Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Life Technologies (Grand Island, NY, USA). RPMI 1640 medium, L-malic acid, adenosine 5’ diphosphate sodium salt (ADP), adenosine 5’ triphosphate disodium salt (ATP), dimethyl sulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), digitonin, cholesterol, and LPS, were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). UA and OA were procured from Bidepharm (Shanghai, China) and Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China), respectively. Sodium pyruvate and bovine serum albumin (BSA) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). ATPlite luminescence assay kit was bought from PerkinElmer (Waltham, MA, USA). Bio-Rad protein assay dye reagent concentrate was obtained from Bio-Rad (Hercules, CA, USA). All other chemicals were of analytical grade.

A H9c2 cell line derived from embryonic rat heart tissue was purchased from American Type Culture Collection (Rockville, MD, USA). H9c2 cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS (HIFBS) and 1 mM sodium pyruvate. Cells were cultured in an atmosphere of 5% CO₂ at 37˚C in a 100 mm culture plate. The medium was replaced every 2 - 3 days and cells were split at a subcultivation ratio of 1:10 2 - 3 days after medium replacement.

Drug pre-incubation and ATP Generation

H9c2 cells were seeded in four 24-well plates (25,000 cells/0.5mL). After stable attachment, the cells were pre-incubated for 4 hours with a medium containing 2 - 10 μM of UA or OA [dissolved in DMSO, 0.2% (v/v) final concentration]. Following the incubation, the cells were washed with phosphate-buffered saline (PBS, 200 μL/well; 136 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.2) and treated with 50 μg/mL digitonin at 37˚C for 3 minutes, except for the protein measurement plate, which was stored at 4˚C until protein determination. For the plate used for measuring basal ATP level, cells were incubated at 4˚C with ATP incubation buffer (120 mM KCl, 5 mM KH₂PO₄, 2 mM EGTA, 10 mM HEPES, 0.1 mM MgCl₂, 0.5% BSA, pH7.4, 150 μL/well) and 30% (w/v) perchloric acid (PCA, 30 μL/well). For the plates used for ATP generation, cells were supplemented with 0.18 mM ADP, 5 mM pyruvate, and 15 mM L-malic acid (all dissolved in ATP incubation buffer, 100 μL for each reagent/well) and incubated at 37˚C for 7.5 and 15 minutes, respectively, followed by the addition of 30% PCA (60 μL/well). The plates (for measuring basal ATP and ATP generation) were then centrifuged at 540 ×g for 20 minutes at 4˚C. After centrifugation, aliquots (80 μL) of supernatants were neutralized with 60 μL 1.4 M KHCO₃ in 1.5 mL micro-centrifuge tubes, followed by vortex mixing and centrifugation at 2150 ×g for 10 min.

ATP assay

To account for ATP contamination in the ADP preparation, an ADP blank was prepared by aliquoting ADP, pyruvate, L-malic acid and 30% PCA, as described earlier, to 1.5 mL in a micro-centrifuge tube, followed by vortex mixing and centrifugation at 20,000 ×g for 5 min. ATP standards (ranging from 0.0005 - 1.0 mM) were prepared by 10-fold serial dilutions. Aliquots (300 μL) of standard solutions were added to 60 μL 30% PCA, followed by vortex mixing, and 80 μL of the standard solutions, as well as the ADP blank, were neutralized with 60 μL 1.4 M KHCO₃, followed by vortex mixing and centrifugation at 20,000 ×g for 5 min. Fifty micro-liters of ATP standards (0.0005 - 1 mM) or tested samples were applied to 96-well white plates with 50 μL 10-fold diluted luciferase enzyme prepared in luciferase dilution buffer (250 mM Tricine, 5 mM EDTA (free acid), 5 mM dithiothreitol, 50 mM MgSO₄, pH 7.8 adjusted by 1 M KOH). The luminescence was read using a microplate reader. ATP content was estimated from an ATP standard calibration curve. ATP-GC was expressed in nmol/mg protein and the area under the curve (AUC1) of the graph plotting ATP generation against incubation time was then computed (Figure 2). These AUC1 values were used to calculate the percentage of control (% Control1), and then the AUC2 of the graph plotting % Control1 against incubation time was computed. These AUC2 values were used to calculate the percentage of control (% Control2) for comparison.

Protein Measurement

Proteins were extracted from the cultured cells using 0.1% Triton X in PBS (200 μL/well), followed by 10-minutes of shaking at room temperature. Aliquots (10 μL) of samples, as well as BSA standards, were mixed with working protein-dye solution (200 μL/well) and the reaction mixtures were allowed to stand at room temperature for 5 minutes. The absorbance of the samples was then read at 600 nm.

Splenocyte isolation

Splenic tissue obtained from adult female ICR mice (25 - 40 g body weight) was pressed through a mesh stainless steel sieve using a glass pestle in 25 mL RPMI-1640 medium to obtain a single-cell suspension. The cells were placed on ice for 5 min, and the suspension was centrifuged at 600 ×g for 5 min, followed by washing with red blood cell lysis buffer (0.1 M Tris-HCl, pH 7.2, 0.8% NH₄Cl) and RPMI-1640 medium. After centrifugation, the pellet was resuspended in RPMI-1640 medium supplemented with 10% HIFBS for cell counting, using 0.4% trypan blue. The cells were then adjusted to 5 × 10⁶ cells/mL and 80 μL of cell suspensions were seeded in each well of a 96-well plate.

Drug incubation and LPS stimulation in vitro

UA or OA (dissolved in DMSO) was added at final concentrations of 3 - 100 μM. LPS was added at final concentrations of 0.5, 1, 2, 4, and 7.5 μg/mL. Splenocytes were then cultured for 72 h at 37˚C in a humidified atmosphere of 5% CO₂ in air. Thereafter, the extent of cell proliferation was assessed.

MTT-based cell proliferation assay

An aliquot (10 μL) of MTT (5 mg/mL in PBS) was added to each well. After 4 h of incubation, 100 μL of solubilization buffer (10% sodium dodecyl sulfate, 45% dimethylformamide, pH 4.7 adjusted by glacial acetic acid) was added, and the mixtures were incubated in 5% CO₂ at 37˚C overnight to dissolve the colored crystals. The extent of splenocyte proliferation was determined by measuring the absorbance at 600 nm using a microplate reader. The extent of LPS-stimulated proliferation of isolated splenocytes was estimated by computing the AUC of a graph plotting the percentage of initial absorbance (mean absorbance of cells stimulated with LPS/mean absorbance of cells not stimulated with LPS × 100%) against LPS concentration (Figure 3). The extent of LPS-stimulated proliferation of isolated splenocytes was estimated by comparison with the control.

H9c2 cells

Cultured cells were pre-incubated with cholesterol (dissolved in absolute ethanol) at a final concentration of 10 μM for 4 h. Cells were then washed with PBS buffer and subjected to drug pre-incubation, which was followed by the measurement of ATP generation.

Mouse splenocytes

The cell suspension was pre-incubated with absolute ethanol or 10 μM cholesterol (final concentration, dissolved in absolute ethanol) in RPMI-1640 medium supplemented with 10% HIFBS for 4 hours, followed by washing with RPMI-1640 medium supplemented with 10% HIFBS and then centrifuging at 540 ×g for 7 minutes. The cells were then adjusted to 5 × 10⁶ cells/mL and were seeded in a 96-well plate.

Data, which were expressed as mean ± SEM, were analyzed by one-way ANOVA, followed by Tukey’s test to detect significant differences between groups when p < 0.05.