A Comparative Study Of Anti-aging Properties And Mechanism: Resveratrol And Caloric Restriction Ⅱ

Effect of resveratrol and CR on the mRNA expressions of SIRT1, p53, and Foxo3a in tissues of aging rats 

Oxidative stress conditions often induce SIRT1 expression and activity. SIRT1 modulates the functions of key survival factors including p53 and Foxo3a [9]. To determine the effect of resveratrol and CR on the mRNA levels of SIRT1, p53, and Foxo3a in D-Gal-induced aging rats, quantitative RT-PCR assays were performed. Compared to the corresponding negative control group, the levels of SIRT1 and Foxo3a mRNA expression were significantly decreased, but p53 level was significantly increased in D-Gal group (P < 0.01; Figure 9A and 9B). Furthermore, simultaneous treatment resveratrol or CR with D-gal in rats caused an increase in the levels of SIRT1 and Foxo3a mRNA expressions but decreased p53 levels as compared with the model control group (P < 0.05). However, compared with CR treatment, the level of SIRT1 mRNA expression was significantly increased in livers of high dose of resveratrol + D-gal group, the level of p53 mRNA expression was significantly decreased in livers and brains of high dose of resveratrol + D-gal group (P < 0.05).

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Effect of resveratrol and CR on the expressions of main SIRT1-associated protein in brain tissues of aging rats 

FOXO3a and p53, which are regulated by SIRT1, are the downstream molecules of SIRT1. Simultaneously, SIRT1 activity is regulated by its upstream molecules, for instance, DBC1 (also known as KIAA1967), AROS (also known as RPS19BP1) and the tumor suppressor HuR (also known as ELAVL1) [9, 10]. To determine the effect of resveratrol and CR on the protein levels of p53, FOXO3a, HuR, AROS, and DBC1 in D-Gal-induced aging rats, western blot assays were performed. Compared to the corresponding negative control group, the levels of FOXO3a, AROS and HuR protein expressions were significantly decreased, but p53 and DBC1 levels were significantly increased in D-gal group (P < 0.01; Figure 10). Furthermore, simultaneous treatment resveratrol or CR with D-gal in rats caused an increase in protein expressions of FOXO3a, AROS and HuR but decreased p53 and DBC1 levels as compared with the model control group (P < 0.05). However, compared with CR treatment, the level of HuR protein expression was significantly increased but DBC1 level was significantly decreased in brains of a high dose of resveratrol + D-gal group (P < 0.05). There were no differences between the resveratrol + D-gal group and CR + D-gal group in levels of p53, FOXO3a and AROS protein expression (P > 0.05).

FOXO3a and p53 expressions in brain and liver tissues

The expression status of FOXO3a and p53 were determined in brain and liver tissues by immunohistochemistry. Immunohistochemical analysis of FOXO3a and p53 revealed that FOXO3a expression in brain and liver tissues was significantly decreased, and p53 expression was significantly increased in D-Gal group, compared with the negative control group (P < 0.05, Figure 11). Moreover, simultaneous treatment resveratrol or CR with D-gal in rats caused an increase in FOXO3a expression but decreased p53 expression as compared with the model control group (P < 0.05). However, there were no differences between the resveratrol + D-gal group and CR + D-gal group in p53 and FOXO3a expressions (P > 0.05).

DISCUSSION

The basic chemical process underlying aging was first advanced by the free radical theory of aging [11]，and oxidative stress is believed to be a primary factor in the normal process of aging. Free radical initiator AAPH was used to induce oxidative stress and establish a model of oxidative stress-induced cellular senescence in human IMR-90 cells [12, 13]. The advantages of this method are that the AAPH decomposes thermally to generate radicals without biotransformations or enzymes, and the rate of radical generation could be easily controlled by adjusting the concentration of the initiator [12]. AAPH was found to inhibit the proliferation of human IMR- 90 cells in a concentration and time-dependent manner. Results showed that 2 d incubation with AAPH (1 mM) significantly increased the percentage of SA β-Gal positive ratio, the population of apoptotic cells, reduced the mRNA expression of SIRT1, and induced an enlarged and flattened morphology. These indicated that oxidative stress resulted by AAPH led IMR-90 cells to senescence. Based on this cellular senescence model, 10 μM resveratrol treatment more efficiently inhibited AAPH-induced senescence and apoptosis than CR treatment. 

D-gal animal model is an internationally recognized aging animal model and has been widely used in the field of anti-aging medicines. D-gal is a physiological nutrient that is normally metabolized by D-galactokinase and galactose-1-phosphate in animals. However, an over-supply of D-gal, which is not metabolized by above enzymes but accumulates in the cells, leads to oxidative stress and cellular damage [14]. Thus, long-term administration of D-gal induces changes that resemble natural aging in animals, such as a shortened lifespan, cognitive dysfunction, neurodegeneration, oxidative stress, decreased immune responses, and advanced glycation endproduct (AGE) formation [15]. The present study indicated the successful establishment of the mimetic aging model, evidenced by remarkable learning and memory impairment, MDA production, lipofuscin accumulation, decline in T-AOC and SOD, and down-regulation of telomerase activity in brain. Meanwhile, resveratrol or CR treatments protected the D-gal-induced rats against oxidative stress by elevating the activity of antioxidant enzymes, decreasing the levels of lipid peroxidation products, and keeping the balance of oxidative and antioxidative systems. In behavioral and telomerase activity tests, the treatment of resveratrol or CR could reverse D-gal-induced cognitive impairment and telomerase activity. Moreover, aging model rats treated with high doses of resveratrol showed relatively more significant effect on the behavior than those treated with CR.

Figure 11: Immunohistochemical analysis of p53 (A, B) and FOXO3a (C, D) expressions in liver (A, C) and brain (B, D) tissues of aging rats. The magnification was 20×. NG, negative control group; MG, model control group; RESL, low dose of resveratrol group; RESH, high dose of resveratrol group; CR, caloric restriction group

Studies have demonstrated that CR, a reduction of 10–40% in intake of a nutritious diet, could retard aging and increase lifespan, thus one commonly used level of CR (40% reduction in food intake) was applied in CR group rats in the present study. Nevertheless, some investigations revealed that no beneficial effect of CR on longevity in naturally aged primates [16]. Similarly, although resveratrol treatment was shown to induce CR-like effects on energy metabolism and metabolic profile in obese humans [17], it did not improve metabolic function in non-obese women with normal glucose tolerance [18]. The reason for the differences between these studies is not clear but it might be that CR and resveratrol work to restore homeostasis in metabolically compromised individuals, and less so in healthy individuals, a possibility consistent with known functions of SIRT1 in animal studies [19]. Therefore, D-gal-induced aging rat models were applied for comparing the anti-aging activities of resveratrol or CR , rather than the naturally aged rat model.

SIRT1, an (NAD+)-dependent deacetylase, has gained much attention because of its multiple roles in life span extension, stress resistance and apoptosis reduction [20, 21]. Accumulating studies have strongly implied that SIRT1 was a key target of resveratrol and CR [22]. There are vast numbers of downstream molecules of SIRT1, including p53, Foxo1, Foxo3, Foxo4 and E2F1, which are regulated by SIRT1. Simultaneously, SIRT1 activity is regulated by its upstream molecules, for instance, p53, HIC1, E2F1, DBC1, HuR and AROS. Acute nutrient withdrawal activates a transcriptional program at the SIRT1 promoter that is directed by the transcription factors Foxo3a and p53. As p53 represses SIRT1 gene expression, its removal by Foxo3a activates SIRT1 transcription [23]. DBC1 and AROS were recently described as direct negative and positive regulators of SIRT1 activity, respectively, HuR shows the effect of reducing levels of SIRT1 expression in aged senescent cells [9, 10]. 

CR retards aging and extends the median and maximal life span in a variety of species, including rats, mice, fish, flies, worms, and yeast. However, such rigorous dietary programs in free-living persons has raised ethical and methodologic issues [24]. At present, increasing the health span might be more important than simply prolonging the lifespan. Recent investigations suggest that resveratrol is one of the most potent instigators of SIR2 activity among all plant polyphenols [25], and it influences longevity through similar mechanisms as seen in calorie restriction. These results showed that CR and resveratrol had similar activities of recovering SIRT1 mRNA expression levels, increasing the protein expressions of FOXO3a, AROS and HuR and decreasing p53 and DBC1 levels. Meanwhile, multiple lines of compelling evidence indicate the beneficial effects of resveratrol on the neurological, hepatic, and cardiovascular systems.

MATERIALS AND METHODS

Cell culture, stress and treatments

The human diploid fibroblast strain IMR-90 was obtained from ATCC. Cells were grown in minimum essential medium (MEM) (Gibco, UK) supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37°C in 5% CO2 . After confluence had been reached, the cells were seeded into 6-well culture plates. One day later, cells were treated for 48 h with 1 mM 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) (Sigma, USA) diluted in MEM + 10% FBS. A 48 h incubation with free radical initiator AAPH establishes a model of oxidative stress-induced cellular senescence [12, 13]. Controls cells were incubated in culture medium alone. After AAPH treatment, IMR-90 were washed with cold phosphate buffer saline (PBS) pH 7.4 and incubated with fresh culture medium or culture medium containing resveratrol (resveratrol group) or MEM supplemented with 3% FBS (CR group) for an additional 48 h before harvest.

SA β-gal staining activity 

The appearance of biomarkers of senescence was checked (decrease of the proliferative potential and increase in SA β-gal-staining) [26] from the day after treatment. Then the cells were stained following the manufacturer’s instruction of the SA β-gal Staining Kit (CST, USA). After staining, at least 300 cells in several fields were examined and SA β-gal positive cells were counted. These experiments were repeated three times, and the results were presented as the mean values with standard deviations (SD). To avoid nonspecific staining possibly due to cell confluency, SA β-gal histochemical staining was performed with subconfluent cells.

Scanning electron microscopy analysis

Cells were grown on coverslips in 6-well culture plates as described above. After resveratrol and CR were treated, coverslips were removed from the plates, then the cells fixed in 2.5% phosphate-buffered glutaraldehyde and post-fixed in 1% buffered osmium tetroxide. Fixed specimens were immersed in t-butyl alcohol after dehydration through a graded series of ethanol. The specimens were freeze-dried from t-butyl alcohol, then evaporative-coated with gold using auto fine coater (JFC-1600, JEOC, Japan), and examined using a scanning electron microscope (JEOL, JSM-6510LV, Japan). 

Apoptotic analysis

After resveratrol or CR incubation, all cells were harvested with trypsin and washed twice with PBS, followed by resuspended in 400 μL Annexin V binding buffer. Then the cells were stained following the manufacturer’s instruction of the Annexin V-FITC cell apoptosis detection kit (BestBio, China). A FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA) was used to detecte fluorescence, and the percentage of apoptotic cells was calculated by the internal software system of the FACSCalibur. Approximately 104 cells were analyzed for each trail.

Animal procedures

Male albino Wistar rats, weighing approximately 200 ± 10 g, were obtained from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology (SCXK 2010-0009). The animals were acclimated for 2 weeks before dosing in the Experimental Animal Center of the Hubei University of Chinese Medicine, during which time they had free access to food and water ad libitum. After acclimation, 50 rats were selected and divided into five groups of ten each. Animals were maintained per the national guidelines and protocols approved by the Institutional Animal Ethical Committee (IAEC).

Group I (Negative control group) rats received the vehicle alone (1 mL/kg bodyweight of 0.9% of saline) for 6 weeks. Group II (Model control group) rats received D-gal (200 mg/kg body weight, Sigma Chemical, St. Louis, MO) for 6 weeks. Group III (Low dose of resveratrol group) rats received D-gal and resveratrol (50 mg/kg body weight) dissolved in 5% dimethyl sulfoxide (DMSO) for 6 weeks. Group IV (High dose of resveratrol group) rats received D-gal and resveratrol (100 mg/kg body weight). Group V (CR group) rats received D-gal and CR (fed a diet that was 40% lower in calories than the ad libitum-fed rats [27]) for 6 weeks. D-gal were given intraperitoneally, whereas resveratrol were given orally for the entire duration of the experiment. The rats were sacrificed on the last day of treatment, then the blood, sera and organs were immediately collected for bioassay or stored at −80°C for later use.

Behavioral tests 

The Morris water maze test was designed to assess the rats’ capacity of spatial learning and memory after 6 weeks post-injury [28]. The Morris water maze test consisted of 4-day learning and memory training and a probe trial on day 5. Animals were trained in a circular pool (155 cm in diameter) with visual cues. An escape platform (9.0 cm in diameter) was submerged 1.0 cm below the surface of the pool water, which was maintained at 25 ± 2°C. The location of the platform remained in the center of the northwest quadrant throughout the 4-day training period. On each day, rats were trained for one morning block and one afternoon block. The rat swam freely until it found the platform within 120 s. If failed, it was placed on the platform for 10 s. The escape latency (finding the submerged escape platform) and the swim speed were recorded. The probe trial was made by removing the platform and allowing each rat to swim freely for 120 s inside the pool. The number of platform crossings in the trained quadrant (where the platform was removed) were recorded with a computerized video system. The visible platform version of water maze was not performed after a significant difference in swim speed was observed between rats treated with D-gal and vehicle.

Detection of oxidative stress-associated biological indicators 

The levels of MDA, SOD, and T-AOC in blood and tissues were determined photometrically in accordance with the manufacturer’s protocol by using commercially available enzymatic assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing City, P. R. China). All the experiments described in this section were performed in triplicate to obtain means and SD.

Lipofuscin level was determined by the Sohal method [29]. In brief, we weighed 200 mg cerebral cortex, added 2 ml chloroform-methanol (2:1) extract, homogenized and filtered, then washed the residue with the extract, combined the filtrates, added the extract to 5 ml and measured the fluorescence intensity on a fluorescence spectrometer. The emission wavelength was 435 nm and the excitation wavelength was 365 nm. The spectrofluorometer was standardized to give a deflection of 50 at the above wavelengths with a 1 μg/ml solution of quinine bisulfate in 0.1 M H2 SO4 . The results were expressed as relative fluorescent units/ml chloroform/g wet tissue weight.

Detection of TE activity 

For telomerase extraction approximately 30 mg of tissue was washed twice in ice-cold PBS, and finally homogenized in about 150 ml of PBS. TE activity was measured using a TE ELISA kits according to the manufacturer’s instructions (Nanjing Sen Beijia Biological Technology Co., Ltd., Nanjing, China). The limits of sensitivity of the assays were 0.8 IU/L for TE.

RT-PCR analysis 

The mRNA expression levels of SIRT1, Foxo3 and p53 in liver and brain tissues were analyzed using RT PCR analysis. The total RNA was extracted using Trizol, and their quantity and purity were assessed by a ultra microspectrophotometer (Thermo Fisher Scientific, USA) based on the absorbance measurement at 260 and 280 nm. After quantification, cDNA was synthesized using a FastQuant RT kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. The levels of SIRT1, Foxo3, p53 mRNA expression were measured by RT-PCR using TIANGEN SuperReal PreMix Plus (Tiangen Biotech, Beijing, China) with specific primers (Table 2). Quantitative real-time PCR was performed with a LightCycler 480°C PCR system (Roche, Basel, Switzerland) using 20 μL as the total volume of each reaction. PCR amplification was initiated by 15 min of denaturation at 95°C, and then followed by 40 cycles of 95°C for 10 s, 59–63°C (annealing temperature) for 20 s and 72°C for 30 s, and a final incubation at 72°C for 5 min. The obtained cycle threshold number of each gene (Ct value) was normalized into fold of relative changes according to the equation of 2-∆∆CT method [30]. 

Table 2: Primer list

Western blot analysis 

The protein expression levels of p53, FOXO3a, HuR, RPS19BP1, and DBC1 in brain tissues were analyzed using western blotting analysis. The total protein of brain tissues was extracted using RIPA Lysis Buffer (Beyotime, China) according to the manufacturer’s instructions. Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime, China). 5 mg of proteins were separated using 12% SDS-polyacrylamide gels and transferred onto a PVDF membrane (0.45 μm, Millipore, USA). The blots were blocked with 5% nonfat milk in TBS, then incubated overnight at 4°C with the appropriate dilution of primary antibodies: anti-p53 (SC- 6243, Santa Cruz Biotechnology), anti-FOXO3a (#12829, Cell Signaling Technology), anti-HuR (#12582, Cell Signaling Technology), anti-AROS (Ab201091, abcam), anti-DBC1 (#5857, Cell Signaling Technology), anti-β- action (AC004, ABclonal Technology). After washing the membranes to remove excess primary antibodies, the membranes were incubated for 1 h at room temperature with the appropriate secondary antibodies at a dilution of 1:2000-1:5000 (AC004 or AS003, ABclonal Technology). The membranes were washed 3 times and then visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific). β-action was used as an internal control. 

Immunohistochemistry 

The tissue samples were fixed in 10% neutral-buffered formalin within 1 h after surgical removal and paraffin-embedded using standard procedures. Then the fixed paraffin-embedded specimens were cut into 5-μm-thick sections, which were deparaffinized in xylene, rehydrated in ethanol, and microwave treated for antigen retrieval. The appropriately diluted primary antibody, anti-p53 (SC-6243, Santa Cruz Biotechnology) and anti-FOXO3a (#12829, Cell Signaling Technology), were incubated for 60 min at room temperature in a humidity chamber. Slides were then rinsed in PBS and subsequently incubated with the secondary antibody against anti-rabbit antibody and the visualization of HRP (concentration 1:300, #074-1506, KPL). Then the slides were washed and the sections were developed in the enzyme-substrate diaminobenzidine (DAB, Sigma) solution. Images of immunohistochemically stained sections were captured by the Olympus digital microscope (IX-73P1F, Olympus, Japan).

Statistical analysis 

Results were expressed as means ± SD for at least three independent experiments and were analyzed using the SPSS 18.0 software. Group differences in the escape latency and swim speed in the Morris water maze training task were analyzed using two-way analysis of variance (ANOVA) with repeated measures, the factors being treatment and training day. The other data were analyzed with one-way ANOVA followed by a post-hoc Tukey test. The value of P less than 0.05 were considered significant. 

CONCLUSIONS 

Resveratrol and CR exhibited similar anti-aging activities both in vitro and in vivo, evidenced by their ability to inhibit AAPH-induced senescence and apoptosis, and restore the age-related cognitive impairment caused by D-gal administration. Their anti-aging mechanisms included up-regulating TE activity, decreasing oxidative damage, and regulating SIRT1 pathway. Overall, 10 μM resveratrol in vitro and high dose of resveratrol in vivo exhibited relatively stronger activities of anti-aging and stimulating SIRT1 levels. These results implicated the potential of resveratrol as a CR mimetic.

Abbreviations 

AAPH, 2,2′-azobis (2-amidinopropane) dihydrochloride; AMPK, adenosine monophosphate-activated protein kinase; ANOVA, analysis of variance; AROS, active regulator of SIRT1; CR, caloric restriction; DBC1, deleted in breast cancer 1; D-gal, D-galactose; FBS, fetal bovine serum; Foxo3a, forkhead box 3a; HuR, Hu antigen R; MEM, minimum essential medium; PBS, phosphate buffer saline; PGC-1α, peroxisome proliferator-activated receptor G coactivator-1α; RES, resveratrol; SA β-gal, senescence-associated β-galactosidase; SD, standard deviations; SIRT1, silencing information regulator; TE, telomerase.

Author contributions

Conception and design of the research: Gang Chen; acquisition, analysis and interpretation of data: Juan Li, XiaChun Zhang, Yi-Mei Liu; obtaining financing and critical revision of the manuscript for intellectual content: Gang Chen, Ke-Li Chen; writing of the manuscript: Juan Li. All authors have read and approved the publication of this manuscript. ACKNOWLEDGMENTS AND FUNDING 

This work was funded by China Postdoctoral Science Foundation (2016M601237), the Natural Science Foundation project (81373704, 30873292).

CONFLICTS OF INTEREST 

No conflflict of interest from all participating authors. 

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