Hydroxyurea (HU) and reactive oxygen species (in dimethyl sulfoxide) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) and Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), respectively. Adjudin was provided by Mary M. Wohlford Laboratory (Population Council, New York, USA). Wild-type (WT) C57BL/129 mice and Sirt3-knockout (KO) C57BL/129 mice were acquired from the Jackson Laboratory (Sacramento, CA, USA). Animal procedures for this research were based on the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (Shanghai, China).

MEFs were isolated from embryonic 13.5 (E13.5) WT or Sirt3-KO mice. This study was approved by the Bioethics Committee of School of Biomedical Engineering, Shanghai Jiao Tong University. The embryos, excluding the head and visceral tissues, were washed with phosphate-buffered saline (PBS), minced with scissor and then transferred into 0.1 mM trypsin/1 mM EDTA solution. After incubation at 37°C for 20 min, twice the amount of medium was added and cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and antibiotics (penicillin, 100 U/ml; 100 µg/ml, streptomycin) at 37°C in a humidified incubator with 5% CO₂.

Cell viability was determined using Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Briefly, ~10,000 MEFs were seeded into one well of 96-well plates. Different concentrations of adjudin were tested both with and without hydroxyurea (24 h, 6 mM: 0, 5, 10, 20 and 40 µM). The cells were pre-treaeted with adjudin for 1 h and then incubated with/without hydroxyurea for 24 h. Following treatment, CCK-8 solution was added to each well at the dilution of 1:10. After incubation for ~1.5 h, the absorbance at 450 nm was measured using a microplate reader (Synergy2; BioTek Instruments, Inc., Winooski, VT, USA).

Cellular senescence was determined by SA-β-gal staining with senescence-associated β-galactosidase staining kit (Beyotime Institute of Biotechnology, Haimen, China). Cells were fixed with 4% paraformaldehyde for 15 min and washed with PBS three times. Subsequently, cells were incubated overnight at 37°C in darkness with the working solution containing 0.05 mg/ml 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside (X-gal).

Western blotting was performed as previously described (25). Cells were lysed in radioimmuno-precipitation assay lysis buffer (EMD Millipore, Billerica, MA, USA) containing Complete Protease Inhibitor Cocktail (1:100) and 2 mM phenylmethylsulfonyl fluoride. The protein concentration was quantified by bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Total protein (30 µg) was separated by 10% SDS-PAGE and then transferred to a 0.45 µm nitrocellulose membrane (EMD Millipore). Following the incubation with primary antibodies at 4°C overnight, the membrane was hybridized with horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution; 111-035-003; Jackson Laboratory) at room temperature for 1 h. Protein signals were visualized by enhanced chemiluminescence detection. The primary antibodies used were as follows: p16 (1:1,000 dilution; ab51243) and p21 (1:1,000 dilution; ab109199; both from Abcam, Shanghai, China); Sirt3 (1:1,000 dilution; 5490S; Cell Signaling Technology, Inc., Danvers, MA, USA); Sirt6 (1:1,000 dilution; ab191385; Abcam); MAPK family antibody [extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK); 9926; Cell Signaling Technology, Inc.]; phospho-MAPK family antibody [phospho-ERK, phospho-p38, phospho-JNK; 1:1,000 dilution; Cell Signaling Technology, Inc.)]; manganese superoxide dismutase (SOD2; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); Akt (1:1,000; ab8805), phospho-Akt (1:1,000; ab38449; both from Abcam); forkhead box O3a (Foxo3a; 1:1,000; 12829; Cell Signaling Technology, Inc.); and β-tubulin (1:1,000; ab6046; Abcam).

Total RNA was isolated from MEFs with the use of RNAiso Plus (Takara Biotechnology Co., Ltd., Dalian, China), and first strand cDNA was synthesized from 1 µg of total RNA using a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd.). RT-qPCR was performed on an ABI 7900HT (Thermo Fisher Scientific, Inc.) by using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.) according to the following protocol: 95°C for 30 sec; 40 cycles consisting of 95°C for 5 sec and 60°C for 30 sec, 95°C for 15 sec and 60°C for 1 min; and 95°C for 15 sec. Primers used were as follows: mSirt1 (sense, 5′-TAG TCC TTC CTA CCC CAA TTTCC-3′ and antisense, 5′-TTG GTC CTT AGC CAC TCC TTC-3′); mSirt2 (sense, 5′-GCC TGG GTT CCC AAA AGGAG-3′ and antisense, 5′-GAG CGG AAG TCA GGG ATACC-3′); mSirt3 (sense, 5′-ATC CCG GAC TTC AGA TCCCC-3′ and antisense, 5′-CAA CAT GAA AAA GGG CTT GGG-3′); mSirt4 (sense, 5′-GTG GAA GAA TAA GAA TGA GCGGA-3′ and antisense, 5′-GGC ACA AAT AAC CCC GAGG-3′); mSirt5 (sense, 5′-CTC CGG GCC GAT TCA TTTCC-3′ and antisense, 5′-GCG TTC GCA AAA CAC TTCCG-3′); mSirt6 (sense, 5′-ATG TCG GTG AAT TAT GCA GCA-3′ and antisense, 5′-GCT GGA GGA CTG CCA CATTA-3′); mSirt7 (sense, 5′-AGC ATC ACC CGT TTG CATGA-3′ and antisense, 5′-GGC AGT ACG CTC AGT CACAT-3′); mSOD2 (sense, 5′-CAG ACC TGC CTT ACG ACT ATGG-3′ and antisense, 5′-CTC GGT GGC GTT GAG ATT GTT-3′); m ribosomal protein, large P0 (mRplp0; sense 5′-AGA TTC GGG ATA TGC TGT TGGC-3′ and antisense, 5′-TCG GGT CCT AGA CCA GTG TTC-3′). RT-qPCR was performed out in triplicate and the results are presented as the Cq values. The mean Cq value was calculated, and the ΔCq value was determined as the mean Cq value for the target gene minus the mean Cq value for mRplp0. Relative mRNA expression was calculated using the 2^-ΔΔCq method.

Following treatment, cells were washed with PBS. Dicholorofluorescein diacetate (DCF-DA; Beyotime Institute of Biotechnology) was diluted in FBS-free DMEM to 10 µM and then added to each well. After incubation for 0.5 h, cells were washed with PBS three times, and the fluorescence was detected using a FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA) flow cytometer at an excitation wavelength of 488 nm and an emission wavelength of 535 nm.

Data are presented as the mean ± standard deviation. Multiple comparisons were analyzed by one-way analysis of variance followed by Tukey's post hoc test. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Cells were treated with HU, which inhibits ribonucleotide reductase activity (26,27), to produce an in vitro senescence model. HU-induced senescence has been widely used to mimic cell aging (28-34). MEFs were pretreated with adjudin for 1 h, followed by a 24-h incubation period with 6 mM HU. As presented in Fig. 1A, cell viability following treatment with the indicated concentrations of adjudin was not significantly different from the control group in the absence of HU, while a slight elevation in cell survival was observed when treated with 20 and 40 µM adjudin in the presence of HU.

Subsequently, whether adjudin could affect cellular senescence was determined. The results demonstrated that pretreatment with adjudin (10, 20 and 40 µM) for 1 h reduced the percentage of HU-induced SA-β-gal-positive cells at a dose-dependent manner (Fig. 1B and C). Furthermore, levels of p16 and p21, two cyclin-dependent kinase inhibitors considered as senescence markers (35,36), were detected by western blot. HU increased the expression of p16 and p21, which was notably decreased by pretreatment with 40 µM adjudin compared with HU alone (Fig. 1D).

Previous studies have implicated sirtuins as key mediators of caloric restriction, which is known to inhibit senescence (37). The effect of adjudin on sirtuin expression was examined, and adjudin significantly upregulated mRNA levels of Sirt3 and Sirt6, but there was no change in Sirt6 protein level between different groups (data not shown).

Adjudin has been reported to protect cochlear hair cell via Sirt3-ROS pathway (24) and Sirt3 mediates antioxidant defense and metabolic adaptation that greatly influences mammalian lifespan (37), so we speculate whether adjudin delays senescence through Sirt3-mediated inhibition of ROS production. Indeed, Sirt3 protein level exhibited a 56% decline following HU treatment. In addition, Foxo3a and SOD2, two target proteins of Sirt3 closely associated with ROS, also presented a 73 and 65% decline in senescent cells, relatively. However, pretreatment with 40 µM adjudin significantly counteracted those changes in the presence of HU in MEFs (Fig. 2A). Similar results were also observed at the mRNA level for Sirt3 and SOD2 (Fig. 2B and C).

Subsequently, it was examined whether adjudin could reduce ROS production by using the DCF-DA method. HU stimulation resulted in accumulation of intracellular ROS, which was alleviated by adjudin treatment (Fig. 2D).

To validate the role of Sirt3 in the anti-senescence effects of adjudin, MEFs from WT and Sirt3-KO mice were isolated. Both WT and Sirt3-KO MEFs exhibited strong SA-β-gal activity following exposure to HU. Notably, adjudin co-treatment with HU caused a 30% reduction in SA-β-gal-positive cells in Sirt3-KO MEFs compared to a 60% reduction in SA-β-gal-positive cells in WT MEFs, indicating that Sirt3 may be involved in the anti-aging property of adjudin (Fig. 3A and B).

In agreement with the SA-β-gal staining results, p16 and p21 levels were also induced by HU in WT and Sirt3-KO MEFs. Adjudin treatment suppressed the upregulation of p16 and p21 expression by HU in WT MEFs, while the effect was not as potent in Sirt3-KO MEFs, demonstrating that Sirt3 mediated, at least partially, the anti-senescence effect of adjudin (Fig. 3C).

Sirt3 deficiency prevented adjudin from elevating Foxo3a and SOD2 levels with HU treatment compared with the effects in WT MEFs (Fig. 4A-C). To determine whether Sirt3 influences ROS production, experiments were performed using DCF-DA. Intracellular ROS levels were higher in Sirt3-KO MEFs than in WT MEFs following treatment with HU and adjudin, suggesting that Sirt3 is, at least partially, required for adjudin to perform antioxidant activity (Fig. 4D). This may suggest that other signaling pathways may be involved in this process.

ROS generation activates the MAPK pathway, thus, it was determined whether adjudin affects the MAPK pathway. As presented in Fig. 5A and B, stimulation with HU induced phosphorylation of p38 at the early time point (1 h), which was suppressed by adjudin in WT and Sirt3-KO MEFs, indicating that Sirt3 and p38 play independent roles. However, levels of phosphorylated ERK or JNK were not affected by treatment with adjudin (Fig. 5A). Phosphorylation of Akt was not affected either (Fig. 5A).