Cocoa Polyphenol Extract Inhibits Cellular Senescence via Modulation of SIRT1 and SIRT3 in Auditory Cells

Cocoa, rich in polyphenols, has been reported to provide many health benefits due to its antioxidant properties. In this study, we investigated the effect of Cocoa polyphenols extract (CPE) against oxidative stress-induced cellular senescence using a hydrogen peroxide (H2O2)-induced cellular senescence model in three auditory cells lines derived from the auditory organ of a transgenic mouse: House Ear Institute-Organ of Corti 1 (HEI-OC1), Organ of Corti-3 (OC-k3), and Stria Vascularis (SV-k1) cells. Our results showed that CPE attenuated senescent phenotypes, including senescence-associated β-galactosidase expression, cell proliferation, alterations of morphology, oxidative DNA damage, mitochondrial dysfunction by inhibiting mitochondrial reactive oxygen species (mtROS) generation, and related molecules expressions such as forkhead box O3 (FOXO3) and p53. In addition, we determined that CPE induces expression of sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3), and it has a protective role against cellular senescence by upregulation of SIRT1 and SIRT3. These data indicate that CPE protects against senescence through SIRT1, SIRT3, FOXO3, and p53 in auditory cells. In conclusion, these results suggest that Cocoa has therapeutic potential against age-related hearing loss (ARHL).


Introduction
Cellular aging or senescence has emerged as an important factor in age-related diseases, and it is a potential target for new therapies [1]. Numerous examples of senescence at sites of aging pathology have been reported, as in cataracts [2], glaucoma [3], osteoarthritis [4], the diabetic pancreas [5], or age-related hearing loss (ARHL) [6]. ARHL is one of the most common and important health social problems [7]. It is characterized by an age-associated decrease of hearing that is due to the dysfunction and loss of hair cells in the Organ of Corti of the cochlea [8]. There is agreement that cumulative Reactive Oxygen Species (ROS) by mitochondrial dysfunction induce hearing loss.
Cellular senescence is a process that induces proliferative arrest on cells in response to different stressors, including DNA damage, oxidative stress, oncogene activation, and metabolic alterations [1]. Oxidative stress and DNA damage are associated with the modulation of mammalian Sirtuin (SIRT), nicotinamide adenosine dinucleotide (NAD)dependent protein deacetylase, which participates in the regulation of numerous cellular processes, including senescence, metabolism, and cell cycle [9,10]. Although still not completely defined, growing evidence has shown that SIRT is a pivotal factor in retarding cellular senescence and prolonging organismal life.

Induction of Cellular Senescence
To induce senescence, auditory cells were treated with hydrogen peroxide (H 2 O 2 ) (VWR Inc., West Chester, PA, USA) at 100 µM for 1 h, a method previously described by our group [38]. Untreated cells were considered as controls.

Cocoa Polyphenol Extraction and Treatment
For this study, cocoa powder with a high content of polyphenols (Chococru, London, UK) was used. Briefly, the cocoa sample was washed with 2N hydrochloric acid (for 1 h at room temperature and constant agitation) and, subsequently, with acetone-water under the same incubation conditions [39]. After centrifugation, the extract was desiccated by rotary evaporation R-14 (Büchi Labor-Technik AG, Flawil, Switzerland) and lyophilized in a LyoQuest lyophilizer (Telstar, Terrassa, Spain). The polyphenol content of the extract was quantified by BQC Phenolic Quantification Assay Kit (Bioquochem, Oviedo, Spain) based on Folin-Ciocalteau method [40] using Gallic acid as standard determined the total polyphenol content.
For the cocoa treatment, 5, 10, and 20 µg/mL of CPE were diluted in a serum-free culture medium and filtered through a 0.2-µm membrane. After 20 h of CPE treatment, a 100 µM of H 2 O 2 concentration was applied to the auditory cells belonging to H 2 O 2 group for 1 h to induce cellular senescence, and post-treatment analysis was performed at 24 h to evaluate the protective effect of the CPE against aging.

MTT Cell Proliferation Assay Kit
The cell proliferation and viability were measured using CyQUANT™ MTT Cell Proliferation Assay Kit (Invitrogen™, Waltham, MA, USA). Auditory cells were cultured in 96-well plates at 5 × 10 4 cells/well and incubated for 24 h. Following that, cells were treated with CPE and/or H 2 O 2 . After a corresponding incubation period, 10 µL of MTT solution was added to wells. At 4 h, 100 µL of the SDS-HCl solution was added to wells for 4 h at 37 • C. The absorbance was measured at 570 nm using a FLUOstar Omega (BMG Labtech, Ortenberg, Germany). The control group (untreated) was considered as 100% viability.

Senescence-Associated β-Galactosidase Assay
To quantify senescence, the Galactosidase Detection Kit (Abcam, Cambridge, UK) was used after the end of treatments agreed to the fabricator's instructions. The number of blue (marker of cellular senescence) and not colored cells were counted in a minimum of three random fields for each sample. Cells were observed in an Olympus BX51 microscopy (Olympus Corporation, Hamburg, Germany).

Morphological Analysis
The morphological changes and characteristics of auditory cells were analyzed for by a light microscope (Olympus CKX41) post-treatment (Olympus Corporation, Hamburg, Germany).

Determination of Population Doubling Rate
To measure the rate of population doublings, 1 × 10 5 cells/well were plated in 6 well plates for 24 h, pre-treated with CPE and/or H 2 O 2 , and then incubated. Cells were counted post-treatments using a neubauer chamber. After the quantification, 1 × 10 5 cells were replated for the next count, every 72 h, for up to 3 passages. The cell populationdoubling rate was calculated by the formula: [log (No. of cells counted)-log (No. of cells seeded)]/log 2, as previously described [41].

Indirect Inmmunofluorescence
After treatments, the cells were fixed and blocked. Then, cells were incubated overnight at 4 • C with primary antibodies: SIRT1 (1/50), SIRT3 (1/50), 8 were administrated to the cells. Subsequently, the nuclei cells were marked with DAPI (Sigma-Aldrich, San Luis, CA, USA). Cells were visualized on an Olympus BX51 microscope (Olympus Corporation, Hamburg, Germany), and the ImageJ program was used to quantify the fluorescence intensity of the micrographs.

Mitochondrial Superoxide Anion Detection
MitoSOX Red fluorochrome (Invitrogen, Waltham, United States) were used to determine the mitochondrial superoxide anions (O 2 •− ). Cell lines were plated at 2 × 10 4 cells per well. The MitoSOX assay was performed 24 h after the treatments, adding 2.5 µM of the MitoSOX-Red reagent to the cells at 33 • C for 45 min in the dark. The fluorescence was visualized with the Olympus BX51 microscope (Olympus Corporation, Hamburg, Germany).
Finally, to corroborate the mitochondrial localization of MitoSOX-Red, cells were then stained with MitoTracker Green staining kit (Abcam, Cambridge, UK). The mitochondria labeled can be observed by fluorescence microscopy. The fluorescence intensities (green, red, and merge) were quantified using the Image J software.

Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction
The miRNeasy Tissue/Cells Advanced Mini Kit (QIAGEN, Hilden, Germany) was used to extract the total RNA derived from the auditory cells following the manufacturer's instructions. Total RNA was quantified spectrophotometrically in NanoDrop One (Ther-moFisher Scientific, Waltham, MA, USA). Afterwards, using the StaRT kit (AnyGenes, Paris, France) and Veriti Thermal Cycler (Applied Biosystems, Waltham, MA, USA), the total RNA (1 µg) was reverse-transcribed. To study gene expression, quantitative real-time reverse transcription-polymerase chain reaction was performed in the 7500 Fast real-time PCR detection system (Applied Biosystems, Waltham, MA, USA). Complementary DNA (cDNA) templates were added to Perfect Master Mix SYBRG (AnyGenes Paris, France). The transcript levels were normalized to β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative expression levels were calculated using the comparative "Ct" method. The specific primers determined in this study were SIRT1, SIRT3, FOXO3, and p53 (AnyGenes, Paris, France). The genes references sequences are the following: NM_011640.3 for p53, NM_019740.2 for FOXO3, NM_019812.2 for SIRT1, and NM_001177804.1 for SIRT3 (AnyGenes, Paris, France).

ELISA
After the end of treatments, cells were lysate in a buffer T-Per (Pierce, Thermo Scientific, United States) with Complete TM Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Cells proteins concentrations were measured using a BCA TM Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) and were adjusted at the same concentration.

Statistical Analysis
Statistical values were calculated by test paired samples T test using the SPSS 19.0 software (IBM, Armonk, NY, USA). Data are shown as mean ± standard deviation (SD). p < 0.05 values indicate statistical significance.

Quantification of Polyphenols in Cocoa Extract
After extraction, a concentration of 574.65 µg/mL of total polyphenols in cocoa powder was obtained. The post-treatment polyphenol content of cell culture supernatants was also extracted ( Table 1). The results obtained show significant differences in the content of polyphenols between those treated and not treated with CEP. Thus, cells treated or not with H 2 O 2 had a polyphenol concentration of 0 µg/mL, while in those treated with CPE, increasing concentrations of polyphenols were obtained depending on the initial dose of CPE (Table 1).  Figure 1 showed that H 2 O 2 (100 µM) decreased cell viability in auditory cells to 41 ± 1.36% in HEI-OC1, to 51 ± 2.22% in OC-k3, and to 48 ± 2.10% in SV-k1 cells compared to the control group (not H 2 O 2 -treated group); this cytotoxic effect was reversed by pretreatment with CPE (5 µg) ( Figure 1B,D,F).

Protective Effect of Cocoa Extract on Cell Viability in Senescent Cells
We also observed changes in the morphology of the three cell lines studied post-H 2 O 2 administration ( Figure 1A,C,E). In general, cells exhibited marked morphological changes characteristics of senescent cells, ranging from longer and thinner cells to ramified and enlarged cells and alterations in the organelles [42]. It should be noted that after CPE treatment to 5 µg/mL the cells recover their usual morphology ( Figure 1A,C,E). CPE was used at 5 µg/mL concentrations in the following experiments, as it did not present cytotoxic effects on auditory cells.

Cocoa inhibits Cellular Senescence in Auditory Cells
We showed that pretreatment with CPE decreased the senescent phenotype as indicated by the data of the SA-β-gal assay ( Figure 2). After treatment with H 2 O 2 , 71% of HEI-OC1, 55% of OC-k3, and 66% of SV-k1 cells were SA-β-gal-positive, which was reduced to 38% in HEI-OC1, 15% in OC-k3, and 25% in SV-k1 cells in the presence of CPE ( Figure 2). These data indicated that CPE protected the auditory cells against H 2 O 2 -induced cellular senescence.

Cocoa Extract Elevated Population Doubling Rate Post H 2 O 2 -Treatments
The next step was to determine the cell-doubling rate to test the cell proliferation capacity. Senescence cells exhibit lower rates, indicating a slower speed of cell growth. Controls group cells (not H 2 O 2 treated) proliferated normally during passages, keeping the doubling rate in all cell types ( Figure 3). However, a decrease in the doubling rate was obtained in cells treated with H 2 O 2 alone (Figure 3). On the contrary, the CPE-treated cells progressively increased their duplication capacity at all passages in senescent cells (Figure 3).

Cocoa Increases SIRT1 and SIRT3 Expression in Auditory Senescent Cells
The immunofluorescence identification of SIRT1 protein (red color) post-treatment shows an objective increase in CPE-treated senescent cells ( Figure 4A-F) compared with Results are means ± SD (n = 3). * p < 0.05 vs of CTR group (0 μg of CPE); # p < 0.05 vs. H2O2 group (0 μg of CPE).
We also observed changes in the morphology of the three cell lines studied post-H2O2 administration ( Figure 1A,C and E). In general, cells exhibited marked morphological changes characteristics of senescent cells, ranging from longer and thinner cells to ramified and enlarged cells and alterations in the organelles [42]. It should be noted that after CPE treatment to 5 μg/mL the cells recover their usual morphology ( Figure 1A,C and E). CPE was used at 5 μg/mL concentrations in the following experiments, as it did not present cytotoxic effects on auditory cells. SIRT3 identification had similar behavior to SIRT1 ( Figure 5A-F). In auditory cells, red fluorescence increased in the senescent group treated with CPE was compared with non-treated senescent cells. The fluorescence was quantified by Image J software, and the differences were statistically significant (p < 0.05) ( Figure 5G). The real-time PCR and ELISA assay show similar data, with a significant increase in SIRT3 expression when the senescent cells were treated with CPE ( Figure 5G,H). CPE at 5 µg/mL shows a statistically significant increase in SIRT3 expression (p < 0.05).

Cocoa inhibits Cellular Senescence in Auditory Cells
We showed that pretreatment with CPE decreased the senescent phenotype as indicated by the data of the SA-β-gal assay (Figure 2). After treatment with H2O2, 71% of HEI-OC1, 55% of OC-k3, and 66% of SV-k1 cells were SA-β-gal-positive, which was reduced to 38% in HEI-OC1, 15% in OC-k3, and 25% in SV-k1 cells in the presence of CPE ( Figure  2). These data indicated that CPE protected the auditory cells against H2O2-induced cellular senescence.  capacity. Senescence cells exhibit lower rates, indicating a slower speed of cell growth. Controls group cells (not H2O2 treated) proliferated normally during passages, keeping the doubling rate in all cell types (Figure 3). However, a decrease in the doubling rate was obtained in cells treated with H2O2 alone (Figure 3). On the contrary, the CPE-treated cells progressively increased their duplication capacity at all passages in senescent cells ( Figure  3). In both cases, SIRT1 and SIRT3, no significant differences were found in the CTR group between cells treated or not with CPE (Figures 4 and 5).

Cocoa Attenuated mtROS Generation in Senescence Auditory Cells
To evaluate the effects of CPE on mitochondrial redox status, a fluorescent probe was used for the detection of the O 2 •− generated in mitochondria, the MitoSOX Red. As shown in Figure 6, H 2 O 2 increased O 2 •− production (H 2 O 2 group) compared with the control group (CTR group), which was markedly reduced by CPE pretreatment of the three auditory cells lines (p < 0.05). Additionally, cells were stained with Mitotracker-Green to check active mitochondria and the total quantity of mitochondria. We found growth of the number of mitochondria in cells treated with H 2 O 2 vs. non-treated cells. H 2 O 2 treatment increased the mass of mitochondria ( Figure 6), but the preventive treatment with CPE decreases it.

Cocoa Extract Induces FOXO3 Activation in Senescence Auditory Cells
To evaluate the effects of CPE treatment on FOXO3 protein expression in senescent cells, we measured its expression by immunofluorescence, real-time PCR, and ELISA assay twenty-four hours after stimulation with H 2 O 2 or H 2 O 2 + CPE. As shown in Figure 7A-F, H 2 O 2 alone did not increase FOXO3 expression. In contrast, the addition of CPE together with H 2 O 2 increased FOXO3 expression significantly ( Figure 7A-F). In concordance with this data, we found a rise of FOXO3 gene expression and protein levels 24 h after treatment with CPE ( Figure 7G,H).

Cocoa Extract Decrease p53 Expression in Senescent Cells
As p53 is a key factor in the induction of senescence [43], we investigated the relationship of p53 to H 2 O 2 -induced cellular senescence in auditory cells and the effect of CPE. In Figure 8A, we showed that after H 2 O 2 treatment, there was increased phosphorylation of p53, which was decreased after CPE treatment. We also found that p53 gene expression was down-regulated with CPE treatment in H 2 O 2 -treated auditory cells ( Figure 8B). These data suggested that CPE protects cells from cellular senescence via modulation of p53 phosphorylation and expression.    lysates post-treatments. Treatments groups: CTR group (0 and 5 μg/mL of CPE for 20 h) and H2O2 group (CPE-pre-treatment at 0 and 5 μg/mL for 20h and H2O2-post-treatment at 100 μM for 1 h). Data are means ± SD of Arbitrary Units (AU) (B,D,F) and fold change (G) in triplicate. * p < 0.05 vs. of CTR group (0 μg of CPE); # p < 0.05 vs. H2O2 group (0 μg of CPE). (n = 4). Reference genes: GAPDH and β-actin.

Cocoa Extract Decrease p53 Expression in Senescent Cells
As p53 is a key factor in the induction of senescence [43], we investigated the relationship of p53 to H2O2-induced cellular senescence in auditory cells and the effect of CPE. In Figure 8A, we showed that after H2O2 treatment, there was increased phosphorylation of p53, which was decreased after CPE treatment. We also found that p53 gene expression was down-regulated with CPE treatment in H2O2-treated auditory cells ( Figure 8B). These data suggested that CPE protects cells from cellular senescence via modulation of p53 phosphorylation and expression.

Cocoa Prevents H 2 O 2 -Induced Oxidative DNA Damage in Auditory Cells
A significant increase in oxidative DNA damage occurs during aging and age-related diseases. We observed the same in our senescent cells [44,45]. Therefore, we studied whether CPE has any effect on the oxidative DNA damage gendered by H 2 O 2 . As shown in Figure 9, the cells were pre-treated with CPE for 20 h before exposure to 100 µM of H 2  diseases. We observed the same in our senescent cells [44,45]. Therefore, we studied whether CPE has any effect on the oxidative DNA damage gendered by H2O2. As shown in Figure 9, the cells were pre-treated with CPE for 20 h before exposure to 100 μM of H2O2 for 1h, showing a significant decrease of 8-oxoguanine (a marker of oxidative DNA damage) compared to cells treated only with H2O2. These data clearly indicate that CPE markedly protects the cells against DNA damage under H2O2.

Discussion
The polyphenols of cocoa contribute to several health benefits, such as reducing the risk of myocardial infarction, stroke, or diabetes [46,47], skin health, including acne [48], and other less explored beneficial properties of cocoa, including antimicrobial and anticancer effects [49][50][51]. However, to date, the role of cocoa in aging and age-related diseases is still unknown. The antioxidant properties of cocoa are mainly attributed to its ability to scavenge ROS, induce antioxidant enzymes, and inhibit pro-oxidant enzymes [52]. This study showed that CPE inhibits cellular senescence (H 2 O 2 -induced) and increases SIRT1, SIRT3, and FOXO3 expression, which played a pivotal role in suppressing the senescent phenotypes as well as inhibiting p53 in auditory cells.
As anticipated, CPE prevented cellular senescence, as shown by attenuation of the SA-β-gal stain and restoration of cell proliferation and morphology. A previous study by Ramirez-Sanchez et al. [53] proposed that (−)-epicatechin (cocoa product) might inhibit senescence in bovine coronary artery endothelial cells.
Our experimental results showed that SIRT1 and SIRT3, which mediate the otoprotective effects of CPE against H 2 O 2 damage, are in agreement with the importance of Sirtuins in aging [54,55]. In particular, SIRT1 is a crucial factor in protection against cellular oxidative stress and DNA damage [56]. Our data showed that CPE increased SIRT1 expression and reduced ROS production and oxidative DNA damage in auditory senescent cells, which avoided H 2 O 2 -induced senescence. Previous research has determined that increased SIRT1 expression inhibited oxidative stress-induced senescence in human endothelial cells [57]. Zhu et al. [58] observed that Luteolin, a sort of flavonoid, in HEI-OC1 cells induces SIRT1 expression, which prevented H 2 O 2 -induced senescence; also, SIRT1 siRNA abolishes the protective effect of Luteolin on H 2 O 2 -induced DNA damage [59,60]. Polyphenols also protect cells against oxidative stress by SIRT1 overexpression [61][62][63][64]. Interestingly, different studies have highlighted that a diet rich in polyphenols has a protective function against cardiovascular, neurodegenerative, metabolic, inflammatory diseases, and cancer by increasing SIRT1 expression [65], and it has been reported that polyphenols, such as quercetin, catechins, and resveratrol, activate SIRT1 in vivo and in vitro studies [65][66][67][68]. Therefore, it would be promising to control SIRT1 activity by cocoa flavanol as a therapeutic application against oxidative stress-induced cellular senescence.
Recent studies suggested that SIRT3 is crucial to regulate mitochondrial ROS (mROS) homeostasis [69]. SIRT3 acts through the deacetylation of a variety of substrates in the mitochondrial matrix where oxidative metabolism takes place, such as the oxidative phosphorylation cycle (OXPHOS) and tricarboxylic acid (TCA). [70,71]. Moreover, it has been reported that SIRT3 functions stimulate deacetylation of enzymes implicated in mtROS homeostasis and activation of FoxO3A-mediated mitochondrial DNA (mtDNA) transcription to reduce oxidative damage [69,72,73]. Consistent with previous studies, our data indicated, for the first time in senescent auditory cells, that CPE through regulation of mtROS homeostasis, mediated by activation of the SIRT3 signaling pathway, reduces oxidative damage. The current study applied MitoSOX Red probe to detect mtROS, and thus differentiate them from ROS generated in the cytoplasm or from sources exogenous to the cell. We determined that CPE treatment decreased excessive mtROS in mitochondria of the auditory senescent cells. In agreement with our results, the study by Zhou et al. [73] showed that resveratrol attenuated tert-butyl hydroperoxide (t-BHP)-induced oxidative injury in HUVEC mediated by activation of the SIRT3 signaling pathway, which reduced mtROS production. Additionally, Zeng et al. [74], using a D-galactose-induced aging rat model that provides a mechanistic of central auditory dysfunction in ARHL, showed that decreased SIRT3 might have less ability to increase superoxide dismutase 2 (SOD2) activity, translating into an elevation in ROS generation. As a result, ROS accumulates associated with aging, inducing mitochondria dysfunction and increasing apoptosis of auditory cells, ultimately resulting in ARHL.
Considering p53 protein is a critical factor that regulates numerous pathways involved in senescence [75], we investigated whether CPE alters the phosphorylation of p53 in the auditory senescent cells. Our study showed that the phosphorylation of p53 was higher in auditory senescent cells compared with non-senescent, which was notably decreased with preventive administration of CPE. These results are in line with previous studies showing that regulation of p53 phosphorylation by Luteolin is at least partially mediated by SIRT1 in HEI-OC1 senescent cells [58] or the protective effect of Salidroside by regulation of redox and p53/p21 expression in senescence [76]. The SIRT1/p53 signaling pathway is closely related to transcriptional alterations of cell cycle inhibitors downstream of various cellular processes, such as cell proliferation and senescence [77].

Conclusions
In conclusion, the results of this study provide relevant insights supporting that cocoa polyphenol extract exerts protective effects against H 2 O 2 -induced cellular senescence. The enhancement of SIRT1, SIRT3, and FOXO3 expression by CPE plays a key role in inhibiting phosphorylation of p53 and suppressing the senescent in the auditory cells. These data present cocoa as a potential natural compound to attenuate the health problems derived from age-related diseases, such as ARHL.