Extract preparation and HPLC
Flowers of C. indicum L. were extracted using water for 8 h at 90°C. The extract was filtered with Whatman paper No. 6 and concentrated in a rotary evaporator under reduced pressure. The concentrate was lyophilized (Ci) and stored at -20°C until further use. Fruits of C. officinalis Siebold & Zucc were extracted using water for 8 h at 90°C. The extract was filtered with Whatman paper No. 6 and concentrated in a rotary evaporator under reduced pressure. The extract was dried using hot air with dextrin (50%) (Co) and stored at -20°C until further use. Ci and Co were mixed in a ratio of 1:2 (FSH-CC) and stored at -20°C until further use. And then, we analyzed luteolin and loganin of Ci and Co, and FSH-CC by high-performance liquid chromatography (HPLC) using Agilent 1260 infinity II HPLC system (Santa Clara, CA, USA).
Cell culture and treatments
The human liver cancer (HepG2) cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in Dulbecco’s minimal essential medium (DMEM; Hyclone Laboratories, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories), 100 mg/L penicillin-streptomycin, and 2 mmol/L glutamine (Hyclone Laboratories) at 37°C in a humid atmosphere of 5% carbon dioxide (CO2).
To obtain the primary mouse renal proximal tubule cells, kidney was isolated from Balb/c mice (22–25 g, 6 weeks, male). The kidney was minced using Hank's balanced salt solution (HBSS) containing trypsin, with the addition of 1 mg/mL deoxyribonuclease (DNAse) and 2 mg/mL collagenase Type I (Sigma-Aldrich, USA). After 30 min, the solution was passed through an 80-mesh and 1709-mesh sieve (Fisher Scientific, Pittsburgh, PA) to remove the cell debris and glomeruli. Proximal tubule cells remained on the sieve filter and were collected by washing the sieve filter with HBSS. The proximal tubule cell suspension was centrifuged for 10 min at 1000 revolutions per minute (rpm) at 4°C and the cell pellet was collected.
HepG2 cells and primary mouse renal proximal tubule cells were cultured with Ci, Co, and FSH-CC for 24 h and treated with 4 mM hypoxanthine. After 2 h, assays were performed to measure the activity of xanthine oxidase, mRNA expression of xanthine dehydrogenase, and the expression levels of OAT1, OAT3, GLUT9, and URAT1 proteins.
Animals
The Institutional Animal Care and Use Committee of Kyung Hee University approved the protocol (KHGASP-20-410) for the use of animals in this study. The animals were cared for in accordance with the “Guidelines for Animal Experiments” established by the university.
Six-week-old male C57 black 6 (C57BL6) mice were purchased from SaeRon Bio (Uiwang, Korea) and housed in cages under automatically controlled temperature (22 ± 2°C), humidity (about 50%), and lighting (12:12-h light-dark cycle) conditions. The mice in the control group with normal diet were fed a commercial pelleted chow (AIN-93G rodent purified diet, Orient Bio, Korea) and water ad libitum. All the mice were randomly divided into eight groups of eight mice per group as follows: normal control (NC), control (C; hyperuricemia-induced mice), positive control (PC; hyperuricemia-induced mice with oral supplementation of allopurinol, xanthine oxidase inhibitor, 10 mg/kg body weight (b.w)), Ci 300 (hyperuricemia-induced mice with oral supplementation of Ci, 300 mg/kg b.w), Co 300 (hyperuricemia-induced mice with oral supplementation of Co, 300 mg/kg b.w), FSH-CC 150 (hyperuricemia-induced mice with oral supplementation of FSH-CC, 150 mg/kg b.w), FSH-CC 300 (hyperuricemia-induced mice with oral supplementation of FSH-CC, 300 mg/kg b.w), and FSH-CC 600 (hyperuricemia-induced mice with oral supplementation of FSH-CC, 600 mg/kg b.w). The extracted samples were orally administered for 21 d. To induce hyperuricemia, an intraperitoneal injection of 200 mg/kg b.w. potassium oxonate (Sigma-Aldrich Co, MO, USA) was given. After 2 h, the mice urine was collected and the mice were anesthetized with isoflurane.
Levels of uric acid and creatinine in the urine and serum
Blood was centrifuged at 3000 rpm for 10 min and the serum was separated. The levels of uric acid in the urine and serum were determined using the uric acid assay kits (BioVision Inc., CA, USA), while the levels of creatinine in the urine and serum were determined using the creatinine assay kit (BioVision Inc., CA, USA).
Activity of xanthine oxidase
The activity of xanthine oxidase was determined from the HepG2 cells in the culture medium and serum from mice using the Xanthine Oxidase Activity Assay Kit (Sigma-Aldrich Co, MO, USA).
mRNA expression of xanthine dehydrogenase
mRNA was extracted from the HepG2 cells and liver tissues using the RNeasy Mini Kit (QIAGEN, MD, USA). Synthesis of complementary DNA (cDNA) using mRNA was performed using the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Polymerase chain reaction (PCR) consisted of 40 cycles of denaturation (95°C for 15 s), annealing (58°C for 15 s), and extension (72°C for 30 s) using the SYBR Green PCR Master Mix (iQ SYBR Green Supermix; Bio-Rad Laboratories, Inc.) and the following primers: GAPDH (H) forward primer 5′-CCC CAC ACA CAT GCA CTT ACC-3′, reverse primer 5′-TTG CCA AGT TGC CTG TCC TT-3′; xanthine dehydrogenase (H) forward primer 5′-ATT GGT GCT GTG GTT GCT-3′, reverse primer 5′-TGT GAT AAT GGC TGG TAG TTC TTC; GAPDH (R) forward primer 5′-CAT GGC CTT CCG TGT TCC TA-3′, reverse primer 5′-GCG GCA CGT CAG ATC CA-3′; xanthine dehydrogenase (R) forward primer 5′-TGC GAA GGA TGA GGT TAC T-3′, reverse primer 5′-GGA TTG TGA TAA TGG CTG GAA-3′. The cDNA was amplified using the real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and data analysis was performed using the CFX Maestro™ Analysis Software (Bio-Rad Laboratories, Inc.).
Serum triglycerides, cholesterols, aspartate transaminase (AST), and alanine transaminase (ALT)
Concentrations of total triglycerides (TG), total cholesterol (TC), very-low-density lipoprotein (VLDL)/low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol, aspartate transaminase (AST), and alanine transaminase (ALT) in serum were determined by enzyme-linked colorimetric methods using commercial kits (BioVision, CA, USA).
Antioxidant enzyme activity in the liver
The liver tissues were lysed using the CelLytic™ MT lysis reagent (Sigma) and the antioxidant enzyme activity was measured using the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) assay kits (Biomax Inc., Seoul). Malondialdehyde (MDA), a lipid peroxidation marker, was measured using MDA assay kits (BioVision Inc., Mountain View, CA, USA).
Expression levels of OAT1, OAT3, GLUT9, and URAT1 proteins
The primary mouse renal tubular epithelial cells and kidney tissues were lysed using the CelLytic™ MT lysis reagent. Equal amounts of proteins (100 µg/lane) were separated by electrophoresis using 10% Mini-PROTEAN® TGXTM Precast Gels (Bio-Rad Laboratories, Inc.) and transferred to polyvinylidene difluoride membranes using Trans-Blot® TurboTM Transfer system (Bio-Rad Laboratories, Inc.). The membranes were incubated for 1 h in a blocking solution containing 5% non-fat milk in Tris-buffered saline and further incubated for 12 h at 4°C with the antibodies recognizing OAT1 (1:500; MyBioSource), OAT3 (1:1000; Santa Cruz), GLUT9 (1:800; Invitrogen), URAT1 (1:800; MyBioSource), and β-actin (1:1000; Cell Signaling Technology). Thereafter, the membranes were incubated with secondary antibodies (anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked antibody, 1:3000; Cell Signaling Technology, Inc.) for 1 h at room temperature. The immunoreactive protein bands were detected using EzWestLumi plus (ATTO, Tokyo, Japan) and analyzed using Ez-Capture Ⅱ (ATTO) and CS Analyzer v.3.0 (ATTO).
Statistical analysis
All data are presented as mean ± standard deviation (SD). The data were statistically evaluated using Duncan’s multiple range tests after one-way analysis of variance (ANOVA) using SPSS statistical procedures (SPSS PASW Statistic v.23.0, SPSS Inc., Chicago, IL, USA). When the data were subjected to prior investigations before analysis, parametric assumptions including homoscedasticity and normality of observations were satisfied. Differences were considered to be statistically significant at p < 0.05 level.