A total of 24 male green fluorescent protein-expressing mice (6-week-old; 20–22 g) were obtained from the Animal Center of Harbin Medical University (Harbin, China). Mice were housed at 22–24°C and 55–60% humidity with a 12 h light/dark cycle with free access to food and water. Mice were sacrificed via decapitation under anesthesia (35 mg/kg pentobarbital; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) following sacrifice, the skin was incised and inguinal fat pad adipose tissue samples were separated and washed with PBS, which were sliced into sections (5–10 mm) and digested with 0.15% type I collagenase for 30 min at room temperature. The supernatant was separated from pellets by centrifugation at 1,500 × g and the pellets were resuspended with phosphate-buffered saline (PBS). The solution was then filtered with a 200 mm mesh into spin down stromal-vascular fraction cell pellets. The retrieved cell fraction was cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (both from Sigma-Aldrich; Merck KGaA) at 37°C with 5% CO₂ for 3 weeks. Animal experimental protocols were approved by the Second Affiliated Hospital of Harbin Medical University Heilongjiang Laboratory Animal Administration Committee (Harbin, China) and experiments were in accordance with the Institutional Guidelines for Animal Experimentation.

A total of 30 male 6-week-old nude mice (20–22 g) were provided by the Harbin Medical University Heilongjiang Experimental Animal Center. Mice were housed and 22–24°C, 55–60% humidity with access to food and water ad libitum. Mice were randomly divided into three groups (n=10 in each group): Control, model and fullerenol groups. The model and fullerenol groups of mice were administered 1,000 mg/kg D-galactose (Sigma-Aldrich; Merck KGaA) by subcutaneous injection. Following 2 weeks of acclimation, the control and model groups were administered PBS by subcutaneous injection. The fullerenol group mice were administered a subcutaneous injection of 10⁶ GFP-expressing ADSCs and 100 mg/kg fullerenol (Sigma-Aldrich; Merck KGaA), as previously described (10). Following 3 weeks of treatment, mice were sacrificed by decapitation under 30 mg/kg of pentobarbital. Skin was immediately harvested washed and stored at −80°C.

ADSCs were divided into two groups: Control and fullerenol. In the control group, ADSCs were induced with β-glycerophosphate and 10⁻⁷ M dexamethasone; in the fullerenol group, ADSCs were induced using 10 mM β-glycerophosphate and 10⁻⁷ M dexamethasone in medium supplemented with 100 µM fullerenol for 3 weeks. All ADSCs were stained using Oil-Red O staining, which identifies fat, bone and cartilage cells. Differentiation of ADSCs was observed using a BX51 light microscope (Olympus, Tokyo, Japan).

Skin tissue samples were fixed in 4% paraformaldehyde at room temperature for 24 h, dehydrated and paraffin-embedded. Then, hematoxylin and eosin was used to stain skin tissue samples, which were extracted from the cell injection site of the mice at the midline of the dorsum. Skin tissue samples were cut into 6-mm sections and assessed under a BX51 light microscope and imaged using a DP71 digital camera (both from Olympus). The total collagen content was analyzed with the aniline blue staining, which was observed using ImageJ software version 3.0 (National Institutes of Health, Bethesda, MD, USA).

A total of 2–3×10³ ADSC were seeded into 96-well plates and cultured with 0.1, 0.3, 1 or 3 µM fullerenol for 24 h at 37°C. Cytotoxicity was assessed using 1% Triton X-100 and a detergent leading to a complete release of the LDH enzyme (Sigma-Aldrich; Merck KGaA). To assess cell viability, 100 µl sterile MTT dye (0.5 mg/ml; Sigma-Aldrich; Merck KGaA) was incubated with the ADSCs for 4 h at 37°C and 150 µl dimethyl sulfoxide for 15 min. Cytotoxicity and cell viability were measured at 490 nm using a microplate ELISA reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Mice were sacrificed and skin tissue was immediately collected and stored at −80°C. Total ribonucleic acid (RNA) was isolated from skin tissue samples using the RNeasy^® Mini kit in accordance with the manufacturer's instructions (Qiagen, Valencia, CA, USA). Using the Reverse Transcription System kit in accordance with the manufacturer's instructions (Promega Corporation, Madison, WI, USA), 1 µg total RNA was reverse transcribed into cDNA. The gene expression of Runx2, ALP and OCN were measured using Real-time PCR with the QuantiTect^® SYBR Green PCR master mix in accordance with the manufacturer's instructions (Qiagen). The primer sequences and product sizes are presented in Table I. GAPDH was used as a reference gene. The PCR amplification was initiated at 94°C for 3 min, followed by 40 cycles of 95°C for 35 sec, 60°C for 45 sec, 72°C for 30 sec and a final step at 72°C for 5 sec. The experiment was repeated three times.

Skin tissue samples were homogenized in an ice-cold lysis buffer and total protein levels were quantified using a BCA protein assay kit (both from Beyotime Institute of Biotechnology, Haimen, China). For each sample, 50 µg total protein was run on 10–12% SDS-PAGE and electro-transferred to nitrocellulose membranes (Thermo Fisher Scientific, Inc.). The membranes were blocked using Tris-buffered saline and Tween-20 (50 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween-20) with 5% bovine serum albumin (Shanghai Sangong Pharmaceutical Co., Ltd., Shanghai, China) for 1 h at room temperature. The membranes were washed with TBST, and incubated with anti-peroxisome proliferator-activated receptor-γ (PPAR-γ, sc-9000; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti- forkhead box protein O1 (FoxO1, ab52857; Abcam, Cambridge, UK) and β-actin (Santa Cruz Biotechnology, Inc.) overnight at 4°C. The membranes were incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (A0208, 1:2,000; Beyotime Institute of Biotechnology) for 1 h at room temperature followed by incubation at 37°C for 1 h with chemiluminescent substrate for horseradish peroxidase antibody and enhancer solution (Thermo Fisher Scientific, Inc.). Protein blank was analyzed by Quantity One software 3.0 (Bio-Rad Laboratories, Inc.). Three replicates were performed.

The results of the quantitative and morphometric analyses were calculated as the mean ± standard error of the mean by SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Statistical analysis was conducted using one-way analysis of variance followed by Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.

ADSCs induced by fullerenol following 3 weeks were observed using a microscope. There was a significant inhibition of adipose differentiation of ADSC in model group, compared with the fullerennol group (P<0.05; Fig. 1). As presented in Fig. 1, fullerenol induced adipose differentiation of ADSC significantly when compared with the control group (P<0.05).

The retention rate of the D-galactose model group was lower than that of control group. ADSCs and 100 mg/kg fullerenol markedly increased the D-galactose-inhibition of retention rate in mice (Fig. 2).

Following fullerenol treatment, the thickness of the dermal portion of skin and collagen ratio was significantly decreased compared with the control group rats (P<0.05; Fig. 3). As presented in Fig. 3, treatment with fullerenol significantly promoted D-galactose-inhibition of the thickness of the dermal portion of skin and collagen ratio mice.

The anti-aging effect of fullerenol on the cytotoxicity (Fig. 4A) and cell viability (Fig. 4B) of D-galactose-induced mice was identified and Fig. 4 indicates the cytotoxicity and cell viability rates for all groups treated with 0.1, 0.3, 1 or 3 µM fullerenol. No significant difference was observed between groups (P>0.05; Fig. 4).

To investigate the anti-aging effect of fullerenol on Runx2 of D-galactose-induced mice, the expression of the Runx2 gene was detected using RT-qPCR. As presented in Fig. 5, Runx2 gene expression in the D-galactose-induced model group was significantly lower than that of control mice (P<0.05). Treatment with fullerenol was effective in significantly decreasing the expression of Runx2 in D-galactose-induced mice (P<0.05; Fig. 5).

In order to confirm that fullerenol has a protective anti-aging effect on skin, ALP gene expression in D-galactose-induced mice was assessed. Gene expression of ALP in D-galactose-induced mice was significantly lower than that of control mice (P<0.05; Fig. 6). Treatment with fullerenol significantly increased the expression of the ALP gene in D-galactose-induced mice (P<0.05; Fig. 6).

To confirm the anti-aging effect of fullerenol on OCN in ADSCs, OCN gene expression was measured using RT-qPCR. Compared with control mice, there was a significant decrease in OCN gene expression of D-galactose-induced model mice (P<0.05; Fig. 7). As presented in Fig. 7, fullerenol treatment significantly inhibited the D-galactose-inhibition of OCN gene expression in D-galactose-induced mice compared with that of D-galactose-induced model group, leading to a significant increase in the expression of OCN (P<0.05; Fig. 7).

The anti-aging effect of fullerenol on PPAR-γ of D-galactose-induced mice was investigated by assessing the protein expression of PPAR-γ using western blot analysis. Western blot analysis demonstrated that PPAR-γ protein expression in D-galactose-induced mice model group was significantly increased compared with that of the control group (P<0.05; Fig. 8). Pretreatment with fullerenol significantly decreased the D-galactose-induced PPAR-γ protein expression in mice, compared with mice in the D-galactose-induced model group (P<0.05; Fig. 8).

To study the potential mechanistic pathways underlying the promotion of fullerenol, FoxO1 protein expression was assessed using western blot analysis. FoxO1 protein expression in D-galactose-induced mice was significantly reduced in the model group, compared with the control group (P<0.05; Fig. 9). However, treatment with fullerenol significantly increased D-galactose-induced inhibition of FoxO1 protein expression in mice (P<0.05; Fig. 9).