Low-dose quercetin positively regulates mouse healthspan
Aging is the leading risk factor for many chronic diseases, accounting for almost 60% of all deaths worldwide. How to achieve healthy aging, alleviate aging-related diseases, and extend healthspan has become a main topic of biomedical research (He et al., 2019). Geroprotective compounds, such as metformin and rapamycin, have been shown to improve both healthspan and lifespan in mice (Martin-Montalvo et al., 2013; Bitto et al., 2016), whereas nicotinamide partially improves healthspan in mice (Mitchell et al., 2018). In addition, senolytics, compounds that eliminate senescent cells, have been proven to improve physical function and increase lifespan in mice (Xu et al., 2018). Although none have proven to be clinically reliable in delaying aging or treating frailty in humans, these compounds have already provoked enthusiasm for identifying a potential “elixir”. Therefore, the exploration of more geroprotective compounds, especially natural active compounds, holds great potential for the development of geriatric medicines.
Quercetin (Que) is a natural bioflavonoid found in fruits and vegetables such as apples and onions. Que (50 mg/kg) in combination with dasatinib (5 mg/kg) (abbreviated as D + Q) has been shown to effectively eliminate senescent cells via induction of apoptosis, thus alleviating senescence-related phenotypes and improving physical function and lifespan in mice (Zhu et al., 2015; Xu et al., 2018). In addition, Que (10 mg/kg) in combination with dasatinib (5 mg/kg) has been reported to reduce hepatic steatosis (Ogrodnik et al., 2017). In each of these in vivo studies, however, Que was used at high doses ranging from 10 to 50 mg/kg body weight, which raises concerns about dose-dependent side effects such as headaches and limb tingling (Shoskes et al., 1999). As a selective tyrosine kinase receptor inhibitor, dasatinib is associated with warnings and precautions including pulmonary arterial hypertension and low blood cell counts. Therefore, high-dose Que and extra side effects of dasatinib would hamper potential clinical applications of Que in geriatric medicines. Through natural products screening using Werner syndrome (WS) human mesenchymal stem cells (hMSCs), we recently identified Que as a geroprotective agent that counteracts accelerated and natural aging of hMSCs at a concentration of as low as 100 nmol/L, which is 100 times lower than the concentration of Que (10 μmol/L) previously used in combination with dasatinib as senolytic drugs to eliminate senescent cells in human umbilical vein cells (HUVECs) (Zhu et al., 2015; Geng et al., 2018).
To investigate how Que improved healthspan in mice, we collected 11 different kinds of tissues from 10-week young male mice (Y-Ctrl) and vehicle (O-Veh)- and low-dose Que-treated 22-month old male mice (O-Que). No significant difference was observed in organ weights between O-Veh and O-Que (Fig. S2A). Given that exercise endurance and diastolic function were improved by Que, we particularly examined the changes in skeletal muscles (SKM), white adipose tissues (WAT), brown adipose tissues (BAT) and hearts. Upon Que treatment, the arrangement of muscle fibers became more regular and compact with less fibrosis and senescence (Figs. 1F and S2B). In WAT, the increases in adipocyte size and senescence-associated β-galactosidase (SA-β-Gal)-positive area during aging were both alleviated upon Que treatment (Fig. 1F). In BAT, although adipocyte size was unaffected, there was a decreasing trend of the SA-β-Gal-positive area upon Que treatment (Figs. 1F and S2B). By comparison, we did not observe any significant differences in mouse hearts by histological analysis and SA-β-Gal staining (Fig. S2B). Therefore, these data suggest that long-term low-dose Que administration may delay aging of SKM, WAT, and BAT in mice.
To further explore the molecular mechanisms of the beneficial effects of Que, we performed whole-transcriptome RNA sequencing (RNA-seq) of SKM, WAT, and BAT from Y-Ctrl, O-Veh, and O-Que mice. Global gene expression profiling revealed that most protein coding genes were unaffected after long-term low-dose Que administration (Fig. 1G). Accordingly, we inferred that low-dose Que might exert its senostatic effect by regulating the expression of non-protein-coding RNAs.
In senescent cells, the activation of RTEs (such as L1) leads to genome instability and accumulation of cytosolic DNA that further binds to cytosolic sensor cGAS and activates TBK1 and IRF3, which subsequently promote senescence-associated secretory phenotype (SASP) (Takahashi et al., 2018; De Cecco et al., 2019). In addition, NF-κB/RelA in cGAS-STING-mediated NF-κB pathway acts with IRF3 and other transcription factors to induce the expression of inflammatory cytokines such as IL-6, the most prominent SASP cytokine (Chen et al., 2016). Notably, both p-TBK1 and p-IRF3 were increased in old mouse tissues compared to the young ones and were repressed upon Que treatment (Fig. 2E), indicating the effect of Que on inhibiting cGAS-STING pathway (Kato et al., 2017). Similarly, RelA (p65) was upregulated in aged mouse tissues and repressed upon Que treatment (Fig. 2E–G). Consistently, the inflammatory cytokine IL-6 was increased in old mice compared to young mice and Que antagonized the increase of IL-6 in both WS-hMSCs and old mouse SKM and BAT (Fig. 2A and 2B). Thus, our data suggest that Que may block SASP through the axis of heterochromatin-RTEs (L1)-innate immune response pathway (Fig. 2H).
In this study, we reported for the first time a geroprotective effect of low-dose quercetin alone that improved the healthspan of aged C57BL/6J male mice. Que-treated mice showed less hair loss, greater athletic endurance, enhanced diastolic function, and less muscle fibrosis, as well as alleviated cellular senescence in multiple tissues. Interestingly, these changes appear to be rarely associated with transcriptional alterations of protein-coding genes but are linked to heterochromatin stabilization and RTE silencing. Que treatment prevented L1 from hyperactivation, thereby inhibiting SASP. In contrast to the reported senolytic effect of high-dose D+Q (Xu et al., 2018), where Que exerts geroprotective effects via the induction of apoptosis of senescent cells, low-dose Que (0.125 mg/kg body weight) alone was sufficient to exert senostatic effects in mice by affecting heterochromatin stability through repression of RTEs activity in this study. In a translational context, low-dose Que monotherapy may be helpful to minimize the dose-dependent side effects compared to high-dose administration and avoid drug interference when used in combination, probably representing a potential therapeutic option for future clinical application (Shoskes et al., 1999). Of note, here we reported the geroprotective effect of Que in male mice and its effect remains to be studied in female mice.
The possible mechanism of low-dose Que in mice may be associated with its function as a heterochromatin stabilizer and its direct inhibitory activity against reverse transcriptase (Ono et al., 1990; Geng et al., 2018). Loss of heterochromatin architecture and genomic instability are two hallmarks of aging (Zhang et al., 2015). In advanced age, the expression of RTEs is often increased, which may in turn contribute to genomic instability and aging-associated cellular defects (De Cecco et al., 2013). Activation of L1 has been implicated in a variety of age-related disorders, including cancer and neurodegenerative diseases. The activation of L1 (and possibly other RTEs in mice) promotes the expression inflammatory factors, a feature of cellular senescence (De Cecco et al., 2019; Simon et al., 2019). Recently, it has been reported that nucleoside reverse-transcriptase inhibitors (NRTIs), such as lamivudine, stavudine, inhibit L1 retrotransposition and thus improve the healthspan and/or lifespan of SIRT6-knockout and physiologically aged mice (De Cecco et al., 2019; Simon et al., 2019). Que has been proven as a potential inhibitor of reverse transcriptase from Rauscher murine leukemia virus (RLV) and human immunodeficiency virus (HIV) by enzyme kinetic analysis, whereas its reverse transcriptase inhibition activity against RTEs in hMSCs and rodents has not been reported (Ono et al., 1990). Our data provide important evidence supporting the role of low-dose Que in safeguarding genomic stability (i.e. inhibition of retrotransposition), which at least in part contributes to its geroprotective activity in rodents.
We thank Lei Bai, Jing Lu, Ying Yang, Qun Chu, Shikun Ma, and Ruijun Bai for administrative assistance. We thank Jingyi Jia (Xuan Wu Hospital), Sai Yang (IBP, CAS), Qi Wei (IBP, CAS), Mengfei Wang (IBP, CAS), and Xinyi Wu (IBP, CAS) for breeding and management of laboratory animals and Lei Zhou (IBP, CAS) for providing veterinary care. We thank Qing Xu (Capital Medical University) for Doppler tissue imaging. High-throughput sequencing data were processed on the “Era” petascale supercomputer of Computer Network Information Center of Chinese Academy of Sciences. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16010100), the National Key Research and Development Program of China (2015CB964800, 2018YFC2000100, 2017YFA0102802, 2017YFA0103304, 2018YFA0107203), the National Natural Science Foundation of China (Grant Nos. 81625009, 81330008, 91749202, 91749123, 31671429, 81671377, 81771515, 31601158, 81701388, 81601233, 81571385, 31601109, 81822018, 81870228, 81801399, 31801010, 81801370, 81861168034), Beijing Municipal Commission of Health and Family Planning (PXM2018_026283_000002), Advanced Innovation Center for Human Brain Protection (3500-1192012) and the State Key Laboratory of Membrane Biology.
The authors declare no conflicts of interest.
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