6-8-week old male C57BL/6 mice (20–25 g) were purchased from the Medical and Laboratory Animal Center of Chongqing (no. SCXK yu. 2012-0001), and housed in specific pathogen free conditions at a temperature of 20–25°C, a 50–70% humidity, a light/dark cycle of 12/12 h, and with free access to water and food. All procedures complied with the guiding principles for the care and use of animals and were approved by the Committee on Ethics of Animal Experimentation at Chongqing Medical University (Chongqing, China). The 60 mice were divided into four groups at random (control, Rg1, Rg1 + D-gal and D-gal). In the D-gal group, D-gal (120 mg/kg/day) was injected subcutaneously into the mice daily for 42 days. In the D-gal + Rg1 group, Rg1 (20 mg/kg/day) was injected intraperitoneally concomitantly for 28 days, from day 15 of the D-gal injections. In the control group, saline was given in the same volume subcutaneously and intraperitoneally, respectively. In the Rg1 group, saline at the same volume as the D-gal injection was injected subcutaneously for 42 days, and Rg1 (20 mg/kg/day) was injected intraperitoneally for 28 days from day 15 of saline injection. On the 43rd day, the mice were sacrificed by cervical dislocation.

Ginsenoside Rg1 (cat. no. RSZD-121106; purity, 98.3%) was purchased from Xi'an Haoxuan Bio-Tech Co., Ltd. (Xi'an, China) (Fig. 1), dissolved in PBS at a concentration of 20 mg/ml, and sterilized by ultrafiltration. D-gal (purity, >99%) was acquired from Shanghai Bioengineering Co., Ltd. (Shanghai, China). The superoxide dismutase (SOD) kit, methane dicarboxylic aldehyde (MDA) kit, total antioxidant capacity (TAC) kit, primary antibody dilution buffer and goat anti-rabbit (cat. no. A0208) and goat anti-mouse (cat. no. A0216) secondary antibodies were obtained from Beyotime Institute of Biotechnology (Haimen, China). The interleukin (IL)-1β kit (cat. no. EMC001B), IL-6 kit (cat. no. EMC004) and tumor necrosis factor (TNF)-α kit (cat. no. EMC102a) were purchased from Neobioscience Co., Ltd. (Shenzhen, China). The senescence-associated β-galactosidase (SA-β-gal) staining kit was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) Apoptosis Assay kit was purchased from Jiamay Biotech (Beijing, China). Anti-S-phase kinase-associated protein (p19) rabbit polyclonal (cat. no. 10272-2-AP), anti-cellular tumor antigen p53 (p53) and anti-cyclin-dependent kinase inhibitoru 1 (p21) mouse monoclonal antibodies (cat. nos. 10442-1-AP and 60214-1-Ig) were obtained from ProteinTech Group, Inc. (Chicago, IL, USA).

Mouse serum obtained from the angular vein was centrifuged (3,000 × g, 10 min, 4°C). The level of serum testosterone was analyzed using an automatic biochemical analyzer (cat. no. DXI800; Beckman Coulter, Inc., Brea, CA, USA).

Testis index was calculated using the equation testis index=testes weight (mg)/body weight (g), and the testes was stored in a solution of 4% paraformaldehyde (pH 7.4) for 24 h at 4°C, dehydrated in graded (50–100%) alcohol and embedded in paraffin. Sections (5–8 µm) were cut and stained with hematoxylin and eosin (hematoxylin staining for 5 min at room temperature; eosin staining for 3 min at room temperature). The pathological structure of the testes and the differential counts of spermatogenetic cells were measured using a microscope image analysis system (Olympus Corporation, Tokyo, Japan) (9).

The SA-β-gal staining and apoptosis detection were performed according to the manufacturers' protocols. Frozen sections (10-µm; −25°C) of mouse testis tissue were prepared for SA-β-gal staining. Senescent cells appeared blue under light microscopy (magnification ×40). Paraffin-embedded sections (4-µm) were prepared for TUNEL staining. The pre-processing included dewaxing, anhydration and antigen retrieval and blocking using normal goat serum for 30 min to prevent nonspecific staining. Antigen retrieval was performed by immersing sections in citrate buffer and then heating the sections for 15 min at 90°C. Following cooling, the sections were washed three times in PBS and then immersed in 3% hydrogen peroxide for 15 min at room temperature. Following this, the sections were again washed using PBS. The sections were stained with TUNEL Staining Solution (solution A: solution B, 1:9) for 60 min at room temperature. The sections were color-developed with diaminobenzidine and the nuclei were stained with hematoxylin for 5 min at room temperature. At least three different fields of vision per section and per animal were observed and analyzed with Image Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD, USA).

The testes of each mouse were weighed and homogenized with cold saline, and centrifuged (10,000 × g, 4°C, 20 min) to obtain the supernatant for further experiments. The SOD activity, MDA content and TAC were evaluated by chemical colorimetric analysis, according to the manufacturer's protocols. The levels of inflammatory cytokines (IL-1β, IL-6 and TNF-α) in each group were measured by ELISA analysis, according to the manufacturer's protocol.

Tissue homogenate in each group was prepared as described above. The total protein was extracted using radioimmunoprecipitation assay lysis buffer mixed 1% protease inhibitor cocktail (Beyotime Institute of Biotechnology), and the concentration was measured by a bichinchoninic acid assay. Samples containing 40 µg proteins were separated via 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked using skimmed milk powder (5%, dissolved in TBS-Tween 20) for 2 h at room temperature, and the membranes were then incubated overnight at 4°C with anti-p53, anti-p21 and anti-p19 antibodies (1:1,000 diluted in primary antibody dilution buffer). The membranes were then incubated with secondary antibodies (1:800 diluted in TBS-Tween 20) for 90 min at room temperature. The membranes were visualized using an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). β-actin (1:1,000 diluted in antibody dilution buffer) was used as an internal control. Integral optical density was quantified using Image Lab 5.2.1 (Bio Rad Laboratories, Inc., Hercules, CA, USA).

Sperm was collected from the cauda epididymis and vas deferens. The epididymis was dissected in PBS and incubated for 10 min in a constant temperature incubator (37°C, 5% CO₂) to allow the sperm to be released. A total of 10 µl diluted sperm from each group was placed in a counting chamber under a light microscope to determine the sperm density and survival rate (10). A total of 5 µl diluted sperm from each group was mixed with 5 µl eosin on microslides for 30 sec at room temperature, and a microscope (×200) was then used to investigate sperm viability (dead sperm appeared red). A total of 10 µl sperm was used to produce the smears. Sperm smears were dried at room temperature, stained with crystal violet (cat. no. C0121; Beyotime Institute of Biotechnology) for 1–3 min and washed using water for 3–5 min. The sperm morphology of each group was then determined.

All data are presented as mean ± standard deviation. All statistical analyses were performed using one-way analysis of variance followed by the Fisher's least significant difference test with SPSS v20.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Following the injection of D-gal, the mice developed withered and caducous hair, decreased skin elasticity, listlessness, a tendency to cluster together in groups, decreased food intake, and decreased activity and responsiveness. The control group, Rg1 group and Rg1 + D-gal group exhibited none of these signs. There were no complications detected in any of the mice. In previous studies, complications were not identified in other organs (4,5,11,12).

Testosterone is one of the steroid hormones produced primarily by Leydig cells in the testes. Testosterone is responsible for the development of the male reproductive system and is involved in the maintenance of male secondary sexual characteristics. Serum testosterone levels have observed to decline gradually with the aging process (13,14). The results of the present study (Fig. 2) demonstrated that the level of serum testosterone was significantly decreased in the D-gal-induced group. Following treatment with Rg1, the level of serum testosterone significantly increased compared with D-gal-induced group. This result demonstrated that Rg1 was able to significantly improve the ability of Leydig cells to secrete testosterone in D-gal-induced aging mice.

A previous study (9) reported that spermatogenetic malfunction may occur during the aging process. Classification and counting of spermatogenetic cells is of diagnostic importance for spermatogenetic malfunction. Tissue examination (Fig. 3A) demonstrated the integrity of the spermatogenetic epithelium and the regular arrangement of spermatogenetic cells in the control group. Compared with the control, structural abnormalities of the seminiferous tubule and a disordered arrangement of spermatogenetic was observed in the D-gal group. In the Rg1 + D-gal group, the pathological alteration of abnormal structure of the seminiferous tubule was not noted. No significant differences were observed in morphology or organ index among the four groups (D-Gal group, 0.0060±0.00081; Rg1 + D-Gal group, 0.0065±0.0012; Rg1 group, 0.0063±0.00037; and control group, 0.0064±0.00048). Subsequently, the differential counts of spermatogenetic cells were measured. The results (Fig. 3B and C) revealed that spermatogenetic cell/Sertoli cell number was decreased in the D-gal group, and increased following treatment with Rg1.

SA-β-gal is a widely-used biomarker for aging cells. By adjusting to pH 6, blue molecules (Fig. 4A) may be visualized in the cytoplasm of the aging cell (2). The intensity of SA-β-gal staining was evaluated via the relative optical density (ROD) value of SA-β-gal-positive cells (Fig. 4A and B). The results demonstrated that the ROD value of the SA-β-gal staining was increased in the D-gal-induced group. By contrast, the ROD value of the SA-β-gal staining was decreased following treatment with Rg1 in the Rg1 + D-gal group. These results suggested that Rg1 may protect the testes against senescence.

Previous studies have indicated that the degeneration of spermatogenesis is caused by apoptosis (15,16). TUNEL staining is a commonly used method, which has been proven to be useful for detecting DNA fragmentation resulting from apoptotic signaling cascades. TUNEL staining relies on the DNA ends that can be identified by TdT and the addition of dUTPs, which is a secondarily labeled marker catalyzed by an enzyme. In the present study, TUNEL was used to detect the level of spermatogenetic cellular apoptosis; positive cells displayed brown-yellow granules in the nucleus (Fig. 5A). Compared with the control group, positive cell number and the apoptotic index were increased in the D-gal group (P<0.05). The apoptotic index and the level of spermatocyte apoptosis were decreased following treatment with Rg1 (Fig. 5B).

Oxidative stress is one of the causes of senescence. A previous study reported that the oxidation-reduction system may be damaged by aging (17). SOD is an enzyme that removes the free radicals generated by oxidative stress in aging. Thus, SOD activity is an important target for estimating antioxidant capacity. MDA may reflect the level of oxidative injury in the body, and level of MDA is an important target for estimating the level of oxidative damage. In the present study, the results demonstrated that SOD activity and TAC were decreased significantly, and MDA content was elevated significantly, in the D-gal group compared with the control. Conversely, in the Rg1 + D-gal group, SOD activity and TAC were significantly increased compared with the D-gal group. In addition, MDA content was decreased in the Rg1 + D-gal group (Table I). These results demonstrated that another factor of the protective effect of Rg1 on the testes may be an increase in the activity of SOD and a decreased in the oxidation product MDA.

Chronic inflammation is increased and the secretion of inflammatory cytokines is upregulated during the aging process. It has been reported that inflammatory cytokines, including TNF-α, IL-1β and IL-6, are increased during natural aging (18). The results of the present study demonstrated that the levels of TNF-α, IL-1β and IL-6 were significantly increased in the testes of the D-gal group. However, compared with the D-gal group, the levels of these three inflammatory cytokines were significantly decreased in the Rg1 + D-gal group (Fig. 6). These results indicated that the effect of D-gal on the levels of inflammatory cytokines may affect the aging of the testes, and that Rg1 was able to reduce the levels of these inflammatory cytokines.

The p19/p53/p21 pathway serves an important role in aging. Following oxidative damage, the p19/p53/p21 pathway is activated and the expression of the proteins is upregulated (8). According to the western blot analysis in the present study, the levels of p19, p21 and p53 in the D-gal group were significantly increased compared with the control group. Following treatment with Rg1, the levels of these proteins were decreased significantly (Fig. 7). The results of the present study demonstrated that the p19/p53/p21 pathway was downregulated following treatment with Rg1.

One of the most common ways of tracking male fertility is semen analysis (10). Therefore, male reproductive status and spermatogenesis monitoring are of importance (Fig. 8). Sperm parameters, including sperm density, normal sperm morphology percentage and activity rate; are meaningful to monitor male reproductive function. In the present study, the sperm density (Fig. 8D), survival rate (Fig. 8E), normal sperm morphology percentage (Fig. 8F) and activity rate (Fig. 8G) were significantly decreased in the aging model. Following treatment with Rg1, sperm parameters improved significantly (Fig. 8). These results suggested that the reproductive function of aging males may be improved by treatment with Rg1.