Cyanidin-3-O-glucoside was extracted from mulberry. The mulberry was purchased from Guangdong Academy of Agricultural Sciences, and dried in an oven at a low temperature about 60°C. Dried mulberry was soaked in 24 volumes of 60% ethanol containing 0.1% trifluoroacetic acid for 2 h and then homogenized. Ultrasonic Cell Disruptor was used to assist the extraction. The extraction was repeated for three times. All the extracts were mixed and the ethanol was removed at 42°C in a rotary evaporator. The crude extract was then partitioned against petroleum ether to remove the lipid components. Then the aqueous extract was purified on an Amberlite XAD-7HP resin column (Rohm and Haas, Philadelphia, PA, USA), and freeze-dried. The resultant powder was rich anthocyanins. C3G was purified using a middle-pressure liquid chromatography (MPLC, Labomatic, Switzerland) with C¹⁸ adsorption column, the fraction containing C3Gwas then lyophilized and stored under -20°C until use.

HPLC-DAD analysis was performed in a Water-DAD system. C3G sample was injected into a Venusil ASB-C¹⁸ HPLC column (250 × 4.6 mm; i.d., 5 μm; Agela Technologies) with a DAD detector at 280 and 560 nm with the column temperature being maintained at 30°C. The gradient mobile phase composed of solvent A (5% formic acid) and solvent B (formic acid: water: methanol, 5:45:50; v/v/v). Gradient elution was programmed at first, the ratio of A to B was 60:40, 0–3 min, at a flow rate of 0.4 mL/min; then changed to linear gradient elution, B increased from 40 to 60% in 24 min, at a flow rate of 0.4 mL/min; B increased from 60 to 100% in 5 min, at a flow rate of 0.8 mL/min; finally B decreased to 40% and held for additional 8 min, and the whole program lasted for 40 min. From the results of the full wave scanning (Figure 1A), C3G has two absorption peaks at 280 and 517 nm, respectively, just as the characters of anthocyanins published before. Compared to the peak area and the sum of relative peak area (Figure 1B), the purity of C3G was higher than 95%.

The Rat Leydig cells (R2C), obtained from the ATCC (Manassas, VA, USA), were cultured in DMEM-F12 medium (Gibco, Rockville, MD, USA) supplemented with sodium pyruvate, NaHCO₃, 15% horse serum (HS), 2.5% fetal bovine serum (FBS), and a 1% penicillin/streptomycin mixture. The cells were incubated at 37°C with 5% CO₂.

MTT assay was used to evaluate cell viability. The R2C cells were seeded in 96-well plates (Costar, Cambridge, MA, USA) at a density of 4 × 10⁴ cells per well, cultured at 37°C for 24 h. First, the cells were treated with the designated concentrations of 1,3-DCP (0, 1, 2, 4, 6, and 8 mmol/L) for 24 h, then added with 10 μL of MTT (5 mg/mL in PBS). After continued incubation at 37°C for additional 4 h, the medium was removed, 200 μL DMSO was added to each well and mixed for 10 min at room temperature. The absorbance was measured using a microplate reader (Thermo Scientific, Chantilly, VA, USA) at 570 nm. In addition, cell viability assay was also performed in R2C cells disposed with various concentrations of C3G (0, 1, 5, 10, 20, 50, and 100 μmol/L), or 2 mmol/L 1,3-DCP with or without various concentrations of C3G. Data were then analyzed through Graphpad software.

For morphological observation, the R2C cells were cultured in six-well plates at a density of 2 × 10⁵ for 24 h, then treated with 2 mmol/L 1,3-DCP with or without C3G (5, 10, 20, and 40 μmol/L) for 24 h. The protective effect of C3G on 1,3-DCP-induced cell shape damage was observed using a fluorescence microscope (Olympus. ×71).

R2C cells were cultured in six-well plates at a density of 2 × 10⁵ for 24 h. Treated cells were harvested and washed with PBS, and centrifuged at 300 g for 5 min at room temperature. The supernatant was discarded and the cells were resuspended in 200 μl Binding Buffer. Apoptosis was measured using Annexin V-FITC kit (Abcam) following the manufacturer’s instructions and analyzed by flow cytometry (BD FACSaria, USA).

R2C cells were disposed with 2 mmol/L 1,3-DCP with or without various concentrations of C3G (5, 10, 20, and 40 μmol/L) for 4 and 24 h. Progesterone levels in the culture media were measured by a radioimmunoassay kit (Beijing North Institute of Biological Technology, Beijing, China) according to the manufacturer’s instructions.

After the treatment, cells were washed with PBS, and fixed with 1 ml Dichloro-dihydro-fluorescein diacetate (DCFH-DA) solution (10 μmol/L) at 37°C for 30 min. Then the cells were washed three times with PBS and trypsinized to a single cell suspension. Cellular fluorescence was measured using a fluorometric plate reader at an excitation/emission of 488 nm/525 nm.

The JC-1 (Beyotime Biotech, Nantong, China) staining method was used to measure the mitochondrial membrane potential (MMP; ΔΨm) of R2C cells following the manufacturer’s instructions. In brief, the treated cells were cultured in six-well plates and disposed with JC-1 staining solution (5 μg/mL) for 20 min at 37°C, and rinsed twice with JC-1 staining buffer and analyzed on a flow cytometer (BD FACSaria, USA) using 488 nm excitation with 670 nm emission filters.

Total proteins were extracted according to the method described previously by Sun et al. (2014). In brief, cells were lysed in RIPA lysis buffer (Cell Signaling, Beverly, MA, USA) on ice, the supernatants were collected after centrifugation at 12,000 g for 15 min at 4°C. Protein concentration was measured by BCA assay. Total proteins were separated on a 12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes. Membranes were then blocked for 1–2 h at room temperature and then incubated at 4°C overnight using relative primary antibodies: rabbit anti-rat StAR antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:1000, rabbit anti-rat 3β-HSD antibody (Santa Cruz Biotechnology) at 1:500, and rabbit anti-rat GAPDH antibody (Cell Signal Technology, Beverly, MA, USA) at 1:1000. The membranes were then incubated for 2 h at room temperature in diluted horseradish peroxidase-conjugated secondary antibody, and detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) following the manufacturer’s protocol.

Cyclic adenosine monophosphate (cAMP) accumulation in cells was determined using cAMP-Glo assay kit (Promega, Madison, WI, USA). In brief, R2C cells were plated in F12 medium on 96-well dishes at a density of 1 × 10⁴ for 24 h. The cells were disposed with 2 mmol/L 1,3-DCP with or without various concentrations of C3G (5, 10, 20, and 40 μmol/L) for 4 h. After 20 min of treatment with induction buffer [PBS containing the phosphodiesterase inhibitors IBMX (0.5 mM) and Ro 20-1724 (0.1 mM); Sigma–Aldrich, St. Louis, MO, USA], the reaction was stopped by cAMP-Glo Lysis Buffer. Then, the kinase reaction was performed using 40 μL cAMP Detection Solution for 20 min. At the end of the kinase reaction, 80 μL Kinase-Glo Reagent was added and incubated for 10 min, then a plate-reading luminometer (Biotek, Synergy HT, Winooski, VT, USA) was used for measurement.

Values are means of triplicate measurements for all experiments. The significance of difference was assessed using one-way ANOVA. p < 0.05 was considered significant in statistic.

The cell viability decreased with the increase of 1,3-DCP concentration of 0, 1, 2, 4, 6, and 8 mmol/L (Figure 2A) At the dosage of 2 mmol/L, the cell viability decreased to 48.7%. Therefore, 2 mmol/L was chosen as the proper concentration of 1,3-DCP for R2C cell injury model in further experiments. In addition, cytotoxicity of C3G was also determined, C3G showed protective effect on R2C cell viability at the concentrations of 0–40 μM (Figures 2B,C). Then the R2C cells were disposed with 2 mmol/L 1,3-DCP with or without various concentrations of C3G (5, 10, 20, and 40 μM) for 24 h. The results showed that C3G intervention, especially at the concentration of 20 and 40 μM, significantly reversed 1,3-DCP -induced cytotoxicity in R2C cells (Figure 2D).

The morphological changes of the cell surface were observed (Figure 3A). When treated with 2 mmol/L 1,3-DCP, the R2C cells were becoming shrinking and floating, and the filamentous-shaped cells increased. After intervention with various concentration of C3G, especially 20 and 40 μmol/L, the cell morphology changed less compared with the control. The cell morphological changes were consistent with cell viability data.

Cell apoptosis was detected by flow cytometry. 1,3-DCP significantly increased the percentage of apoptotic cells. After intervention with various concentrations of C3G, especially 10–40 μmol/L, the apoptosis ratio decreased significantly (Figures 3B,C). Thereby it was further proved that C3G could protect against 1,3-DCP-induced R2C cell injury.

After treatment with 2 mmol/L 1,3-DCP for 4 h, the progesterone production of R2C cells decreased compared with control, but C3G intervention could not significantly increase the progesterone secretion at the same time. However, after 24 h treatment, the addition of C3G (20–40 μmol/L) significantly increased the progesterone secretion in 1,3-DCP treated cells. The data suggested that C3G protected against 1,3-DCP-induced reduction of progesterone synthesis in a time- and dose-dependent manner (Figure 4).

The results showed that ROS generation increased immediately after 1,3-DCP treatment, and much more increased after 4 h treatment (Figures 5A,B). The addition of C3G, ranging from 10 to 40 μmol/L, reduced the ROS production significantly in 1,3-DCP treated cells immediately or 4 h later. Furthermore, the ROS level was also examined in 1,3-DCP-treated cells with or without 20 μmol/L C3G from 0 to 120 min (Figure 5C). The data showed that 20 μmol/L C3G obviously reduced the ROS level at different time points, while a rising trend of ROS was observed in C3G supplement group from 90 to 120 min.

JC-1 dye was used as an indicator of MMP in R2C cells. The MMP level was detected by flow cytometry, JC-1 aggregate image was represented by red fluorescence, and JC-1 monomer was represented by green fluorescence, mitochondrial depolarization was indicated by green/red fluorescence intensity ratio (or called MMP damage ratio). The results showed that 1,3-DCP increased the MMP damage ratio from 2.84 to 18.33%, and addition of C3G decreased the ratio in a dose-dependent manner (Figure 6).

Cyclic adenosine monophosphate plays an important role in progesterone synthesis, there is also a positive correlation between cellular cAMP level and androgen production. The results showed that the 1,3-DCP treatment alleviated the intracellular cAMP level after 24 h treatment, but not obviously after 4 h. Addition of various concentrations of C3G, especially 20 μmol/L, increased cAMP level after 24 h (Figures 7A,B).

StAR and 3β-HSD are the key proteins in progesterone synthesis. As depicted in Figure 8, 1,3-DCP down-regulated the protein levels of StAR and 3β-HSD, the addition of C3G reversed the trend, especially 20 μmol/L C3G up-regulated both StAR and 3β-HSD protein levels significantly.