Grifolic acid was obtained from R&D Systems Inc. (Minneapolis, MN, USA). Rabbit anti-FFAR4 polyclonal antibody (cat. no. SAB4501490) and cellular adenosine 5′-triphosphate (ATP) assay kit were purchased from Sigma-Aldrich (Merck KGaA; Darmstadt, Germany). Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining kits were purchased from BD Pharmingen (BD Biosciences, San Jose, CA, USA). Mouse FFAR4 Silencer Select siRNA (cat. no. S200889), Silencer Negative Control siRNA (cat. no. AM4613), Lipofectamine^® RNAiMAX reagent, Opti-MEM medium, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), JC-1 dye and rabbit anti-b-actin polyclonal antibody (cat. no. PA1-183) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

Mouse RAW264.7 cells were purchased from ATCC (Manassas, VA, USA) and cultured in a humidified incubator with 5% CO₂ at 37°C in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. The medium was changed every 3 days, and RAW264.7 cells were subcultured at 80% confluency and seeded to plates or dishes for each experiment.

RAW264.7 cells were cultured in 96-well plates and treated with grifolic acid (1.25–20 µmol/l for 2–24 h at 37°C) at 90% confluency in serum-free medium. The control cells were treated with the solvent without grifolic acid. Subsequently, MTT was added into medium (0.5 mg/ml) and cells were incubated for 4 h at 37°C. The medium was discarded and 100 µl isopropanol with 0.01 M HCl was added to each well and plates were agitated to fully dissolve MTT crystals. The absorbance of each well was measured at 560 nm with an ELISA microplate reader (Thermo Fisher Scientific, Inc.). The background absorbance at 690 nm was also measured and subtracted from the values measured at 560 nm. The experiments were repeated three times.

The Annexin V-FITC/PI Apoptosis Detection kit was used to measure cell death. Following incubation with 0 µmol/l (control), 10 or 20 µmol/l grifolic acid, RAW264.7 cells were detached from dishes by 0.05% trypsin/EDTA. Cells were washed with cold PBS and resuspended in the binding buffer supplied in the kit at 1×10⁶ cells/ml. Annexin V-FITC and PI were added to cell suspension at a dilution of 1:20 and incubated for 15 min at room temperature in the dark. Subsequently, cells were diluted in binding buffer and analyzed by flow cytometry with the CFlow Plus software (BD Biosciences), and the FITC⁺/PI⁺ double-positive cells were counted. The experiments were repeated three times.

RAW264.7 cells (1×10⁷ cells per sample) were treated with grifolic acid (10 or 20 µmol/l for 2 h). Subsequently, cells were lysed and intracellular ATP levels were measured using ATP assay kits (Sigma-Aldrich; Merck KGaA). Briefly, cell lysates from different groups were obtained by incubating with detergent and agitation at ~12 Hz for 5 min at room temperature. The constituted substrate solutions given in the kits were added to each sample and incubated for 5 min in the dark. The luminescence of each sample was measured with a luminescence plate reader (Thermo Fisher Scientific, Inc.). ATP levels of each sample were calculated according to the constructed standard curve. The total protein of each sample was extracted using the radioimmunoprecipitation lysis buffer from the Beyotime Institute of Biotechnology (Beijing, China) and quantified by bicinchoninic acid (BCA) assay. The cellular ATP content was corrected to the total protein levels. The experiments were repeated three times.

RAW264.7 cells (5×10⁵ cells/well) were plated into 35 mm dishes and stained with JC-1 (5 µg/ml) in serum-free medium for 15 min at 37°C. Subsequently, cells were washed with serum-free medium, and the fluorescence was recorded prior to (0 min) and following grifolic acid treatment every 10 min using a Leica TCS SP8 confocal microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA) in live cell station at 37°C. For cyclosporine A treatment, the cells were incubated with 20 µmol/l cyclosporine A for 10 min prior to stimulation with grifolic acid. The red fluorescence (excitation 560 nm; emission 600 nm) and green fluorescence (excitation 488 nm; emission 535 nm) were measured synchronously, and the ratio of fluorescence intensity for each cell was analyzed by LAS LITE version 3.3 (Leica Microsystems, Inc., Buffalo Grove, IL, USA). The experiments were repeated three times.

RAW264.7 cells were grown to 80% confluency at the time of transfection. The mouse FFAR4 siRNA (100 pmol, Thermo Fisher Scientific, Inc.) or negative control siRNA (100 pmol; Thermo Fisher Scientific, Inc. was diluted in 50 µl Opti-MEM medium (Thermo Fisher Scientific, Inc.), and Lipofectamine RNAiMAX (1 µl; Thermo Fisher Scientific, Inc.) was diluted in 50 µl Opti-MEM, and they were incubated for 15 min at room temperature. Subsequently, the siRNA and Lipofectamine RNAiMAX solutions were mixed and incubated for 15 min at room temperature to allow complexes to form. The complexes were added to each well at a dilution of 1:4. Cells were incubated at 37°C in a humidified 5% CO₂ incubator for 24 h to induce gene knockdown. Following 24 h incubation with Opti-MEM medium the cells were used for experiments. The experiments were repeated three times.

Total protein was extracted from RAW264.7 cells (1×10⁷ cells per sample) using the ReadyPrep Protein Extraction kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and quantified by bicinchoninic acid assay. Proteins (40 µg per sample) were separated using a 12% SDS-PAGE and were transferred to nitrocellulose membranes using a Trans-Blot SD semi-dry electrophoresis transfer cell (Bio-Rad Laboratories, Inc.). Membranes were subsequently blocked at room temperature with 5% skimmed milk for 2 h at room temperature. Then membranes were incubated with a rabbit-anti FFAR4 antibody (1:1,000) or rabbit-anti β-actin antibody (1:1,000) at 4°C, overnight. Following three washes with TBS, the membranes were incubated with a horseradish peroxidase-conjugated goat-anti rabbit immunoglobulin G antibody (1:1,000; cat. no. 31460; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Following three washes with TBS, Clarity Max^™ Western enhanced chemiluminescence Substrate (Bio-Rad Laboratories, Inc.) was added and membranes were incubated at room temperature for 4 min. The luminescence from the membranes was imaged by a ChemiDoc MP gel imaging and analysis system (170–8280; Bio-Rad Laboratories, Inc.). The experiments were repeated three times.

Data are presented as the mean ± standard error of the mean. Comparisons of means of multiple groups with each other or against one control group were analyzed with one-way analysis of variance followed by Bonferroni post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Grifolic acid at 20 µmol/l significantly inhibited RAW264.7 cell viability at 2 h and led to almost total loss of cell viability following 6 h incubation (P<0.01). Observations were obtained at 2, 6, 12 and 24 h for each concentration following preliminary experiments, which demonstrated that these incubation lengths were optimal for use in the present study (data not shown). Grifolic acid at 10 µmol/l exhibited significant inhibition of viability following 6 h incubation and achieved maximal effects at 24 h (P<0.05). Grifolic acid at 5 and 2.5 µmol/l inhibited RAW264.7 cell viability from 6 and 12 h, respectively and demonstrated significantly increased levels with the increased duration of incubation Treatment with grifolic acid at 1.25 µmol/l did not exhibit inhibitory effects at any time points investigated (Fig. 1).

Based on the MTT assay results, at 10 and 20 µmol/l grifolic acid demonstrated more significant effects on RAW264.7 cells and were used in the experiments of Annexin V/PI staining. Observations were obtained at 2 and 6 h for each concentration following preliminary experiments, which demonstrated that longer incubations resulted in cell lysis whereas little effect was observed at shorter incubation times (data not shown). Grifolic acid (20 µmol/l) treatment for 2 or 6 h lead to a significant increase in the number of both Annexin V- and PI-staining positive RAW264.7 cells compared with the control cells that were not treated with grifolic acid (P<0.01; Fig. 2A-D). Similarly, grifolic acid treatment at 10 µmol/l for 6 or 12 h also demonstrated a significant increase in the number of Annexin V-FITC⁺/PI⁺ cells compared with the control cells (P<0.01; Fig. 2E).

The cellular ATP content in untreated control RAW264.7 cells was 32.31±5.32 nmol/mg protein. ATP content significantly reduced to 10.29±4.76 nmol/mg protein when cells were treated with 20 µmol/l grifolic acid for 2 h (P<0.01). Grifolic acid treatment at 10 µmol/l for 2 h also significantly reduced cellular ATP content to 18.26±4.41 nmol/mg protein (P<0.01; Fig. 3).

Mouse FFAR4 siRNA transfection resulted in a notable reduction in FFAR4 protein expression level compared with the untransfected control cells (Fig. 4A). FFAR4 knockdown did not exhibit a significant influence on grifolic acid-induced inhibition of cell viability and apoptosis, as indicated by MTT assay and Annexin V-FITC/PI staining (Fig. 4B and C, respectively). Similarly, the decrease in cellular ATP content in grifolic acid-treated RAW264.7 cells was not affected by FFAR4-knockdown (Fig. 4D).

JC-1 emits green fluorescence in the cytoplasm and exhibits membrane potential-dependent accumulation in mitochondria with a shift of emission wavelength from green to red (10,11); MMP reduction is indicated by a decrease in the red/green fluorescence intensity ratio. In the present study, grifolic acid treatment resulted in a decrease in red/green fluorescence intensity ratio in a dose- and time-dependent manner (Fig. 5). Cells treated with 20 µmol/l grifolic acid exhibited a decrease in MMP at 5 min, with the maximal effects achieved within 20 min (Fig. 5E). Grifolic acid treatment at 10 µmol/l significantly reduced MMP at 10 min and achieved the maximal effects in 40 min (Fig. 5E). MMP was also significantly decreased compared with the control 60 min following treatment with 5 and 2.5 µmol/l grifolic acid at 40 and 50 min, respectively, but not with 1.25 µmol/l treatment (Fig. 5E).

Cyclosporine A is able to attenuate mitochondrial permeability transition and improve mitochondrial respiratory function (12,13). Therefore, the effects of cyclosporine A on the grifolic acid-induced decrease in MMP were investigated. When RAW264.7 cells were pretreated with cyclosporine A at 20 µmol/l for 10 min, grifolic acid-induced MMP loss was significantly attenuated compared with control cells that were treated with grifolic acid but not with cyclosporine A (P<0.01; Fig. 6A). Untreated control cells demonstrated no changes during measurement (data not shown). In addition, cyclosporine A treatment (10 µmol/l) also inhibited grifolic acid-induced decrease in cell viability of RAW264.7 cells (Fig. 6B).