Bergenin (purity >99%) was purchased from JingZhu Biological Technology (Nanjing, China). Uric acid, allopurinol, yeast polysaccharide, and HEPES were purchased from Sigma-Aldrich (United States). Potassium oxonate (PO), rosiglitazone, GW9662, WR-1065, and Pifithrin-β were purchased from MedChemExpress (United States). Antibodies against acetylated-p53, p53 and PPARγ were obtained from Cell Signaling Technology (United States). Antibodies against Lamin A/C, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were obtained from Santa Cruz Biotechnology (United States). Antibodies against SLC2A9 and ABCG2 were obtained from Novus (United States) and Abcam (United States), respectively. Penicillin/streptomycin and TRIzol reagent were purchased from Invitrogen Life Technologies (United States).

Male C57BL/6 mice (6–8 weeks old) were provided by the Academy of Medical Sciences of Zhejiang Province. Mice were given ad libitum access to food and water. All experiments were conducted in accordance with the Animal Care and Use Committee guidelines of Zhejiang province. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University.

Control mice were fed a standard diet. To induce hyperuricemia, mice were given 25% yeast polysaccharide (YP) mixed in daily diet and intraperitoneal injected of PO (250 mg/kg) at 8:00 a.m. every day. Mice of the treatment group were subjected to intragastric administration of either of two concentrations (40 or 80 mg/kg) of bergenin, while the same volume of normal saline (NS) was used as a control. Intragastric administration of 25 mg/kg allopurinol was performed as a positive control (Figure 1).

To measure the serum uric acid levels (SUA), we collected blood samples 2 h after treatment on days 0, 7, 14, and 21. The day before sacrificing, urine samples were collected from mice with metabolic changes within 24 h. Mice were sacrificed 2 h after the last treatment, and blood samples were collected. Uric acid and creatinine levels in serum and urine were determined using the phosphotungstic acid method and a Jaffe reaction kit, respectively (Nanjing Jiancheng Biological Technology Co., Ltd., China). Fractional excretion of urate (FEur) was calculated as per a previously reported method (Perez-Ruiz et al., 2002): FEur = (Uur Scr)/(Sur Ucr) × 100%. Sur, serum urate level; Scr, serum creatinine level; Uur, Urinary urate level; Uur, Urinary creatinine level.

Intestinal urate excretion was determined according to a previously described method (Ichida et al., 2012). Briefly, after overnight fasting, mice were anesthetized with 2% isoflurane inhalation using an isoflurane delivery system 2 h after the last treatment. Subsequently, mice were cannulated with polyethylene tubing at the upper duodenum and middle jejunum, making an intestinal loop at the upper half of the intestine. The intestinal contents were slowly removed by saline and air. Efflux buffer (saline containing 0.3 mM PO) was added into the intestinal loop; the bugger was collected at the indicated time points, and urate concentrations were quantified. Intestinal urate excretion was calculated using the following equation: intestinal urate excretion = (urate concentration in the intestinal loop) × (volume of efflux buffer in the intestinal loop) (length of the whole small intestine/length of the intestinal loop). Urate concentration was determined using the QuantiChrom Uric Acid Assay Kit (Bioassay Systems, United States).

After mice were sacrificed, kidney tissues were collected and cleaned, followed by fixation in 4% paraformaldehyde for at least 48 h at room temperature. Specimens were embedded in paraffin wax, and 3-mm thick sections were prepared. After mounting the sections onto polylysine-coated slides, hematoxylin and eosin, as well as Masson’s stainings were performed on consecutive tissue sections. Images were obtained using a microscope.

Xanthine oxidase (XO) activity in liver tissues was measured using a Xanthine Oxidase Activity Assay Kit (Sigma, United States) according to the manufacturer’s instructions. XO activity was determined by a coupled enzyme assay, which results in a colorimetric (570 nm) fluorometric product, which is proportional to the hydrogen peroxide generated. XO activity was expressed as nanomoles of uric acid per min per mg of total protein (mU/mg).

Blood samples were collected 2 h after the last treatment. The concentrations of IL-1β, TNFα, IL-6, IL-10, and IL-1Ra were determined using the respective ELISA kits (BOSTER, China for IL-1Ra and NEOBIOSCIENCE, China for the rest) according to the manufacturer’s instructions.

HK-2, Caco-2, and RAW264.7 cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). HK-2 cells were maintained in Dulbecco’s modified Eagle medium (DMEM)/F12 medium (Gibco, United States) containing 10% fetal bovine serum (FBS; Gibco, Australia). Caco-2 and RAW264.7 cells were cultured in high-glucose DMEM (Gibco, United States) supplemented with 10% FBS (Gibco, Australia). Cells were maintained in a humidified incubator containing 5% CO₂ at 37°C.

Inhibitors were dissolved in DMSO or double-distilled water (ddH₂O). Prior to treatments, we cultured cells overnight in serum-free medium to induce growth arrest. Cells were treated with bergenin and indicated inhibitors in a humidified incubator containing 5% CO₂ at 37°C, with or without stimulation with soluble uric acid for an additional 12 h. The final concentrations and incubation times were as follow: rosiglitazone (30 μM, 2 h), GW9662 (10 μM, 2 h), WR-1065 (1 mM, 2 h), and Pifithrin-β (10 μM, 2 h). Following the addition of HEPES at a final concentration of 25 mM, cells were treated with uric acid or the solvent (10 mM NaOH). The solution was filtered through a 0.22-μm pore size filter (Millipore, Shanghai, China) before use.

The effect of bergenin on the viability of HK-2 and Caco-2 cells was evaluated using the CCK8 assay. Cells were treated with different concentrations of bergenin, followed by incubation with 10% CCK-8 (Dojindo, Kumamoto, Japan) in 100 μL of high-glucose serum-free DMEM for 4 h at 37°C. Absorbance at 450 nm was measured on a microplate reader (ELX808; BioTek, Winooski, VT, United States).

For total protein extraction, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with proteasome inhibitors (Beyotime, Shanghai, China).

Nuclear and cytoplasmic extracts were prepared using the NE-PER Nuclear Cytoplasmic Extraction Reagent Kit (Pierce, Rockford, IL, United States) according to the manufacturer’s instructions. Briefly, cells were washed in PBS. Ice-cold CER I buffer was added to the cell pellet and vortexed vigorously for 15 s. After a 10-min incubation on ice, ice-cold CER II buffer was added to the cells. Samples were incubated on ice for 1 min, followed by centrifugation for 5 min at 16,000 × g. Subsequently, the supernatant containing the cytoplasmic extract was immediately transferred to a pre-chilled tube.

Equal amounts of protein were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore). Membranes were blocked in 5% non-fat dry milk for 2 h at room temperature, followed by overnight incubation at 4°C with the appropriate primary antibody: GAPDH (1:2000), ABCG2 (1:1000), SLC2A9 (1:1000), PPARγ (1:1000), p53 (1:1000), acetylated-p53, β-actin (1:1000), or Lamin A/C (1:1000). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse IgG (1:5000; Cell Signaling Technology) secondary antibody was applied for 1 h at room temperature. Membranes were covered with enhanced chemiluminescence solution (Millipore) and exposed to film. Signal intensity was measured using the Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, United States).

HK-2 and Caco-2 cells were seeded onto 24-well plates. After treatment, cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized with or without 0.1% Triton X-100 (Beyotime) for 30 min. After blocking in 10% goat serum for 60 min, slides were incubated with a rabbit p53 antibody (1:200) overnight at 4°C. Samples were then incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Invitrogen) for 2 h, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma, United States). Samples were observed under a fluorescence microscope (Leica, Solms, Germany).

Total RNA was isolated using TRIzol reagent (Invitrogen) and quantified by measuring the absorbance at 260 nm (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, United States). Complementary single-stranded DNA was synthesized from total RNA by reverse transcription (PrimerScript RT Master Mix, TaKaRa, Kyoto, Japan). RT-qPCR reactions were prepared using the SYBR Premix Ex Taq Kit (TaKaRa) in a total volume of 20 μL. All reactions were prepared in duplicates and were run on an ABI StepOnePlus System (Applied Biosystems, Warrington, United Kingdom). The PCR temperature cycling conditions were as follows: 95°C for 30 s followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. Relative gene expression was analyzed using the 2^–ΔΔCt method. The primer sequences used are provided in Supplementary Table S1.

Cells were seeded onto 6-well plates and cultured overnight in DMEM/F12 or DMEM without FBS and antibiotics. siRNA transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, 10 μL of siRNA and 5 μL of Lipofectamine 2000 reagent were combined in a total of 300 μL of Opti-MEM I (Gibco, Invitrogen). Thereafter, 700 μL of Opti-MEM I was added to the mixture, and the mixture was added to each well. After incubation for 6 h, fresh DMEM or DMEM/F12 containing 10% FBS was added to each well. Cells were incubated for an additional 48–72 h. PPAR-γ siRNAs and the negative control siRNAs were purchased from GenePharma (Shanghai, China).

Cells were transfected with luciferase reporter constructs PPARγ-Luc (Promega, United States) or p53-Luc (Promega, United States) as previously described (Ye et al., 2019). Cells were then treated with uric acid (UA, 8 mg/dL) and/or bergenin for 12 h. Subsequently, luciferase activity was measured using a luciferase assay system (Promega, United States).

Statistical analysis was performed using the SPSS statistical software for Windows, version 19.0 (IBM, Armonk, NY, United States). All experiments were performed at least in triplicate, and the data were expressed as mean ± standard error of the mean (SEM). Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Fisher’s Least Significant Difference (LSD) test when comparing more than two groups. P values ≤ 0.05 were considered statistically significant. The correlation between variables was evaluated by the Pearson correlation test. Two-sided P values < 0.05 were considered statistically significant.

The procedure followed to develop a hyperuricemia mouse model is illustrated in Figure 1. Compared with control mice, SUA levels were significantly higher in hyperuricemia mice after day 7, and remained elevated between day 14 (407.41 ± 79.09 μmol/L vs. 194.07 ± 25.85 μmol/L; P < 0.01) and day 21 (439.39 ± 48.11 μmol/L vs. 220.60 ± 35.28 μmol/L; P < 0.01). SUA elevation was suppressed by bergenin (80 mg/kg) and allopurinol administration. While bergenin at 80 mg/kg profoundly decreased SUA levels (253.18 ± 31.74 μmol/L) compared to hyperuricemia mice, no significant difference was observed at day 21 after administration of 40 mg/kg bergenin (397.39 ± 52.69 μmol/L; Figure 2A). This finding suggests that the effects of bergenin are dose-dependent to some degree. It is worth noting that SUA level of the Bergenin (80 mg/kg) group stabilized at baseline levels from day 1 to day 21. As expected, SUA levels were significantly lower in allopurinol-treated mice (positive control) compared with control mice. No significant differences in SUA levels were observed between the mice in the bergenin (80 mg/kg) group and the allopurinol group.

Scr, FEur, intestinal urate excretion rate, and hepatic XO activity were assessed at day 21. Scr levels were higher in hyperuricemia mice compared with control mice; however, the difference did not reach statistical significance (P > 0.05) (Figure 2B). In hyperuricemic mice, FEur was decreased; bergenin (40 mg/kg, 80 mg/kg) treatment rescued FEur. Furthermore, urate excretion from the intestine was significantly increased in mice treated with bergenin (80 mg/kg) compared with hyperuricemic mice (P < 0.01; Figure 2D). While allopurinol reduced hepatic XO activity (P < 0.01), it did not affect uric acid excretion in the kidneys and intestine (P > 0.05) compared with hyperuricemia mice (Figures 2C–E). Additionally, bergenin had no effect on XO activity in liver and jejunum (Figure 2E, Supplementary Figure S1).

Histological analysis revealed that the ultrastructure of the kidneys was intact in mice treated with PO and YP. Compared with control mice, degeneration and necrosis were observed in tubular epithelial cells of hyperuricemic mice (Figure 2F). Bergenin (80 mg/kg) and allopurinol treatment ameliorated PO- and YP-induced pathological lesions. No histological changes were observed in the intestine of hyperuricemic or bergenin-treated mice (Supplementary Figure S2).

To determine the effects of bergenin on uric acid excretion in the kidneys, we analyzed the expression of urate transporters involved in urate export and reuptake in the kidney. We found no significant differences in the mRNA levels of the genes encoding urate transporter 1 (Urat1) and PDZ domain-containing 1 (Pdzk1) among the different groups (P > 0.05; Figure 3A). Abcg2 expression was lower in hyperuricemic mice compared with control mice; nevertheless, its expression in hyperuricemic mice was rescued by bergenin (40 mg/kg, 80 mg/kg) treatment, both at the mRNA and protein levels (P < 0.01) (Figures 3A,E). Bergenin at 80 mg/kg and 40 mg/kg resulted in 2-fold and 1.5-fold increases in Abcg2 mRNA levels, respectively.

In contrast, Slc2a9 expression was higher in hyperuricemic mice compared with control mice, and bergenin (80 mg/kg) suppressed Slc2a9 expression, which was evident both at the mRNA and protein levels (P < 0.01). Although bergenin treatment (40 mg/kg) decreased SLC2A9 protein levels (P < 0.05, compared to the model group), no difference was observed at the mRNA level (Figures 3A,E). Hyperuricemic mice exhibited a reduction in PPARγ expression (P < 0.01, compared to control mice), PPARγ expression was restored by bergenin (40 mg/kg, 80 mg/kg; Figure 3E).

RT-qPCR analyses revealed no significant differences in the expression of Pdzk1 and solute carrier family 17 member 3 (Slc17a3) in the intestine among the groups (P > 0.05; Figures 3B–D). In the colon, bergenin (40 mg/kg, 80 mg/kg) significantly increased Abcg2 mRNA and protein levels (Figures 3B,F). Hyperuricemic mice had lower Abcg2 mRNA and protein levels compared with the control group, which were restored by bergenin (40 mg/kg, 80 mg/kg) treatment (Figures 3C,G). In the jejunum, the expression of Abcg2 was increased in hyperuricemic mice, and its expression was elevated by bergenin in a dose-dependent manner, both at the mRNA and protein levels (Figures 3D,H). These RT-qPCR and western blot findings were also confirmed by immunofluorescence (Supplementary Figure S3).

Compared to control mice, hyperuricemic mice exhibited decreased expression of Slc2a9 in the ileum and jejunum, both at the mRNA and protein levels (P < 0.01). Administration of bergenin significantly restored Slc2a9 expression in the ileum and jejunum (Figures 3C,D,G,H). The mRNA levels of Slc2a9 were increased by approximately 2-fold and 4-fold after administration of bergenin at 40 and 80 mg/kg, respectively, suggesting a dose-dependent effect. On the other hand, RT-qPCR and western blot analyses showed no changes in Slc2a9 expression in the colon (Figures 3B,F). SLC2A9 expression in intestine was further confirmed by immunofluorescence (Supplementary Figure S3).

PPARγ was expressed at lower levels in the ileum and jejunum of hyperuricemic mice (P < 0.01, compared with control mice), but not in the colon. Bergenin treatment enhanced PPARγ expression in a dose-dependent manner (Figures 3F–H).

Several studies suggested that hyperuricemia and soluble uric acid levels are associated with systemic inflammation (Joosten et al., 2020). Therefore, we investigated the effects of bergenin on the serum levels of several inflammatory cytokines. IL-1β, TNF-α, and IL-6 serum levels were significantly increased in hyperuricemic mice; bergenin treatment reduced the serum levels of these cytokines. Notably, bergenin at 80 mg/kg reduced the serum levels of IL-1β, TNF-α, and IL-6 by 16%, 25%, and 57%, respectively (Figures 4A,C,E). In contrast, no significant difference was observed in the serum levels of IL-1Ra or IL-18 (Figures 4G,I). Additionally, SUA levels were positively correlated with IL-1β, TNF-α and IL-6 serum levels and were negatively correlated with those of IL-1Ra (Figures 4B,D,F,H). No correlation between SUA and IL-18 (Figure 4J).

To gain further insight into the effects of bergenin on inflammatory responses, we treated RAW267.4 cells with UA (8 mg/dL) or bergenin (50 μM). Exposure to UA polarized RAW267.4 cells toward an inflammatory (M1) phenotype with upregulation of inducible nitric oxide synthase (iNos) and downregulation of Il-10. In contrast, bergenin treatment polarized RAW267.4 cells toward an anti-inflammatory (M2) phenotype, with high expression of Arginase-1 (Arg-1), Il-10, and CD206 and low expression of Tnf-α and iNos. RAW267.4 cells pretreated with bergenin prior to UA exposure acquired an M2 phenotype, with increased Arg-1, Il-10, and CD206 expression and low Tnf-α and iNos expression (Figure 4K).

We found that bergenin at 10–100 μM did not affect the viability of HK-2 cells (Figure 5A). Hence, we pretreated HK-2 cells with 10, 30, 50, or 100 μM bergenin for 2 h, followed by treatment with 8 mg/dL UA for 10 h. UA decreased ABCG2 expression at the mRNA and protein level (P < 0.05, compared to the control group). UA-mediated ABCG2 downregulation was rescued by bergenin treatment (50 and 100 μM). In contrast, SLC2A9 mRNA and protein levels were increased after UA treatment (P < 0.05, compared to the control group). Pretreatment with bergenin (50 and 100 μM) suppressed UA-induced SLC2A9 upregulation (Figures 5B,C); however, lower concentrations of bergenin did not affect SLC2A9 expression. No significant increase was observed in ABCG2 and SLC2A9 expression in cells treated with 30 μM bergenin alone (Figures 5B,C).

Luciferase reporter assays revealed that UA significantly reduced the expression and transcriptional activity of PPARγ, both of which were restored by bergenin (Figures 5C,D). siRNA-mediated PPARγ silencing decreased the mRNA and protein levels of ABCG2. Moreover, bergenin treatment failed to enhance ABCG2 expression after PPARγ knockdown. PPARγ silencing did not affect the expression of SLC2A9, regardless of the presence of bergenin (Figures 5E,F).

Similarly to bergenin, pretreatment of HK-2 cells with the PPARγ agonist rosiglitazone before stimulation with UA increased the expression of PPARγ and ABCG2. In contrast, pretreatment with the PPARγ antagonist GW9662 enhanced the UA-mediated downregulation of PPARγ and ABCG2 (Figure 5G). Additionally, bergenin failed to restore ABCG2 expression in cells treated with GW9662 and UA (Figure 5G). These results suggest that bergenin regulates the expression of ABCG2 by activating PPARγ.

As the previous research indicated SLC2A9 was a direct target gene of p53 (Itahana et al., 2015), we next investigated the effects of bergenin and UA on p53 signaling. Although bergenin and UA had no effects in the total p53 and acetylated p53 levels (Figure 5H), UA significantly decreased p53 levels in the cytoplasm and increased its levels in the nucleus. These results suggest that UA promotes p53 protein translocation from the cytoplasm to the nucleus. Interestingly, bergenin suppressed nuclear translocation of p53 (Figure 5H). Immunofluorescence and luciferase reporter analyses confirmed these results (Figures 5I,J).

Pifithrin-β (PFT-β), a potent p53 inhibitor, significantly suppressed the UA-induced SLC2A9 upregulation. On the other hand, the p53 agonist WR-1065 reinforced SLC2A9 upregulation. Bergenin treatment failed to suppress SLC2A9 expression in HK-2 cells treated with WR-1065 and UA (Figure 5K).

We next used Caco-2 cells as an in vitro model of human intestinal epithelial cells to examine the effects of bergenin on intestinal urate transporters. We found that treatment with bergenin at 10–100 μM for 12 h had no effects on the viability of Caco-2 cells (Figure 6A). We then pretreated Caco-2 cells with bergenin for 2 h, followed by treatment with UA at 8 mg/dL for 10 h. UA increased ABCG2 mRNA and protein levels and downregulated SLC2A9 levels. Treatment with bergenin alone increased the protein levels of ABCG2 and SLC2A9. Compared to cells treated with UA alone, ABCG2 and SLC2A9 were significantly upregulated when cells were treated with UA and 100 μM bergenin (P < 0.01) (Figures 6B,C).

Similar to HK-2 cells, UA significantly inhibited PPARγ activity in Caco-2 cells, which was restored by bergenin (Figures 6C,D). Interestingly, siRNA-mediated PPARγ silencing reduced the expression of both ABCG2 and SLC2A9. In addition, bergenin treatment failed to enhance ABCG2 and SLC2A9 expression after PPARγ silencing (Figures 6E,F). PPARγ activation by rosiglitazone significantly enhanced the UA-mediated ABCG2 and SLC2A9 upregulation (P < 0.01). In contrast, pretreatment with GW9662 enhanced the UA-mediated downregulation of PPARγ, ABCG2, and SLC2A9. Furthermore, bergenin failed to restore ABCG2 and SLC2A9 expression in cells treated with GW9662 and UA (Figure 6G).

Bergenin treatment did not alter the total p53 and acetylated p53 levels in Caco-2 cells (Figure 6H). Additionally, immunofluorescence analyses indicated that bergenin did not promote nuclear translocation of p53 (Figure 6I).