Hesperetin (95%–99%; Chemical Abstracts Service (CAS) number: 520-33–2) was purchased from Chengdu Purifa Technology Development (Chengdu, China). Xanthine oxidase (XOD), allopurinol, uric acid, sodium urate (SU) (BCCB4889); xanthine (CAS-69-89–6), and yeast extract (YE) (2340951-02) were obtained from Sigma-Aldrich (Saint Louis, MO, United States). Potassium oxonate (PO) (01045378) was from Damas-beta (Shanghai, China). Hypoxanthine (H108384) was purchased from Aladdin (Shanghai, China). XOD kit and UA kits were procured from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Solarbio (Beijing, China). WST-1 (W201) was purchased from Dongren Chemicals (Shanghai, China). Anti-XOD antibody [EPR4605] (ab109235), anti-NLRP3 antibody [EPR20425] (ab210491), and anti-IL-18 antibody [EPR19956] raised against mice were purchased from Abcam (Cambridge, UK). Anti-OAT1 (1F2): sc-293323, anti-OAT3 (3C11): sc-293264, anti-OCT1 (12F11): sc-8024 and toll-like receptor (TLR)4: sc-293072 antibodies raised against mice were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Anti-OCT2 (H-3): sc-377476 and NF-κB p65 (C22B4) antibodies were obtained from Cell Signaling Technology (Beverly, MA, United States). Anti-β-tubulin (66240-1-Ig) was purchased from Proteintech (Rosemont, IL, United States). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or anti-rabbit IgG were obtained from R&D Systems (Minneapolis, MN, United States).

Animal studies (animal ethics approval code number: YUAN2019LLWYH003-2) were undertaken according to recommendations set in Guide for the Care and Use of Laboratory Animals [United States National Institutes of Health (Bethesda, MD, United States)]. Male specific pathogen-free (SPF) Institute of Cancer Research (ICR) mice (20–22 g) were purchased from Kunming Medical University (license number: SCXK 2015-0004). In order to adapt to the environment, animals were housed in an SPF-grade laboratory (license number: SYXK 2018-0005) with a 12-h light-dark cycle (20°C–25°C, 60% ± 10% humidity) in the animal house. Free food and water were supplied for 1 week before performing any procedure.

Uric acid (which has maximum optical density at 295 nm (OD₂₉₅)) works as an indicator of xanthine oxidase activity. According to the literature (Qin et al., 2018), a 100-μL reaction system comprising xanthine oxidase (40 U/L), xanthine (250 mmol/L), WST-1 (100 μmol/L), and test compounds at various concentrations in reaction buffer (0.1 mol/L sodium paraphosphate, 0.3 mmol/L ethylenediamine tetraacetic acid, pH 8.3). The OD₂₉₅ of the reaction system was monitored dynamically for 20 min at 25°C with a microplate reader (SpectraMax M3; Molecular Devices, Sunnyvale, CA, United States). The slope of the OD₂₉₅-time curve reflects xanthine oxidase activity. The slope of the OD₄₅₀-time curve represents the O^2- level. The dose-response curve was obtained and the half-maximal inhibitory concentration (IC₅₀) value of each compound was calculated.

Hesperetin and allopurinol were prepared in saline solution containing 1% Tween-80 immediately before the use to obtain a uniform suspension and the administrated volume was 10 mL/kg. Previous studies have shown that intraperitoneal administration of 20 mg/kg HSE can prevent ototoxicity by increasing antioxidant enzymes and reducing oxidation parameters, as well as prevent apoptosis caused by cochlear implant cell proliferation (Kara et al., 2016), and the experimental dose adjustment in this study is based on this.

Refer to the previous methods (An et al., 2021) and dosage regimens (He et al., 2020), mice were randomly assigned into five groups: normal; model; positive-treated (allopurinol, 10 mg/kg); hesperetin (10 mg/kg)-treated; hesperetin (5 mg/kg)-treated. Mice in the normal group underwent intragastric administration with saline solution contain 1% Tween-80 (vehicle) at 10 mL/kg for successive 13 days. Instead, mice in other groups were orally administrated with yeast extract (15 g/kg). After 6 h, hesperetin group was given the corresponding dose (10 and 5 mg/kg), the positive-treated and the normal group accepted the administration of same volume of vehicle. After 13 days, mice were fasted for 10 h, and then the positive allopurinol was intragastrically administered once according to the report of Niu et al. (2018), and the other groups were administered by intraperitoneal injection of the corresponding articles. After 30 min, blood was collected via the orbital vein. Serum samples were obtained by centrifuging whole-blood samples at 4,000 × g for 10 min at 4°C. Serum levels of uric acid, creatinine, and urea nitrogen were determined using an automatic biochemistry analyzer (AU680; Beckman Coulter, Fullerton, CA, United States). Hepatic xanthine oxidase activity was measured using the xanthine oxidase kit. The study flowchart was shown in Figure 2A.

The levels of malondialdehyde (MDA), glutathione peroxidase (GSH-PX), catalase (CAT) activity in the livers of mice induced by yeast extract were determined using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Left upper-lobe liver tissues were embedded in paraffin, cut into 5 μm thick slices, and stained with hematoxylin and eosin staining. We evaluated the general morphology under light microscopy, as reported previously (Xiong et al., 2017).

Mice were randomly assigned into five groups: normal; model; allopurinol (10 mg/kg)-treated; hesperetin (10 mg/kg)-treated; hesperetin (5 mg/kg)-treated. Mice in the hesperetin group were injected once a day for consecutive 6 days. The normal, model group and positive group received the vehicle in the same manner. On the seventh day, HSE group mice, fasted for 10 h, were injected subcutaneously to avoid mutual interference with the modeling reagent. The positive control received one gavage of allopurinol (10 mg/kg). 30 min after administration, all groups were administered potassium oxonate (450 mg/kg, i. p.) except that the normal control group was injected with an equal volume of vehicle. The study flowchart was shown in Figure 2B.

Blood was drawn from the inner canthus after modeling for 1.5 h. After killing, the kidney was frozen in liquid nitrogen and stored at −80°C for later use. Serum samples were obtained by centrifuging blood samples at 4,000 × g for 10 min 4°C. Serum levels of uric acid, creatinine, and urea nitrogen were determined using an automatic biochemistry analyzer (AU680; Beckman Coulter).

L-O2 cells purchased from Shanghai Meixuan Biotechnology (Shanghai, China) and HEK293T cells (American Type Culture Collection, Manassas, VA, United States) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin in an atmosphere of 5% CO₂ at 37°C.

L-O2 cells were seeded in 96-well plates at 5 × 10⁴ cells per well to determine the effect of hesperetin on L-O2 cells. The cells were treated with hesperetin (25, 50, 100, 200, or 400 μmol/L) for 24 h. Then, 20 μL of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to the 96-well plates, and incubation for 4 h in the dark allowed. Dimethyl sulfoxide (200 μL) was added to dissolve the remaining formazan crystals of MTT, and agitation for 15 min carried out. Cell viability was measured at 492 nm using a multimode microplate reader (Flexstation^™ 3; Molecular Devices).

The same method was used to determine the effect of hesperetin on HEK293T cells. Cells were seeded in 96-well plates at 1.5 × 10⁴ cells per well and treated with hesperetin (25, 50, or 100 μmol/L) for 24 h. Then, cell viability was measured as described above.

L-O2 cells were incubated with hesperetin (i.e., 25, 50, or 100 μmol/L) or allopurinol (100 μmol/L) for 24 h. Then, cells were washed gently with phosphate-buffered saline (PBS). After incubated with hypoxanthine (800 μmol/L) for 24 h, xanthine oxidase (4 μL/well) was added at a final concentration of 0.3 U/g, and incubation for 1 h in the dark permitted, followed by extraction of the supernatant or protein. Experiments were repeated at least thrice. The uric acid content of cell supernatants was measured using the uric acid kit.

As reported by W. D. Chen and Y. L. Zhao (Chen et al., 2020), HEK293T cells were resuspended with DMEM and adjusted to 1.5 × 10⁴ cells per well seeded in 96-well plates. Then, the cell supernatants of each well were discarded and hesperetin (6.25, 12.5, 25, or 50 μmol/L) added to the hesperetin group after inoculation for 24 h. The supernatant of each well was discarded 24 h after dosing. Sodium urate (SU) (8 mg/dL) was added to cells. The uric acid content of the supernatants and broken cells was detected after incubation for 24 h in a 37°C incubator. Experiments were repeated at least thrice. The uric acid content of cell supernatants was measured using the uric acid kit.

After pretreatment of cells, they were rinsed with pre-cooled PBS, and lysed in high-efficiency radioimmunoprecipitation (RIPA) tissue/cell lysate (lysate: phenylmethylsulfonyl fluoride (PMSF) = 100:1) on ice for 20 min. After scraping, the cell lysate was centrifuged at 15,000 g for 10 min at 4°C with the supernatant collected. Protein from liver tissues and kidney tissues was extracted using RIPA tissue/cell lysate, homogenized for 2 min, and centrifuged at 1,036 g for 10 min at 4°C. The supernatant fractions from cells and tissues were collected using 1.5-mL centrifuge tubes. The supernatant with equal amount of protein was mixed with SDS-PAGE loading buffer and immediately heated at 100°C for 10 min. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 8%–12% polyacrylamide gels, and transferred to polyvinylidene fluoride (PVDF) membrane. After blocked non-binding sites with 5% skimmed milk for 1 h, the membrane was incubated overnight with specific antibodies in PBS: XOD (1:1,000), NLRP3 (1:1,000), IL-18 (1:200), TLR4 (1:200), NF-κB p65 (1:1,000), OAT1 (1:1,000), OAT3 (1:1,000), OCT1 (1:1,000), OCT2 (1:1,000), FOXO3a (1:1,000), MnSOD (1:1,000) or β-tubulin (1:1,000). Then, the blots washed thrice with tris-buffered saline with 0.1% Tween-20 (TBST) for 5-min each, and incubated with HRP-conjugated goat anti-rabbit IgG (1:5,000) or anti-mouse IgG (1:5,000) diluted with TBST for 1 h at room temperature. After three 5-min washes with TBST, images were acquired using a Pro-light HRP Chemiluminescence Kit (Tiangen Biotech, Beijing, China) on a FluorChem E System (Protein Simple, Santa Clara, CA, United States).

Statistical analyses were undertaken with Prism 5 (GraphPad, La Jolla, CA, United States), using mean ± standard error of the mean. Dunnett’s tests were used for post-hoc evaluations and p < 0.05 was considered statistically significant.

Allopurinol inhibited xanthine oxidase activity in a dose-dependent manner with an IC₅₀ of 6.044 μmol/L, whereas the IC₅₀ of the O^2- level was 11.2 μmol/L (Figures 1B,C). Hesperetin inhibited xanthine oxidase activity with an IC₅₀ of 110.4 μmol/L, whereas the IC₅₀ of the O^2- level was 317.6 μmol/L (Figures 1D,E). The inhibitory activity on xanthine oxidase elicited by hesperetin is consistent with the literature, though the IC₅₀ of hesperetin was higher than that of allopurinol (Haidari et al., 2009). Hesperetin appeared to be a potent inhibitor of xanthine oxidase activity.

Compared with normal mice, mice in which hyperuricemia had been induced using yeast extract showed markedly increased serum levels of uric acid, urea nitrogen, and creatinine and hepatic xanthine oxidase activity, indicating mild damage to kidney function (Figure 3). Serum levels of uric acid, urea nitrogen, and creatinine and hepatic xanthine oxidase activity were reduced markedly after hesperetin treatment (10 and 5 mg/kg) (p < 0.05/0.01), and the effect was similar to that of allopurinol. These results suggested that hesperetin not only reduced the serum uric acid level and hepatic xanthine oxidase activity but also protected the kidneys from damage caused by high-purine diets.

Expression of XOD (Figures 4A, B) and proteins associated with NLRP3 inflammasome signaling, such as TLR4 (Figure 4C), NF-κB (Figure 4D), NLRP3 (Figure 4E) and IL-18 (Figure 4F), was increased in the liver tissues of mice suffering from hyperuricemia induced by yeast extract compared with those in the normal group. Expression of all of these moieties was downregulated after hesperetin treatment for 14 successive days compared with that in the model group (p < 0.05). Similar results were observed in the allopurinol group, of them, the downregulation of IL-18 inflammatory signaling protein was inferior to that of hesperetin.

The model group showed elevated MDA in mice liver compared to the normal group, which was significantly reduced (p < 0.01) (Figure 5A) by HSE administration. Next, we further analyzed the endogenous antioxidant levels in liver. Compare the normal group, the model group remarkably diminished the levels of GSH-PX and CAT activities. Interestingly, 10 mg/kg of HSE obviously increased the GSH-PX (p < 0.01) (Figure 5B) and CAT activities (p < 0.01) (Figure 5C) compared to the model group. As expected, this data suggests that HSE treatment significantly reversed the oxidative stress parameters in the liver and prevented the development of its conditions. In addition, compared with the model group, HSE could significantly upregulate the protein levels of FOXO3a and MnSOD, which further indicated that HSE could improve the level of oxidative stress caused by yeast extract.

Hematoxylin and eosin staining of liver sections (Figure 6) from the yeast extract-induced hyperuricemia group showed obvious pathologic changes: unclear structure of hepatic lobules, degeneration of hepatocyte vacuoles, and inflammatory-cell infiltration. These pathologic changes could be alleviated by treatment with hesperetin (10 and 5 mg/kg) (Figure 6). The positive allopurinol group showed similar improvements in inflammatory cell infiltration and hepatocyte vacuolation in liver tissue. These results demonstrated that hesperetin exerted a hepatoprotective effect in mice with yeast extract-induced hyperuricemia.

Single injection with potassium oxonate significantly increased serum levels of uric acid in mice with hyperuricemia compared with that in normal mice (Figure 7), a result which is consistent with data from Tung Y. T (Tung et al., 2015). Hesperetin (10 and 5 mg/kg) could reduce serum uric acid levels and improve renal injury in mice with hyperuricemia 7 days after intraperitoneal injection (Figures 7A–C). Moreover, hesperetin (10 mg/kg) could upregulate expression of OAT1 (Figure 7E), OAT3 (Figure 7F), OCT1 (Figure 7G), and OCT2 (Figure 7H) proteins. Interestingly, allopurinol reduced serum levels of uric acid and improved renal injury, but weakly regulated expression of uric acid-excretion proteins compared with that of hesperetin at 10 mg/kg. These results indicated that hesperetin could reduce serum uric acid levels markedly through upregulation of expression of OAT1/OAT3/OCT1/OCT2 proteins to promote excretion of uric acid.

Uric acid is produced in the liver. A model of high uric acid in L-O2 hepatocytes was induced by a combination of hypoxanthine and xanthine oxidase. The level of uric acid in the model group was obviously more than that in the control group, so the hyperuricemia model had been established (Figure 8A). Then, the MTT assay was used to evaluate the action of hesperetin against hyperuricemia in L-O2 cells (Figure 8B). Interestingly, hesperetin exerted no obvious inhibition at 200 μmol/L (Figure 8B), but the uric acid level was decreased by 34% at 100 μmol/L of hesperetin (p < 0.001) (Figure 8C). Furthermore, hesperetin reduced expression of XOD protein compared with that in the model group (p < 0.05) (Figures 8D, E). These results indicated that hesperetin could reduce uric acid levels by inhibiting expression of XOD protein in a hyperuricemia model.

We wished to assess the effect of hesperetin on uric acid excretion. Hence, we used a sodium urate-induced model of hyperuricemia in HEK293T cells to detect changes in intracellular and extracellular uric acid levels. Hesperetin (25, 50, or 100 μmol/L) had hardly effect on the survival of HEK293T cells (p > 0.05) (Figure 9A). Compared with the control group, the intracellular uric acid content of the model group was remarkably increased (p < 0.001) (Figure 9B), showing that the model could be used in experimental studies. The intracellular uric acid level decreased gradually as the hesperetin concentration increased (p < 0.001) (Figure 9B), whereas the extracellular uric acid level increased gradually (p < 0.001) (Figure 9C) compared with that in the model group. These results showed that hesperetin could promote uric acid excretion from inside to outside of HEK293T cells.