The Citrus flavanone naringenin prolongs the lifespan in C. elegans and slows signs of brain aging in mice

Aging is one of the main risk factors for neurodegenerative disorders, which represent a global burden on healthcare systems. Therefore, identifying new strategies to slow the progression of brain aging is a compelling challenge. In this article


Introduction
Aging is a main risk factor for neurodegenerative disorders, which are associated with neurological deficits and impaired cognitive abilities.A chronic low-grade production of pro-inflammatory mediators and reactive oxygen species (ROS), termed ox-inflammation, has been identified as a major mechanism of aging.Indeed, ox-inflammation can alter cell function and metabolism, ultimately causing cell death (Santoro et al., 2020).In this context, the enzyme sirtuin 1 (SIRT1) and multiple downstream targets are crucial in the senescence process.SIRT1 plays a central role in the regulation of cellular homeostasis (Chen et al., 2020).Indeed, SIRT1 activates the antioxidant transcription factor Nrf2 (Xu et al., 2021), inhibits the nuclear translocation of the proinflammatory transcription factor NF-κB (Song et al., 2022), and activates the transcription factor forkhead box O-3 (FOXO3) (Bordbari et al., 2022) which is involved in stress resistance and metabolism (Morris et al., 2015).SIRT1 also regulates cellular energy metabolism and mitochondrial function through activation of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-gamma coactivator (PGC1-α) (Canto and Auwerx, 2009).Finally, SIRT1 reduces the expression of senescence-associated genes, such as p16, thus slowing down cellular senescence (Duan et al., 2022), extending lifespan and preventing the development of age-related diseases, including neurological disorders (Song et al., 2022;Razick et al., 2023).However, a progressive reduction of SIRT1 expression and activity has been reported during aging in several tissues (Testai et al., 2020;Xu et al., 2020).
In this context, many nutraceuticals have attracted great interest as they can reduce oxidative stress, improve cognitive abilities, prolong lifespan and promote healthspan in aged animals (Musillo et al., 2021).Among them, polyphenols have been largely studied for the antioxidant, anti-inflammatory and anti-senescence properties (Maria and Ingrid, 2017;Gurau et al., 2018), as well as for the ability to reduce some key components of senescence-associated secretory phenotype (SASP) (Maria and Ingrid, 2017).Resveratrol, but also the Citrus flavanone naringenin (NAR), are two of the most known polyphenols among SIRT1 activators (Borra et al., 2005;Testai et al., 2020).Several studies have demonstrated the preventive effects of NAR against cardiovascular and neurodegenerative disorders (Da Pozzo et al., 2017;Nouri et al., 2019;Piano et al., 2019;Testai et al., 2020;Kesh et al., 2021;Salman et al., 2022;Wang et al., 2023), as well as against the senescence process in the cardiovascular system (Da Pozzo et al., 2017;Testai et al., 2020;Wang et al., 2023).In the study by Testai and colleagues, daily treatment with the SIRT1 activator NAR significantly reduced oxidative stress, inflammation and fibrosis in the myocardium of aged mice (Testai et al., 2020).
In previous work, daily administration of bergamot juice, containing naringin (precursor of NAR), promoted antioxidant and anti-senescence effects in the myocardium of 12-month-old mice, potentially through increased expression of SIRT1 (Da Pozzo et al., 2018).However, other mechanisms could contribute to the potential effects of polyphenols against the aging process.The Citrus flavanone NAR led to the opening of large-conductance calcium-activated potassium channels in mitochondria (mitoBKCa) in young-adult and aged rats (Testai et al., 2017), while hesperetin prevented the senescence process through activation of the CISD2 gene in aged mice (Yeh et al., 2022) and reduction of oxidative stress in Saccharomyces cerevisiae (Guo et al., 2019;Xiao et al., 2023).Finally, several flavonoids have been shown to slow the progression of age-related neurodegenerative diseases through cholinesterase inhibition (Khan et al., 2018;Li et al., 2022).To this regard, NAR promoted positive effects on memory consolidation (Liaquat et al., 2018) and exhibited preventive effects against neurodegenerative processes (Ghofrani et al., 2015;Piano et al., 2019;Kesh et al., 2021;Piano et al., 2022;Salman et al., 2022).Also, an 8-week treatment with flavanone-rich orange juice provided benefits for cognitive function in healthy volunteers (Kean et al., 2015) while long-term dietary flavonoid intake was associated with a lower risk of cognitive decline in men and women (Yeh et al., 2021).Therefore, natural flavonoids could have a high translational potential in clinical practice for the prevention of age-related neurodegenerative diseases, but the molecular mechanisms should be better elucidated.
In this study, based on the promising results on the anti-senescence effects of the SIRT1 activator NAR in the mouse myocardium (Testai et al., 2020), we investigated the potential effects of NAR in slowing down the signs of brain aging in vivo.Many animal models are commonly used to study the potential anti-aging effects of new compounds and/or nutraceuticals, such as the nematode Caenorhabditis elegans (C.elegans) which has about 60-80 % genes with homologs in humans (Caldwell et al., 2020).In fact, many intracellular pathways participating in the onset and progression of age-related neurodegenerative disorders are conserved from C. elegans to mice and humans (Markaki and Tavernarakis, 2020).Recently, it was reported that lifespan timing can affect brain and cognitive functions (Walhovd et al., 2023).Therefore, we first evaluated the effects of daily treatment with NAR on lifespan and healthspan in the short-living nematode C. elegans.Then, we investigated the molecular mechanisms through which daily NAR supplementation could slow down the signs of brain aging in longlived vertebrates with human-like organs and systems (i.e., middle-aged mice) (Brunet, 2020).In particular, we focused on the SIRT1/Nrf2 axis and evaluated the effects of NAR on both mitochondrial function and expression of the senescence marker p16 and SASP-related markers interleukin (IL)-6 and IL-18 (Idda et al., 2020;Saul et al., 2022).

Experiments in C. elegans 2.1.1. C. elegans cultivation and treatment
Wildtype (N2) nematodes were kept at 20 • C on NGM plates supplemented with ampicillin (0.01%w/v) and tetracycline (0.0005%w/v).E. coli HT115 (L4440) was used as a food source.HT115 (L4440) was obtained from Ahringer C. elegans RNAi feeding library (Kamath and Ahringer, 2003) and grown in LB medium supplemented with ampicillin (0.01%w/v) and tetracycline (0.0005%w/v) at 37 • C overnight before spotting on NGM plates.NAR was solubilized in DMSO in a concentration 50-times higher than the final concentration.The compound was then spread on the NGM plates seeded with E. coli HT115 (L4440) to obtain the final desired concentration considering the final volume of the NGM plates.

Lifespan and healthspan
Lifespan and healthspan assays (movement) were carried out in three biological replicas (each with at least 60 animals).Age-synchronous populations of animals were recorded from egg-lay on NGM plates containing NAR 100-400 μM.The treatment with NAR was continued for the entire lifespan.Starting from 1-day-adulthood, the worms were transferred to fresh NGM plates every day (during the fertile period) or every other day (after the fertile period) and the numbers of dead, alive and censored animals were scored.Animals not moving, moving (spontaneously or upon prodding) and censored were scored for the healthspan analysis.Animals with internal hatching, an exploded vulva or which died desiccated on the wall were censored.Survival analysis of pooled populations was performed in OASIS 2 (Schiavi et al., 2015;Han et al., 2016).

Chronic treatment of mice in vivo
All procedures were performed in accordance with Italian (D.L. March 2014 n.26) legislation and European (EEC Directive 2010/63).The experimental protocols were reported as indicated by the ARRIVE guidelines (McGrath and Lilley, 2015).
Male 6-month-old C57BL/6J mice (ENVIGO, Italy) were housed in cages (3 mice per cage), and they had free access to food pellets and drinking water, at 22 • C.After the acclimation period, they were divided into two groups (12 mice per group): the first group was treated with NAR (100 mg/kg/day; Sigma-Aldrich, MO, USA) administered in drinking water, while the second one received vehicle (1 % DMSO; Sigma-Aldrich, MO, USA) for 6 months.The allocation of mice in both groups was random.During the whole treatment, water intake was daily monitored, and mice were weekly weighted.NAR was daily prepared by dissolving the powder in DMSO (40 mg/ml) and diluting in drinking water up to a final concentration of 0.4 mg/ml.The selected daily dosage of NAR (100 mg/kg) has been used in previous in vivo experiments, and it was not associated with toxic effects (Testai et al., 2017;Piano et al., 2019).At the end of the 6-month treatment, mice were anesthetized with urethane (30 % w/w, i.p.; Sigma-Aldrich, MO, USA) and quickly sacrificed by collecting blood from the heart; then, brains were taken and stored in liquid nitrogen for functional and biochemical analyses.Untreated 6-, 9-and 12-month-old mice (12 mice per group; mice per cage) represented controls and were used to assess the timecourse of: i) the enzymatic activity of citrate synthase and cytochrome C oxidase; ii) mRNA expression of Sirt1 and Nrf2 and their downstream targets, namely antioxidant (Ho-1, Foxo3), inflammatory (Il-6, Il-18) and anti-senescence (p16) genes.

Measurement of cytochrome C oxidase activity
The oxidation of cytochrome C by the enzyme cytochrome C oxidase (CcO) was detected with a spectrophotometric assay.Briefly, cytochrome C was reduced with a solution of 1,4-dithiothreitol (DTT; Sigma-Aldrich, MO, USA) 10 − 1 M for 15 min at room temperature (1:200).Reduction of cytochrome C appeared as a change in colour from dark red to light purple, and it was confirmed by measuring the ratio between λ = 550 nm and λ = 465 nm with a spectrophotometer (EnSpire; Perki-nElmer, MA, USA).Reduction was considered complete if the calculated ratio ranged from 10 to 20.Then, supernatants were diluted in Assay Buffer (composed by Tris HCl 10 mM and KCl 120 mM, pH 7.0) and reaction was initiated by adding reduced cytochrome C (1:6) in each well.Absorption was measured at 30 • C for 15 min with 30 s intervals (λ = 550 nm) with a microplate reader (EnSpire; PerkinElmer, MA, USA).CcO activity was calculated in brains of 4-6 animals per group by interpolating results with a calibration curve of standard CcO (Sigma-Aldrich, MO, USA) diluted in enzyme dilution buffer (Tris HCl 10 mM, sucrose 250 mM, pH 7.0).

Real-time RT-PCR
Brain tissues from 6-, 9-and 12-month-old untreated mice and 12month-old treated mice (3 mice per group) were lysed with Phenol/ Guanidine-Based QIAZol Lysis Buffer (Qiagen, Germany) and total RNA was extracted by using Rneasy Mini Kit® (Qiagen, Germany) following the manufacturer's protocol.RNA purity was assessed with NanoDrop™ Lite Spectrophotometer (Invitrogen, CA, USA).The cDNA was obtained by retro transcription from extracted RNA by using the iScript cDNA Synthesis Kit (Bio-Rad, CA, USA).50 ng of cDNA, 10 μl of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, CA, USA) and 300 nM each of the forward and reverse primers (Table 1) were used for Real-Time PCR Reaction.Steps temperatures were as following: 95 • C for 15 s (enzyme activation), 98 • C for 30 s (initial denaturation), and primer-specific annealing and extension temperatures for 30 s. Denaturation, annealing and extension phases were repeated for 40 cycles.GAPDH was used as the housekeeping gene to normalize Ct values. 2 − Δct method was used for a relative quantification of mRNA expression.

C. elegans experiments
C. elegans survival assays were performed in triplicate (each trial with at least 60 animals) if not differently specified.Statistical analysis of C. elegans in vivo treatments was executed using the log-rank test.The p-values were corrected for multiple comparisons using the Bonferroni method.Data were expressed as mean ± SEM (see Table in Fig. 1).

Mouse experiments
Each experiment was performed in triplicate in the brains of 3-6 animals per group (N = 3 for gene and protein analysis, N = 4-6 for measurement of CS and CcO activity).Statistical data analysis and graphical presentations were realized with GraphPad Prism® (Graph-Pad Software Inc., San Diego, CA, USA).Statistical analysis was performed by Mann-Whitney t-test for two-group analyses and with oneway analysis of variance (ANOVA) with Bonferroni's corrected test for analyses with three or more groups.A p-value < 0.05 was considered statistically significant.Data were expressed as mean ± SEM.

NAR displays anti-aging properties in vivo in C. elegans
To evaluate the potential anti-aging effect of NAR in vivo, we first used the nematode C. elegans, a powerful 3R-compliant model organism for aging and intervention studies.Dose-response analysis was initially carried out to select the optimal concentration of NAR (data not shown).
Despite having a mild effect, NAR 100 μM consistently showed the highest anti-aging efficacy, since it significantly extended C. elegans lifespan (Fig. 1A).Of note, given that parameters associated with animals' health during aging (healthspan), such as neuromuscular features (e.g.motility or sensory functions) or resistance to stressors, have been shown in C. elegans to correlate with lifespan in a context-dependent manner (Maglioni et al., 2014;Banse et al., 2024), we also analyzed the effect of NAR on animals' ability to move during aging.Consistent with the effect on lifespan, 100 μM NAR also mildly but significantly promoted animals' healthspan (movement ability, Fig. 1B).

NAR enhances CS ad CcO activities in the brain of middle-aged mice
Based on the promising effects observed in the nematode C. elegans, the potential anti-aging properties of NAR were then investigated in longer-lived animals, i.e. middle-aged mice.No systemic toxicity was evident in mice during the 6-month treatment with NAR (100 mg/kg/ day) (data not shown).Then, a preliminary mechanistic investigation of NAR was performed, evaluating the effects on the activity of two key enzymes of cellular metabolism (i.e., CS and CcO) in the brains of middle-aged mice.
CS is a well-recognized marker of the mitochondrial metabolism, whose activity is reported to decrease during aging (Brys et al., 2010;Pellegrini et al., 2020;Testai et al., 2020;Yan et al., 2021).A similar trend was observed in our experimental conditions; indeed, CS activity in brains from 12-month-old mice was about halved than that from 6month-old mice.More in detail, CS activity in the brain tissue was 118.1 ± 18.7 mU/ml in 6-month-old mice, 117.3 ± 18.1 in 9-month-old mice, and 66.0 ± 8.7 mU/ml in 12-month-old mice.Interestingly, CS activity in brains from mice daily receiving NAR for 6 months was significantly higher (117.9 ± 9.5 mU/ml) compared to that of 12month-old mice treated with vehicle, and almost superimposable with that of 6-month-old mice (P = 0.049, F(3,14) = 3.314, DF = 17) (Fig. 2A).
CcO is a mitochondrial enzyme whose activity decrease during aging (Curti et al., 1990;Paradies et al., 1993).CcO activity showed an age-dependent decline also in our study, being 568.3 ± 45.5 mU/ml in 6month-old mice, 445.7 ± 61.8 mU/ml in 9-month-old mice, and 346.7 ± 48.2 mU/ml in 12-month-old mice.As previously observed for CS, also CcO activity was significantly lower (about 50.0 %) in brains from 12-month-old mice compared to that of 6-month-old mice.Conversely, brains from 12-month-old mice receiving NAR for 6 months showed a CcO activity (574.7 ± 57.8 mU/ml) significantly higher if compared with controls of the same age.Once again, this value was almost superimposable with that measured in younger controls (P = 0.0324, F (3,16) = 3.755, DF = 19) (Fig. 2B).

NAR increases the expression of antioxidant response-related genes and proteins in the brain of middle-aged mice
Given NAR promising effects on mitochondrial activity in the brain of middle-aged mice, a time course of transcriptional levels of genes involved in the antioxidant response was performed and the effects of NAR on their mRNA expression were evaluated.In particular, the Sirt1 and Nrf2 pathways were investigated.Indeed, it has been shown that SIRT1 counteracts increased ROS levels by regulating the mitochondrial electrons transport chain (Guo et al., 2014;Wan and Garg, 2021) and promotes Nrf2 transcription, thus exerting an antioxidant effect (Shah et al., 2017).
The results of the time-course transcriptional analysis revealed that NAR treatment increases mRNA expression of genes that showed an agerelated decrease in mRNA levels (Sirt1 and Foxo-3) or, in general, with lower mRNA levels in untreated mice (Nrf2 and Ho-1).For this reason, focusing on groups of mice fed with NAR compared with the untreated 12-month-old mice, protein analysis by Western Blot was performed to confirm the antioxidant effects of the NAR-supplemented diet in 12month-old mice.

Discussion
The increase in life expectancy raised the number of people over 60 and, then, of age-related pathologies, such as metabolic, cardiovascular and neurodegenerative disorders, making aging an important issue worldwide (Franceschi et al., 2018).Many studies have shown that healthy aging is the result of multiple factors, including genetics (or not modifiable) and environmental ones (or modifiable); to this regard, nutrition can represent a powerful strategy for modulating the aging process (Leitao et al., 2022).
In this study, we investigated the potential anti-aging activity of the Citrus flavanone NAR in both invertebrates (C.elegans) and vertebrates (mice).In the short-lived, 3R-compliant model (Brunet, 2020), we observed that daily treatment with 100 μM NAR significantly promoted lifespan and healthspan.Then, we investigated the possible effects of NAR in slowing the aging process in the brain of middle-aged mice to give more clinical translatability to our findings, since mice are longlived vertebrates with human-like organs and systems (Brunet, 2020).6-month-old mice were treated for 6 months with NAR (100 mg/kg/day) to simulate a nutraceutical intervention in middle-aged people.No signs of toxicity were highlighted during the treatment, as the intake of water and food did not change during the experimental protocol and the body weight gain was comparable to that of control and in line with a physiological increase (data not shown).As previously reported (Curti et al., 1990;Paradies et al., 1993;Brys et al., 2010;Pellegrini et al., 2020;  E. Piragine et al.Testai et al., 2020;Yan et al., 2021), CS and CcO activities showed an age-dependent decline; in the brains of 12-month-old mice their activity was almost halved compared to that of young animals.Interestingly, NAR supplementation prevented this trend, suggesting its ability to preserve mitochondrial function and integrity, potentially ensuring adequate energy production for the cell.The mechanisms underlying these NAR-mediated protective effects may be related to those previously demonstrated in the myocardium of senescent animals (Testai et al., 2020).Therefore, we hypothesized that activation of SIRT1 enzyme may play a crucial, but not exclusive, role also in this tissue.Based on this idea, we measured mRNA expression of Sirt1, and downstream genes closely related to Sirt1 (i.e., Nrf2, Ho-1 and Foxo3).We observed a marked decline in the expression of these hallmarks of aging; on the contrary, these levels were significantly increased in the brains of NAR-treated animals.Of note, these transcriptional factors are known to be involved in the antioxidant defences of cells (Li et al., 2014).In agreement with the mRNA results, the protein assessment conducted on brain homogenates, by western blot method, confirmed the increasing trends, both for SIRT1 and for the proteins involved in the antioxidant response, namely Nrf2 and PGC1-α.Indeed, Nrf2 is an evolutionary conserved redox transcription factor, which plays a central role in the modulation of antioxidant responses and has been shown to be involved in the aging process in the nematode C. elegans (Blackwell et al., 2015;Tullet et al., 2017).PGC1-α is instead a transcription coactivator, also known to regulate the aging process in C. elegans (Corton and Brown-Borg, 2005).In particular, it regulates cellular energy metabolism by promoting the mitochondrial biogenesis and modulating both carbohydrate and lipid metabolism (Liang and Ward, 2006).
Therefore, these results agree with our previous findings and suggest a possible contribution of SIRT1 in the anti-aging effects of NAR in the brain.Indeed, SIRT1, regulates several pathophysiological processes, such as senescence and antioxidant response, through the deacetylation of different transcriptional factors or proteins (Chung et al., 2010;Grabowska et al., 2017).SIRT1 activates the FOXO3 pathway to reduce oxidative stress (Kops et al., 2002), as well as the expression of PGC1-α, which is involved in mitochondrial biogenesis and protection from mitochondrial dysfunction (Lin et al., 2004;St-Pierre et al., 2006) that occurs during aging, as confirmed by our data on CS and CcO.Finally, coactivation of Nrf2 promoting mRNA expression of its target gene Ho-1, confirms that NAR increases the antioxidant defence in cells.
Given the increased levels of proteins involved in the antioxidant response, we decided to study the anti-senescence effects of NAR treatment on p16, whose upregulation is involved in cell cycle arrest and, therefore, cellular senescence (Zindy et al., 1997;Krishnamurthy et al., 2004).Through a p16-mediated manner, senescent cells develop a SASP, which is characterized by increased mRNA expression of critical pro-inflammatory cytokines capable of establishing and worsening the chronic inflammation typical of aging and age-related functional alterations, including cognitive decline and shortened lifespan (Saul et al., 2022).The results on the mRNA expression of the senescence marker p16 and of the SASP-related proteins IL-6 and IL-18 seem to confirm the anti-senescence effects of the Citrus flavanone NAR, which can potentially prevent the p16-dependent cell cycle arrest and the release of the cytokines IL-6 and IL-18, whose levels are increased in senescent cells (Saul et al., 2022).
To the best of our knowledge, this is the first study aimed at evaluating the potential effects of chronic treatment with the SIRT1-activator NAR in preventing "physiological" brain aging rather than specific neurodegenerative diseases.Our data provide new evidence on the effects of NAR on lifespan and healthspan in C. elegans, previously reported only for the precursor glycoside-conjugated naringin (Zhu et al., 2020).Furthermore, our results not only confirm the neuroprotective effects shown by NAR in animal models of neurodegenerative diseases (Zbarsky et al., 2005;Ghofrani et al., 2015;Nouri et al., 2019;Salman et al., 2022) but also describe additional mechanisms of action (i.e., improved mitochondrial function and increased expression of Sirt1, Foxo3 and p16 genes) in the brain of middle-aged, healthy mice.Therefore, our findings open new perspectives in the prevention of agerelated cognitive decline and reduced neuronal function, which are prodromal to the development of neurodegenerative disorders in older adults.

Conclusions
Our results demonstrate that daily treatment with NAR prolongs the lifespan and improves the healthspan in short-lived nematodes (C.elegans).In long-lived animals (mice), dietary supplementation with NAR promotes the activity of enzymes involved in cellular metabolism (CS and CcO), prevents the age-dependent decrease of Sirt1 and Foxo3 mRNA expression, and improves that of Nrf2 and Ho-1 in the brain of middle-aged mice.Consistently, NAR treatment increases SIRT1, NRF2 and PGC1-α protein levels and restores mRNA expression of the senescence marker p16 and the pro-inflammatory genes Il-6 and Il-18.In conclusion, daily supplementation with NAR could represent a potential nutraceutical approach to slow the signs of brain aging and prevent the decline in neuronal and metabolic plasticity that occurs during aging.Indeed, from a translational point of view, this work focused on middleaged mice to mimic "preventive" NAR supplementation in young-adult people, aimed at slowing the first signs of cognitive decline and neuronal deterioration.Therefore, studying the effects of NAR administration in aged mice for a shorter period, when neuronal and metabolic plasticity are already compromised, could represent a future direction of our work.Furthermore, studies evaluating functional parameters and behavioural patterns in mouse models of brain aging are also needed to confirm our findings.

Fig. 1 .
Fig. 1.NAR promotes lifespan (A) and healthspan (B) extension in the nematode C. elegans.Mantel-cox survival analysis of wild-type animals treated with vehicle (DMSO) or NAR 100 μM.C. elegans assays were performed in triplicate (each trial with at least 60 animals).Abbreviations: SEM: standard error of the mean; CI: confidence interval.

Fig. 2 .
Fig. 2. NAR increases the activity of metabolic enzymes and the mRNA expression of antioxidant genes in the brain.Histograms show the CS activity (A) and the CcO activity (B), expressed in mU/ml, in the brain of animals of different ages (6-, 9-and 12-month-old) and of 12-month-old mice daily treated for 6 months with NAR (100 mg/kg) (N = 4-6 for each group).Histograms (C-F) show the mRNA expression of Sirt1, Foxo3, Nrf2 and Ho-1 in the brain of animals of different ages (6-, 9-and 12-month-old) and of 12-month-old mice daily treated for 6 months with NAR (100 mg/kg).Each bar represents the mean ± SEM (N = 3 for each group) (* significant vs 12-month-old; * p < 0.05; ** p < 0.01¸ *** p < 0.001).

Table 1
Sequences, annealing temperature and product amplicon size of primers used in RT-PCR.