, western blot, and real-time quantitative polymerase chain reaction were performed to demonstrate that D+Q suppress HUVECs senescence. Methylated RNA immunoprecipitation (MeRIP)-qPCR assay and RIP-qPCR confirmed that RNA m6A plays a key role in the suppression of HUVECs senescence by D+Q. Chromatin immunoprecipitation and mRNA stability assay were carried out to prove that D+Q alleviate HUVECs senescence in a YTHDF2-dependent manner. Results: Here, we demonstrate that D+Q alleviate LPS-induced senescence in HUVECs via inhibiting autocrine and paracrine of the senescence-associated secretory phenotype (SASP). We further confirm that D+Q alleviate HUVECs senescence via the TNF receptor-associated factor 6 (TRAF6)-MAPK pathway. Mechanically, this study validates that D+Q suppress SASP by upregulating m6A reader YTHDF2. Besides, YTHDF2 regulates the stability of MAP2K4 and MAP4K4 mRNAs. Conclusion: Collectively, we first identified that D+Q alleviate LPS-induced senescence in HUVECs via the TRAF6-MAPK-NF-κB axis in a YTHDF2-dependent manner, providing novel ideas for clinical treatment of age-related cardiovascular diseases.

Aging is the primary risk factor in the development of most diseases, including cancer, neurodegenerative diseases, and cardiovascular diseases. Cellular senescence, a physiological and pathological program, is a permanent state of cell cycle arrest that accompanies the acquisition of various secretory phenotypes 1. The features of senescent cells are telomere shortening, arrest of proliferation, DNA damage response caused by dysfunctional telomeres, as well as senescence-associated secretory phenotype (SASP) 2. Increasing evidence has demonstrated that senescent cells can fuel overt age-related diseases 3. Therefore, therapeutic strategies that target the mechanism of senescence may provide novel ideas for clinical treatment of age-related diseases.

The senescence of endothelial cells, one of the hallmarks of vascular aging, has been implicated in the promotion of age-related cardiovascular diseases 4. Many studies have shown that senescent endothelial cells could result in endothelial dysfunction by the increased production of reactive oxygen species, oxidative stress, and inflammation 5. In addition, senescent endothelial cells can inhibit the angiogenic activity of endothelial progenitor cells through SASP 6. Inflammatory and growth factors damage the vascular endothelium through the activation of the TARF6-ARNO-Rac axis which can promote vascular permeability, leukocyte adhesion, and angiogenesis 7. Furthermore, the inhibition of endothelial nuclear factor-κB (NF-κB) signaling has been demonstrated to alleviate the age-associated vascular senescence 8.

Chronic inflammation is associated with senescence 9. Inflammation is characterized by the increased levels of several pro-inflammatory cytokines, including IL-6, IL-18, and tumor necrosis factor-α 10. TLR4 signaling on the plasma membrane is activated by lipopolysaccharide (LPS), a natural adjuvant synthesized by Gram-negative bacteria, thereby stimulating pro-inflammatory signaling pathways dependent on the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) 11. It has been reported that METTL3 could promote LPS-induced microglial inflammation by activating the TRAF6-NF-κB pathway in an N6-methyladenosine (m6A)-dependent manner 12.

m6A, the most prevalent internal mRNA posttranscriptional modification, has been demonstrated to be closely associated with inflammation 13. m6A plays a vital role in the regulation of downstream molecular events and biological functions, ranging from RNA processing, nuclear export, RNA translation to decay 14, 15. m6A modification exerts biological functions via recruiting m6A “reader” proteins, including YTH domain-containing proteins (YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2), heterogenous nuclear ribonucleoprotein A2/B1, and insulin-like growth factor 2 mRNA-binding protein 16. Emerging evidence has suggested that m6A is involved in inflammation and senescence 17. However, the role of m6A in vascular aging and the senescence of endothelial cells remains unknown. Therefore, we sought to elucidate the molecular mechanism of m6A in the senescence of endothelial cells, which may provide a potential target for treating age-related cardiovascular diseases.

Recently, the drug combination of dasatinib and quercetin (D+Q) is a senolytic molecule with significant antiaging effects 18. Dasatinib, a short-acting inhibitor of small-molecule tyrosine kinase, can attenuate the viability of senescent adipocytes and induce apoptosis 19. Besides, quercetin possesses potent antioxidant, anti-inflammatory, immunoprotective, as well as anticarcinogenic effects, thereby reducing lipid peroxidation, platelet aggregation, and capillary permeability 20. The senolytics cocktail D+Q selectively eliminate senescent cells but have no significant effects on non-senescent cells, ultimately leading to the decreased number of naturally occurring senescent cells and the reduced secretion of pro-inflammatory cytokines in human adipose tissue 21. Moreover, it has been reported that D+Q could reduce the burden of senescent cells in skin and adipose tissues, attenuate the accumulation of macrophages, enhance the replication potential of adipose progenitor cells, and decrease the key cyclic SASP factors 22, 23. However, the mechanism of D+Q in endothelial cell senescence-related cardiovascular diseases remains still unclear. Therefore, our work aimed to investigate the specific mechanism through which D+Q could inhibit the senescence of endothelial cells, providing insights regarding whether D+Q are able to provide prophylactic and therapeutic effects for the treatment of endothelial cell senescence-related cardiovascular diseases.

Cell Culture and Treatment

Human umbilical vein endothelial cells (HUVECs) were obtained from American Type Culture Collection. The cells were cultured at 37°C in a 5% carbon dioxide incubator and under ECM with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. HUVECs were transfected with small interfering RNA (siRNA) or negative control siRNA (Shanghai GenePharma, China) using Lipofectamine 3000 Reagent (Invitrogen, USA) as described in manufacturer’s protocols. The siRNA sequences for transcription used are listed in online supplementary Table S4 (see www.karger.com/doi/10.1159/000522656 for all online suppl. material). Dasatinib and quercetin were purchased from MedChemExpress (China).

Senescence-Associated β-Galactosidase Activity

Senescence-Associated β-galactosidase (SA-β-gal) staining was performed according to the manufacturer’s instructions. Cells were fixed with fixative solution for 15 min followed by 3 washes. Next, cells were incubated in staining solution containing staining supplement and X-Gal for 24 h at 37°C incubator. The SA-β-Gal positive blue cells were visualized under a fluorescence microscope (Nikon, Japan) and photographed.

Cell Viability Analysis

Cell viability analysis was performed using the cell counting kit 8 (CCK-8) assay (Beyotime, Beijing, China) according to the manufacturer’s instructions. HUVECs were seeded in 96-well plates for 24 h. Then, we added 100 μL ECM containing CCK-8 at 10:1 dilution ratio to the cells. After 1 h incubation, cell viability was detected by the SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Western Blot

Cells were lysed with RIPA (Beyotime, Beijing, China) buffer containing protease inhibitor phenylmethylsulfonyl fluoride (Beyotime, Beijing, China) on ice. The protein concentration levels were quantified using a BCA Protein Assay kit (Beyotime, Beijing, China). The equal amounts of protein samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked by non-fat dry milk for 1 h at room temperature. Then, the membranes were incubated overnight in specific primary antibodies at 4°C followed by appropriate secondary antibodies for 1 h at room temperature. Finally, imaging was done with Pierce ECL Western Blot Substrate. The antibodies used for Western blot are shown in online supplementary Table S1.

Real-Time Quantitative Polymerase Chain Reaction

Total RNA was isolated using TRIzol reagent (Invitrogen, USA) as manufacturer’s instructions followed by reverse transcription to cDNA. Real-time quantitative polymerase chain reaction (qRT-PCR) was performed using HiScript-II-Q RT SuperMix (Vazyme, Nanjing, China) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Quantitative gene expression data were normalized to the expression levels of control. The expression of genes was analyzed by the method of 2−ΔΔCt. Primers were synthesized by a commercial vendor (Nanjing Generay, China). The primers used in qRT-PCR are listed in online supplementary Table S2.

Immunofluorescence

Cultured cells were fixed in 4% paraformaldehyde for 20 min. Next, cells were permeabilized with 0.5% Triton X-100 for 20 min and blocked with 3% BSA for 1 h, followed by incubation with primary antibodies at 4°C overnight. After three washes in PBS, cells were subsequently incubated with secondary antibodies at 37°C for 1 h, followed by DAPI staining and washes in PBS. Finally, the staining was observed under a confocal laser scanning microscope (LSM800; Zeiss, Oberkochen, Germany).

Quantitative Real-Time PCR for Analysis of Telomere Lengths

DNA was extracted from the samples as per manufacturer’s instructions. We prepared premixes of PCR reagents and T and S primers in separate tubes. For each template DNA, real-time reactions were run for three times. For telomere length analysis, qRT-PCR was used to analyze the relative telomere lengths (telomere/single-copy gene of albumin, T/S) with a minor modification by the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers used are listed in online supplementary Table S3.

Methylated RNA Immunoprecipitation-qPCR Assay

We performed the methylated RNA immunoprecipitation (MeRIP) assay by a Magna MeRIPTM m6A kit (#17-10,499; Merck Millipore, MA, USA) in accordance with a previously reported protocol with minor modifications. Total RNA was purified from HUVECs by using Trizol. Then, RNA was incubated with 2 μg of anti-m6A antibodies or anti-IgG at 4°C overnight. Dynabeads Protein G was next mixed with RNA. After 3 washes, RNA was eluted from the beads after immunoprecipitation (IP). m6A-modified mRNA was then detected through qPCR as described above.

RNA Immunoprecipitation-qPCR

RNA immunoprecipitation (RIP)-qPCR was conducted by using the Magna RIPTM Quad RNA-Binding Protein Immunoprecipitation Kit (17-704; Millipore, Billerica, MA, USA) according to manufacturer’s illustrations. Briefly, magnetic beads were mixed with specific antibodies before the addition of cell lysates at 4°C overnight. Interested RNAs were eluted from immunoprecipitated complex and purified for further analysis using qPCR.

mRNA Stability Assay

mRNA stability assay was carried out as previously described 24. HUVECs were incubated with actinomycin D (5 μg/mL) and subsequently harvested at 0, 2, and 4 h. Then, total RNA was extracted at the indicated times and detected by qRT-PCR.

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) are conducted following the manufacturer’s protocols 25 with cells cross-linked in 1% formaldehyde for 10 min and subsequently quenched by 0.125 M glycine for 5 min. The cells were collected and sonicated to generate DNA fragments. Next, the lysate was immunoprecipitated with primary antibodies at 4°C with rotation overnight. Finally, immunoprecipitated DNAs were extracted and analyzed by qRT-PCR. The ChIP primer sequences are listed in online supplementary Table S5.

Statistical Analysis

Results obtained were presented as mean ± SEM based on three independent experiments. Differences among the treatment groups were assessed with one-way ANOVA followed by Tukey’s test for post hoc comparison when appropriate. Unpaired two-tailed Student’s t test was used for comparisons between two groups. Statistical significance was defined as p value <0.05. Statistical analyses were performed with GraphPad Prism 8.0 software.

Senolytics (D+Q) Alleviate LPS-Induced Senescence in HUVECs

Previous studies have demonstrated that endothelial cell senescence and dysfunction are the key factors leading to cardiovascular injury. Senolytics (D+Q), including the combination of dasatinib and quercetin, selectively eliminate senescent cells and play a protective role in aging-related diseases. In view of the effect of D+Q, we further aim to explore whether D+Q can inhibit the senescence of endothelial cells and the specific mechanism. The chemical structure formulas of quercetin and dasatinib were shown as Figure 1a. Next, we performed the CCK-8 experiment and found that 10 μM quercetin and dasatinib, respectively, had the greatest antagonistic effect on LPS (1 μg/mL)-treated HUVECs. Meanwhile, there was no obvious difference in cell viability of HUVECs treated with 10 μM quercetin and dasatinib for different times (0, 6, 12, 24 h), respectively (Fig. 1b, c). To evaluate the effect of D+Q on the senescence of HUVECs, different concentrations of D+Q (10 nM, 100 nM, 1 μM, 10 μM, and 100 μM) were used to treat LPS-stimulated HUVECs. As shown in Figure 1d, 10 μM D+Q incubating HUVECs for 24 h were chosen as the optimal condition to antagonize LPS-induced senescence. We next found that the number of cells positive for SA-β-gal staining increased in response to LPS and the LPS-induced senescence was attenuated obviously in HUVECs treated with 10 μM D+Q (Fig. 1e). Moreover, BrdU incorporation assay demonstrated that D+Q markedly blocked LPS-induced cell cycle arrest (Fig. 1f). We further performed cell counting and found that the LPS-induced growth arrest of HUVECs (Fig. 1g) was reversed by treatment with 10 μM D+Q for 24 h. Since telomere length is used as a predictor of the self-renewal potential of HUVECs 26. We observed that the telomeres in the LPS-treated group were shortened; the shortening of the telomeres was blocked by D+Q (Fig. 1h). Western blot analysis found that the senescent markers p53, p21, and p16, which were upregulated by LPS, were significantly reduced by treatment with 10 μM D+Q (Fig. 1i). Collectively, these observations above indicated that D+Q have an antagonistic effect against the LPS-induced senescence in HUVECs.

Fig. 1.

Senolytics (D+Q) alleviate LPS-induced senescence in HUVECs. a The chemical structure formulas of quercetin (left) and dasatinib (right). b–d HUVECs were respectively treated with different concentrations of quercetin, dasatinib, D+Q (10 nM, 100 nM, 1 μM, 10 μM, and 100 μM) or vehicle for the control and different time (0, 6, 12, 24, 48, 72 h), then measured by CCK-8 assay. e SA-β-gal and DAPI staining of HUVECs induced by LPS. Quantitative analysis of the percentage of SA-β-Gal positive cells (%) in HUVECs. Scale bar, 50 μm. f Immunofluorescence staining for colocalization of BrdU and DAPI in HUVECs treated with LPS for 48 h, where indicated, treated with (+) or without (−) 10 μM D+Q. Percentage of BrdU-positive cells in HUVECs. g Growth curves for HUVECs cultured with (+) or without (−) 10 μM D+Q. h Relative telomere lengths expressed as T/S ratios in HUVECs. i Western blot analysis of senescent markers p53, p21, and p16. β-Actin was used as the loading control. Data are expressed as mean ± SEM, n= 3. *p< 0.05, **p< 0.01, ***p< 0.001.

Fig. 1.

Senolytics (D+Q) alleviate LPS-induced senescence in HUVECs. a The chemical structure formulas of quercetin (left) and dasatinib (right). b–d HUVECs were respectively treated with different concentrations of quercetin, dasatinib, D+Q (10 nM, 100 nM, 1 μM, 10 μM, and 100 μM) or vehicle for the control and different time (0, 6, 12, 24, 48, 72 h), then measured by CCK-8 assay. e SA-β-gal and DAPI staining of HUVECs induced by LPS. Quantitative analysis of the percentage of SA-β-Gal positive cells (%) in HUVECs. Scale bar, 50 μm. f Immunofluorescence staining for colocalization of BrdU and DAPI in HUVECs treated with LPS for 48 h, where indicated, treated with (+) or without (−) 10 μM D+Q. Percentage of BrdU-positive cells in HUVECs. g Growth curves for HUVECs cultured with (+) or without (−) 10 μM D+Q. h Relative telomere lengths expressed as T/S ratios in HUVECs. i Western blot analysis of senescent markers p53, p21, and p16. β-Actin was used as the loading control. Data are expressed as mean ± SEM, n= 3. *p< 0.05, **p< 0.01, ***p< 0.001.

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D+Q Suppress LPS-Induced SASP

To explore the effect of D+Q on SASP, we next systematically analyzed the secretome of LPS-induced senescent HUVECs by using a qRT-PCR array. The heatmap showed that D+Q downregulated the expression of the majority of LPS-induced SASP genes (Fig. 2a), which indicated that D+Q suppress SASP. The suppressive effect of D+Q on SASP was confirmed through qRT-PCR; as LPS-induced senescent HUVECs were treated with D+Q, the expression of SASP-associated, functionally indispensable factors, such as CXCL1, IL6, IL8, MMP3, and VEGFC, obviously decreased (Fig. 2b). Besides, SASP has been reported to induce paracrine senescence by secreting inflammatory cytokines 27. We performed further experiments to verify whether SASP reinforced senescence and whether the reinforced SASP expression could also be abolished by treatment with D+Q. As the presentation of Figure 2c, D+Q markedly reduced the SA-β-gal activity in HUVECs that were cultured in LPS-conditioned medium (LPS-CM) with LPS-treated senescent HUVECs. Similarly, after treatment with 10 μM D+Q, the elevated expression of representative SASP genes in LPS-CM-induced senescent HUVECs was decreased (Fig. 2d). These collective results demonstrated that D+Q can efficiently suppress the autocrine and paracrine of LPS-induced SASP genes.

Fig. 2.

D+Q suppress LPS-induced SASP. a qRT-PCR analysis of SASP genes in HUVECs treated with LPS combined with (+) or without (−) 10 μM D+Q. Relative expression values are expressed as a color code (red high and blue low value). b mRNA expression levels of SASP genes in HUVECs treated with LPS combined with (+) or without (−) 10 μM D+Q were detected by qRT-PCR. c, d HUVECs treated with conditioned medium from LPS-CM or the normal medium (N-CM) with (+) or without (−) 10 μM D+Q c SA-β-Gal and DAPI staining of HUVECs induced by conditioned medium from LPS-CM. Quantitative analysis of the percentage of SA-β-Gal positive cells (%) in HUVECs. Scale bar, 50 μm. d mRNA expression levels of SASP genes in HUVECs were detected using qRT-PCR. Data are expressed as mean ± SEM, n= 3. *p< 0.05, **p< 0.01, ***p< 0.001.

Fig. 2.

D+Q suppress LPS-induced SASP. a qRT-PCR analysis of SASP genes in HUVECs treated with LPS combined with (+) or without (−) 10 μM D+Q. Relative expression values are expressed as a color code (red high and blue low value). b mRNA expression levels of SASP genes in HUVECs treated with LPS combined with (+) or without (−) 10 μM D+Q were detected by qRT-PCR. c, d HUVECs treated with conditioned medium from LPS-CM or the normal medium (N-CM) with (+) or without (−) 10 μM D+Q c SA-β-Gal and DAPI staining of HUVECs induced by conditioned medium from LPS-CM. Quantitative analysis of the percentage of SA-β-Gal positive cells (%) in HUVECs. Scale bar, 50 μm. d mRNA expression levels of SASP genes in HUVECs were detected using qRT-PCR. Data are expressed as mean ± SEM, n= 3. *p< 0.05, **p< 0.01, ***p< 0.001.

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D+Q Alleviate LPS-Induced Senescence via TRAF6-MAPK Pathway

The activation of TRAF6 signaling pathway has been reported to be involved in controlling many key inflammatory genes identified in cellular senescence 28. Next, we aimed to determine whether D+Q alleviated LPS-induced HUVECs senescence via TRAF6-MAPK pathway. Western blot assay suggested that D+Q reversed the elevated expression of TRAF6 and MAPK in HUVECs treated with LPS (Fig. 3a), which was consistent with the results of qRT-PCR assay (Fig. 3b). Next, the results of IP indicated MAPK and TRAF6 interaction increased in LPS-treated HUVECs and D+Q reduced the binding between MAPK and TRAF6 (Fig. 3c, d). We performed specific siRNAs to knock down TRAF6 and MAPK in HUVECs, respectively (Fig. 3e, f). We found that siTRAF6 inhibited LPS-induced senescence, suggesting that LPS could cause HUVECs senescence through TRAF6 (Fig. 3g). SA-β-Gal staining activity further confirmed that D+Q inhibited the NF-κB pathway activation during LPS-induced senescence and MAPK deficiency reversed this inhibition (Fig. 3h). Therefore, D+Q alleviate LPS-induced senescence in HUVECs via TRAF6-MAPK pathway.

Fig. 3.

D+Q alleviate LPS-induced senescence via TRAF6-MAPK pathway. a Expression of TRAF6 and MAPK was analyzed by Western blot. b Relative mRNA levels of TRAF6 and MAPK in HUVECs treated with or without D+Q were determined by qRT-PCR. c IP analysis of relative amount of TRAF6 binding to MAPK in LPS-treated HUVECs. d IP analysis of relative amount of MAPK binding to TRAF6 in LPS-treated HUVECs. e, f Efficiency of siTRAF6 or siMAPK by RNA interference in HUVECs was measured by Western blot analysis. g, h SA-β-gal and DAPI staining of HUVECs induced by LPS. Quantitative analysis of the percentage of SA-β-Gal positive cells (%) in HUVECs. Scale bar, 50 μm. Data are expressed as mean ± SEM, n= 3. *p< 0.05, **p< 0.01, ***p< 0.001.

Fig. 3.

D+Q alleviate LPS-induced senescence via TRAF6-MAPK pathway. a Expression of TRAF6 and MAPK was analyzed by Western blot. b Relative mRNA levels of TRAF6 and MAPK in HUVECs treated with or without D+Q were determined by qRT-PCR. c IP analysis of relative amount of TRAF6 binding to MAPK in LPS-treated HUVECs. d IP analysis of relative amount of MAPK binding to TRAF6 in LPS-treated HUVECs. e, f Efficiency of siTRAF6 or siMAPK by RNA interference in HUVECs was measured by Western blot analysis. g, h SA-β-gal and DAPI staining of HUVECs induced by LPS. Quantitative analysis of the percentage of SA-β-Gal positive cells (%) in HUVECs. Scale bar, 50 μm. Data are expressed as mean ± SEM, n= 3. *p< 0.05, **p< 0.01, ***p< 0.001.

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D+Q Attenuate SASP by Upregulating YTHDF2

Given that previous studies have reported that m6A functions in inflammation and senescence 29. To explore the role of the m6A-binding proteins of the YTH domain family in the SASP, we first testified the expression levels of YTHDF2, YTHDF1, and YTHDF3 in LPS-induced senescent HUVECs before and after treatment with D+Q. The results of Western blot showed that YTHDF2 was significantly downregulated during LPS treatment and reversed by D+Q (Fig. 4a), which was consistent with the results of qRT-PCR (Fig. 4b) and immunofluorescence analysis (Fig. 4c). To further validate the relationship between YTHDF2 and SASP, we, respectively, established stable YTHDF2 knockdown and overexpression (OE) models of HUVECs (Fig. 4d). The expression levels of CXCL1, IL6, IL8, MMP3, and VEGFC upregulated after YTHDF2 knockdown and subsequently restored after YTHDF2 OE (oeYTHDF2), which was reversed by D+Q (Fig. 4e). Collectively, these data indicated that D+Q attenuate SASP by upregulating YTHDF2.

Fig. 4.

D+Q attenuate SASP by upregulating YTHDF2. a Expression of YTHDF2, YTHDF1 and YTHDF3 was assessed by Western blot. b mRNA expression levels of YTHDF2, YTHDF1 and YTHDF3 were detected by qRT-PCR. c Immunofluorescence staining for colocalization of YTHDF2 and DAPI in HUVECs treated with LPS, where indicated, treated with (+) or without (−) 10 μM D+Q. d Efficiency of YTHDF2 interference in HUVECs and the effect of YTHDF2 OE were measured by Western blot analysis. e mRNA expression levels of SASP genes in LPS-induced HUVECs transfected with siYTHDF2 or oeYTHDF2, with (+) or without (−) 10 μM D+Q were detected by qRT-PCR. Data are expressed as mean ± SEM, n= 3. *p< 0.05, **p< 0.01, ***p< 0.001.

Fig. 4.

D+Q attenuate SASP by upregulating YTHDF2. a Expression of YTHDF2, YTHDF1 and YTHDF3 was assessed by Western blot. b mRNA expression levels of YTHDF2, YTHDF1 and YTHDF3 were detected by qRT-PCR. c Immunofluorescence staining for colocalization of YTHDF2 and DAPI in HUVECs treated with LPS, where indicated, treated with (+) or without (−) 10 μM D+Q. d Efficiency of YTHDF2 interference in HUVECs and the effect of YTHDF2 OE were measured by Western blot analysis. e mRNA expression levels of SASP genes in LPS-induced HUVECs transfected with siYTHDF2 or oeYTHDF2, with (+) or without (−) 10 μM D+Q were detected by qRT-PCR. Data are expressed as mean ± SEM, n= 3. *p< 0.05, **p< 0.01, ***p< 0.001.

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YTHDF2 Regulates the Stability of MAP2K4 and MAP4K4 mRNAs

MAP2K4 and MAP4K4 have been identified as the target genes of YTHDF2 30, 31. We performed a qRT-PCR analysis of MAP2K4 and MAP4K4 using siYTHDF2 or oeYTHDF2 in HUVECs. Interestingly, the mRNA levels of MAP2K4 and MAP4K4 were significantly increased in the siYTHDF2-treated HUVECs and decreased in the oeYTHDF2-treated HUVECs (Fig. 5a, b). Notably, the abundance of m6A in MAP2K4 and MAP4K4 mRNAs was measured through MeRIP-qPCR assay and we found m6A modifications in MAP2K4 and MAP4K4 mRNAs (Fig. 5c). Additionally, RIP-qPCR analysis revealed that YTHDF2 interacted with MAP2K4 and MAP4K4 mRNAs (Fig. 5d). Next, by conducting an mRNA stability assay, we observed that the stability of MAP2K4 and MAP4K4 mRNA transcripts was enhanced by a deficiency in YTHDF2 (Fig. 5e). Meanwhile, we found that oeYTHDF2 has an impact on MAP2K4 and MAP4K4 mRNA stability, which increased the instability of MAP2K4 and MAP4K4 mRNAs (Fig. 5f). Altogether, these results indicated that D+Q increase the instability of MAP2K4 and MAP4K4 mRNAs by upregulating the expression of YTHDF2.

Fig. 5.

YTHDF2 regulates the stability of MAP2K4 and MAP4K4 mRNAs. a, b mRNA expression levels of MAP2K4 and MAP4K4 in HUVECs transfected with siYTHDF2 or oeYTHDF2 were detected by qRT-PCR. c Analysis of m6A abundance in MAP2K4 and MAP4K4 mRNAs determined by MeRIP-qPCR. d YTHDF2 RIP-qPCR analysis of MAP2K4 and MAP4K4 in HUVECs. e, f mRNA expression levels of MAP2K4 and MAP4K4 in HUVECs transfected with NC, siYTHDF2, empty vector and oeYTHDF2 were assessed by qRT-PCR. Data are expressed as mean ± SEM, n= 3. *p< 0.05, **p< 0.01, ***p< 0.001.

Fig. 5.

YTHDF2 regulates the stability of MAP2K4 and MAP4K4 mRNAs. a, b mRNA expression levels of MAP2K4 and MAP4K4 in HUVECs transfected with siYTHDF2 or oeYTHDF2 were detected by qRT-PCR. c Analysis of m6A abundance in MAP2K4 and MAP4K4 mRNAs determined by MeRIP-qPCR. d YTHDF2 RIP-qPCR analysis of MAP2K4 and MAP4K4 in HUVECs. e, f mRNA expression levels of MAP2K4 and MAP4K4 in HUVECs transfected with NC, siYTHDF2, empty vector and oeYTHDF2 were assessed by qRT-PCR. Data are expressed as mean ± SEM, n= 3. *p< 0.05, **p< 0.01, ***p< 0.001.

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D+Q Inactivate MAPK-NF-κB Pathway and Regulate the Extent of NF-κB-Binding to SASP Genes

Since the components of the MAPK and NF-κB pathways can be phosphorylated by upstream phosphatases, we aimed to explore the relationship between D+Q and the activation of MAPK and NF-κB. Western blot and immunofluorescence assay confirmed that D+Q suppressed the NF-κB pathway activation during LPS-induced HUVECs senescence and MAPK deficiency reversed this suppression (Fig. 6a, b). In order to demonstrate the binding of the proteins, as annotated in Figure 6, to their target genes, we further performed a ChIP of the promoter elements (−1 to +1 kb) of SASP genes in LPS-induced HUVECs combined with D+Q treatment. No-antibody control, NF-κB, H3K27ac, and H3 (positive control) were analyzed by ChIP-qPCR. We found that after LPS treatment, the promoter regions of the SASP genes were remarkably enriched for NF-κB (Fig. 6c). NF-κB and H3K27ac were analogously enriched at the promoters in LPS-induced senescence in HUVECs; D+Q could effectively prevent NF-κB and H3K27ac from binding to these loci. The control marker H3 was pulled down evenly (Fig. 6d). These collective results indicated that D+Q have an antagonistic effect against LPS-induced senescence in HUVECs by regulating the extent of NF-κB-binding to the SASP genes. Our findings provided key insights into the mechanism by which D+Q alleviate LPS-induced senescence in HUVECs.

Fig. 6.

D+Q inactivate MAPK-NF-κB pathway and regulate the extent of NF-κB-binding to SASP genes. a Expression of pNF-κB and NF-κB in LPS-induced HUVECs treated with (+) or without (−) 10 μM D+Q combined with siMAPK was assessed by Western blot. b Immunofluorescence staining for colocalization of pNF-κB and DAPI in HUVECs treated with LPS, where indicated, treated with (+) or without (−) 10 μM D+Q. Scale bar, 50 μm. c ChIP-qPCR assay of NA, NF-κB, H3K27ac, and H3 in HUVECs treated with LPS, where indicated, treated with (+) or without (−) 10 μM D+Q. d ChIP-qPCR assay of the control H3 protein. Data are expressed as mean ± SEM, n= 3. *p< 0.05, **p< 0.01, ***p< 0.001. NA, no-antibody.

Fig. 6.

D+Q inactivate MAPK-NF-κB pathway and regulate the extent of NF-κB-binding to SASP genes. a Expression of pNF-κB and NF-κB in LPS-induced HUVECs treated with (+) or without (−) 10 μM D+Q combined with siMAPK was assessed by Western blot. b Immunofluorescence staining for colocalization of pNF-κB and DAPI in HUVECs treated with LPS, where indicated, treated with (+) or without (−) 10 μM D+Q. Scale bar, 50 μm. c ChIP-qPCR assay of NA, NF-κB, H3K27ac, and H3 in HUVECs treated with LPS, where indicated, treated with (+) or without (−) 10 μM D+Q. d ChIP-qPCR assay of the control H3 protein. Data are expressed as mean ± SEM, n= 3. *p< 0.05, **p< 0.01, ***p< 0.001. NA, no-antibody.

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In conclusion, senolytics (D+Q) exhibit an antagonistic effect against LPS-induced senescence in HUVECs via TRAF6-MAPK-NF-κB axis in a YTHDF2-dependent manner. LPS stimulates the activation of TRAF6-MAPK signaling pathway and suppresses the expression of YTHDF2. Meanwhile, D+Q attenuate LPS-induced HUVECs senescence by upregulating YTHDF2. YTHDF2 regulates the stability of MAP2K4 and MAP4K4 mRNAs, which triggers the activation of NF-κB and SASP. D+Q, as a combined antiaging agent, effectively decrease SASP and the senescence of HUVECs by inhibiting the TRAF6-MAPK-NF-κB axis in a YTHDF2-dependent manner (Fig. 7).

Fig. 7.

Model. Senolytics cocktail D+Q alleviate HUVECs senescence via the TRAF6-MAPK-NF-κB axis in a YTHDF2-dependent manner.

Fig. 7.

Model. Senolytics cocktail D+Q alleviate HUVECs senescence via the TRAF6-MAPK-NF-κB axis in a YTHDF2-dependent manner.

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Previous reports revealed that the senescence of endothelial cells, which could lead to endothelial dysfunction, is closely associated with the initiation and development of aging-associated diseases, such as diabetes, atherosclerosis, and cancer 32. Senescent endothelial cells are characterized by reduced cell proliferation, impaired cell migration, as well as increased expressions of senescence markers 33. Moreover, an elevated level of ET-1 expression can activate the endothelin-1 system, thereby promoting endothelial cell senescence 34. Recently, it has been reported that inhibition of senescence of endothelial cells could contribute to alleviating endothelial dysfunction 35. Therefore, novel therapies targeting the endothelial senescence function as an important strategy for the treatment of cardiovascular diseases.

m6A has generated much interest in the field of senescence in recent years. Previous studies have suggested that m6A demethylase FTO, which plays a critical role in ischemia, is decreased in senescent human and mouse hearts 36. Intriguingly, a significant downregulation of FTO expression is detected in the aged mouse hearts which might contribute to the age-related intolerance to myocardial ischemia 37. Moreover, abnormal m6A modifications function in age-related neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease 38. Dysregulation of m6A has been demonstrated to be associated with age-related infertility and heart failure in mice 39. However, the mechanism of m6A in senescent endothelial cells remains to be further investigated. Here, we found that endothelial cell senescence could be alleviated in a YTHDF2-dependent manner. YTHDF2, as a reader, has been identified to facilitate the translation of m6A mRNAs, the destabilization of m6A, and the degradation of m6A mRNAs 40, 41. Recent studies have reported that YTHDF2 could efficiently ameliorate cardiac hypertrophy via Myh7 mRNA decoy in an m6A-dependent manner 42. Here, we revealed that D+Q alleviate HUVECs senescence via TRAF6-MAPK-NF-κB axis in a YTHDF2-dependent manner.

Several studies have shown that endothelial cell inflammation plays an essential role in atherosclerosis, notably during senescence 43. The atherosclerotic lesions of patients with Hutchinson-Gilford progeria syndrome (HGPS) have indicated the presence of inflammation 44. In particular, an increasing expression of the pro-inflammatory transcription factor NF-κB was detected in mouse models of HGPS 45. Concomitantly, the suppression of the inflammatory response via Sirt7 ectopic expression in endothelial cells could significantly improve senescence features in HGPS mice 46. Endothelial progerin expression has been suggested to contribute to an enhanced expression of the adhesion molecules which is involved in the recruitment of macrophage into physiological aging, thereby leading to a pro-inflammatory response 47. In our study, we identified a novel TRAF6-MAPK-NF-κB axis and suggested it as a novel drug target of D+Q for the treatment of cardiovascular diseases.

D+Q have been demonstrated to contribute to selective elimination of senescent cells, decrease the secretion of frailty-related pro-inflammatory cytokines, and alleviate the senescent cell transplantation-induced physical dysfunction in young mice, ultimately improving lifespan and healthspan 48. Moreover, D+Q upregulated spinal glycosaminoglycan levels in progeroid mice, providing an effective treatment for age-related intervertebral disc diseases 49. Previous studies have noted that D+Q restored progenitor dysfunction, attenuated tissue inflammation, and alleviated age-related metabolic dysfunction 50. Notably, the specific effects of quercetin on age-related diseases need to be further investigated. Overall, we found the role of D+Q in treatment with age-related cardiovascular diseases and our findings supported that D+Q have potential as a lead molecule in drug development programs.

An ethics statement was not required for this study type, no human or animal subjects or materials were used.

The authors declare that there are no conflicts of interest.

This work was supported by Zhejiang Medical and Health Science and Technology Plan Project of China (No. 2022KY231); and Zhejiang Pharmaceutical Association Science Foundation of China (No. 2018ZYY06).

Ting Fan: conceptualization, methodology, and validation; Yi Du: formal analysis, investigation, and resources; Mingwan Zhang: data curation, writing – original draft, and visualization; Austin Rui Zhu: supervision and project administration; and Jianjun Zhang: writing – review, editing and funding acquisition.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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