Fisetin Attenuates Zinc Overload-Induced Hepatotoxicity in Mice via Autophagy-Dependent Nrf2 Activation

To establish a Zn overload model, different ZnSO₄ concentrations (150, 200, 250, 300, and 350 μM) and treatment time points (2, 4, and 6 h) were tested to verify cell viability. As shown in Figure 1A–C, exposure to varying concentrations of ZnSO₄ for different durations consistently induced a significant reduction in cell viability. After treatment with 250 μM ZnSO₄ for 4 h, approximately a 50% decrease in cell viability was observed relative to the control. Therefore, 250 μM ZnSO₄ and 4 h of culture were the parameters chosen for subsequent experiments. Afterwards, on the basis of 4 h of culture, various N,N,N′,N′-Tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) concentrations (30, 40, 50, and 60 μM) were tested to establish a Zn deficiency model. As demonstrated in Figure 1D, cell viability presented a significant reduction with the increase in TPEN concentrations. Moreover, approximately a 50% decrease in cell viability was observed relative to the control after treatment with 50 μM TPEN. Accordingly, 50 μM TPEN and 4 h of culture were the parameters chosen for subsequent experiments.

Intracellular Zn²⁺ level was detected by using a flow cytometer to verify whether the Zn imbalance model was successfully established. Compared to the control, treatment with 250 μM ZnSO₄ significantly increased the intracellular Zn²⁺ level while 50 μM TPEN significantly decreased the intracellular Zn²⁺ level, which indicated that Zn imbalance models were effectively established (Figure 1E,F). To assess the influence of Zn imbalance on cell damage, the morphology of AML12 cells was photographed. Treatment with ZnSO₄ and TPEN induced significant changes in morphology and cell numbers (Figure 1G). Next, to further evaluate the damage degree of AML cells, we measured the vitality of supernatant lactate dehydrogenase (LDH), which rapidly releases outside the cell when the plasma membrane is damaged and is a key feature of cellular damage. As presented in Figure 1H, compared to the control group, treatment with ZnSO₄ and TPEN both remarkably increased the vitality of the supernatant LDH.

To delineate the impact of Zn dyshomeostasis on redox imbalance, intracellular reactive oxygen species (ROS) flux was quantitatively profiled using probe 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) fluorescence spectrometry, complemented by a systematic evaluation of antioxidant defense capacity through measurement of total antioxidant capability (T-AOC), total superoxide dismutase (T-SOD), glutathione (GSH), and malondialdehyde (MDA) using standardized commercial assays. It can be presumed from Figure 2A,B that both ZnSO₄ and TPEN treatment drastically escalated cellular ROS accumulation compared to the control group. However, as for MDA, only TPEN treatment significantly increased the level of MDA (Figure 2C). T-AOC and GSH were remarkably decreased after the treatment of both ZnSO₄ and TPEN, while T-SOD had no obvious exposure effect on ZnSO₄ or TPEN (Figure 2D–F). To more precisely examine cellular oxidative stress, the expression levels of antioxidant proteins were examined. TPEN exposure had no effect on the expression of p-Nrf2 and its downstream protein heme oxygenase 1 (HO-1), while ZnSO₄ treatment significantly enhanced the expression of p-Nrf2 and HO-1 (Figure 2G–I).

Annexin V-FITC/PI staining, the protein expression levels of Bax, cleaved caspase3, LC3B, and p62 were measured to investigate the impact of Zn imbalance on apoptosis and autophagy. As shown in Figure 3A, the percentages of apoptotic cells were slightly increased following TPEN treatment compared with the control group (Figure 3A). However, no statistically significant alterations in apoptosis-associated proteins Bax and cleaved caspase-3 expression were observed in either ZnSO₄- or TPEN-treated groups relative to control conditions (Figure 3B–D). Furthermore, quantitative analysis of autophagic flux revealed differential responses among treatment groups. Compared to the control group, TPEN administration significantly reduced p62 protein levels without altering the LC3BII/I ratio. In contrast, ZnSO₄ treatment not only decreased p62 expression but also increased the LC3BII/I ratio, suggesting enhanced autophagosome formation. Autophagy inhibitor chloroquine (CQ) treatment, as expected, markedly increased both p62 and the LC3BII/I ratio (Figure 3E–G). These findings collectively demonstrate that ZnSO₄ treatment effectively enhanced cellular autophagy activity.

To study the alleviated effect of fisetin on the decline in cell viability induced by Zn imbalance, AML12 cells were cultured with ZnSO₄ or TPEN in the presence or absence of fisetin for 4 h before cell viability detection. In the beginning, increasing doses of fisetin (20, 40, and 60 μM) were used for co-incubation with ZnSO₄ to test whether fisetin could minimize the negative impact on cell viability caused by Zn overload. As illustrated in Figure 4A, fisetin treatment at 40 μM demonstrated a more pronounced cytoprotective effect against ZnSO₄-induced cellular viability inhibition compared to 20 μM, though no statistically significant difference was observed between 40 μM and 60 μM concentrations. Notably, co-treatment with all tested fisetin concentrations (20–60 μM) significantly attenuated the detrimental effects of Zn overload on cellular viability. Next, 40 μM fisetin was used to detect its function on TPEN-induced cell viability decrease. Unexpectedly, fisetin failed to recover the decline in cell viability resulting from Zn deficiency (Figure 4B). Collectively, these results uncover that fisetin had a remarkable protective effect on Zn overload-triggered cell viability decrease but did not alleviate the Zn deficiency-evoked effect on cell viability; thus, the influence of fisetin on Zn overload-induced cell injury was mainly studied in subsequent experiments. Next, morphology investigation and supernatant LDH vitality detection of AML12 cells were performed. Fisetin ameliorated significant Zn overload-induced changes in morphology and rendered the AML12 cells close to the normal morphology (Figure 4C). In addition, supernatant LDH vitality decreased after fisetin administration under conditions of ZnSO₄ treatment (Figure 4D). Polyphenols contain abundant phenolic hydroxyl groups which could act as donors in metal–chelator interactions [26]. To rule out that the protective effects of fisetin would be due to the chelation of Zn²⁺ ions, we determined the intracellular Zn²⁺ level. Compared to the control group, ZnSO₄ treatment remarkably increased the Zn²⁺ level. However, fisetin co-treatment exerted no influence on Zn²⁺ level when compared to the ZnSO₄ group (Figure 4E,F). Consequently, the mitigating effect of fisetin on Zn overload was not dependent on chelated Zn²⁺ ions. These results suggest that the detrimental impact of Zn overdose on cell damage could be intensively abolished by fisetin.

Fisetin has been reported to exert antioxidant effects [19], so ROS accumulation and the levels of T-AOC, GSH, and MDA were measured to reveal the response of fisetin to ZnSO₄-induced oxidative stress. We found that ROS accumulation in ZnSO₄-exposed AML12 cells could be mitigated by fisetin co-treatment (Figure 5A,B). In addition, we observed that fisetin co-treatment had no influence on MDA but notably reverted the ZnSO₄-induced decline in T-AOC and GSH levels when compared to the ZnSO₄ group (Figure 5C–E). These results suggest that fisetin is capable of attenuating Zn overload-induced ROS accumulation and antioxidant response suppression in AML12 cells.

To validate these findings and elucidate the causal relationship between autophagic flux modulation and Nrf2-mediated antioxidant response activation, we further implemented CQ and dissected Nrf2 signaling. Notably, co-treatment with fisetin augmented ZnSO₄-induced autophagic flux, as evidenced by exacerbated p62 degradation and the amplified LC3BII/I ratio (Figure 5F–H). Consistently, fisetin further significantly enhanced ZnSO₄-induced Nrf2 activation, while CQ co-treatment significantly attenuated p-Nrf2 levels and HO-1 expression (Figure 5I–K), despite inducing expected autophagosome accumulation (Figure 5F–H). This functional antagonism demonstrates that intact autophagic flux is a prerequisite for fisetin’s amplification of ZnSO₄-induced Nrf2 signaling.

Compared to control mice, excess Zn exposure reduced body weight in mice, whereas fisetin supplementation partially ameliorated this weight loss, although not statistically significantly (Figure 6A,B). Consistent with in vitro findings, fisetin markedly potentiated Zn overload-induced autophagic flux, as evidenced by enhanced degradation of p62 and an elevated LC3BII/I ratio (Figure 6C–E). Oxidative stress analysis demonstrated that fisetin attenuated Zn overload-triggered ROS overproduction and MDA level changes while increasing T-AOC and GSH levels (Figure 6F–I). Moreover, fisetin potentiated the activation of the Nrf2 signaling pathway, demonstrated by enhanced p-Nrf2 and its downstream protein HO-1 (Figure 6J–L).

Mouse hepatocytes AML12 (Keygen Biotech, Nanjing, China) were grown in DMEM/F12 (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, Australia), 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA), 0.5% ITS-G (Pricella, Wuhan, China), and 40 ng/mL dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a humidified 5% CO₂ incubator.

To establish the Zn imbalance cell model, cells were treated with Zn overload by incubating them with various concentrations (150, 200, 250, 300, and 350 μM of Zn concentration) of ZnSO₄ (Sigma-Aldrich, St. Louis, MO, USA) for various durations (2, 4, and 6 h); cells were also treated with Zn deficiency by incubating them with various concentrations (30, 40, 50, and 60 μM) of TPEN (a chelator for Zn; MedchemExpress, Monmouth Junction, NJ, USA) for 4 h.

To investigate the effects of fisetin on liver injury induced by Zn imbalance and its mechanism, the cells were treated with ZnSO₄ or TPEN in the presence or absence of various concentrations (20, 40, and 60 μM) of fisetin (dissolved in dimethyl sulfoxide, HY-N0182, purity 99.99%, MedchemExpress, Monmouth Junction, NJ, USA) for 4 h. Moreover, 20 μM CQ (MedchemExpress, Monmouth Junction, NJ, USA) was used for co-treatment with ZnSO₄, TPEN, and fisetin to explore the role of autophagy in Zn imbalance-induced cellular damage.

Cell viability was determined by using the Cell Counting Kit-8 (CCK-8) assay (MedchemExpress, Monmouth Junction, NJ, USA) according to established protocols. Cells were dispensed in 96-well plates at a density of 10⁴ cells per well and incubated for 24 h. Cell-free wells and cell untreated wells were set as background correction and control groups, respectively. After the indicated treatments, 10 μL of CCK-8 reagent was added to each well and then incubated for 2 h, following which absorbance was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). The data are shown as the percentage of the control.

Cells were seeded in 6-well plates. After the indicated treatments, cells were lysed and centrifuged to collect the cell supernatant, then washed twice with phosphate buffer saline (PBS) before loaded with 1 μM fluozin^TM-3, AM (Invitrogen, Carlsbad, CA, USA) for 1 h at 37 °C, and lastly washed once with PBS. The cell pellet was resuspended in 500 μL PBS and analyzed using a flow cytometer from FITC channel (FACS Calibur, BD Biosciences, San Jose, CA, USA). A minimum of 10,000 cells were analyzed per production.

Cells were seeded in 6-well plates. After the indicated treatments, the cells were photographed immediately under a microscope (Leica, Wetzlar, Germany), and then a 100 μL supernatant sample of every well was collected and LDH was quantified using the LDH assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

CM-H2DCFDA (Invitrogen, Carlsbad, CA, USA) was used to monitor the intracellular accumulation of ROS, which induces oxidative stress. Briefly, cells were seeded in 6-well plates. After the indicated treatments, cells were lysed and centrifuged to collect the cell supernatant, then washed twice with PBS before being loaded with 5 μM CM-H2DCFDA for 10 min in the dark at 37 °C, and lastly washed once with PBS. The cell pellet was resuspended in 500 μL PBS and analyzed using a flow cytometer from FITC channel (FACS Calibur, BD Biosciences, San Jose, CA, USA). A minimum of 10,000 cells were analyzed per production. The quantification of ROS in the liver was conducted in accordance with the methodology outlined by Gabbia et al. [42].

The levels of MDA and T-AOC as well as the activity of reduced GSH and T-SOD levels were measured using a specific commercial assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). After treatment, the detection of cell samples was performed according to the manufacturer’s instructions.

Apoptosis was evaluated using the Annexin V-Enhanced Green Fluorescent Protein (FITC)/Propidium Iodide (PI) staining kit (Keygen Biotech, Nanjing, China). Cells (10⁵ per well) were inoculated into 12-well plates containing sterile cell climbing tablets. After the indicated treatments, the culture medium was removed and cells were washed twice with cold PBS cells. A 500 µL binding buffer was mixed with 5 µL Annexin V-FITC and 5 µL PI, and cells were stained with the mixture for 5 min at room temperature in the dark. Subsequently, the images were acquired by using a fluorescence microscope (Leica, Wetzlar, Germany). Live cells show little or no fluorescence (Annexin V⁻/PI⁻), early apoptosis cells show green fluorescence (Annexin V⁺/PI⁻), and late apoptosis cells and necrosis cells show red and green fluorescence (Annexin V⁺/PI⁺).

Following experimental interventions, cells and liver samples were collected and lysed with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing a protease inhibitor and phosphatase inhibitors. The total protein concentrations in the samples were measured by using a BCA assay kit (Keygen Biotech, Nanjing, China). Equal masses of protein samples were separated on sodium dodecyl sulfate-polyacrylamide gels and transferred from the gels onto polyvinylidene fluoride membranes. After blocking with TBST containing 5% skim milk for 1.5 h at room temperature, the membranes were incubated overnight at 4 °C with appropriate primary antibodies: β-actin (M1210-2, HUABIO, Hangzhou, China), Bax (ET1603-34, HUABIO, Hangzhou, China), cleaved caspase-3 (ET1602-47, HUABIO, Hangzhou, China), Nrf2 (16396-1-AP, Proteintech, Wuhan, China), p-Nrf2 (ab76026, Abcam, Cambridge, MA, USA), HO-1 (HA721854, HUABIO, Hangzhou, China), LC3B (db15555, Diagbio, Hangzhou, China), and p62 (HA721171, HUABIO, Hangzhou, China). On the following day, the membranes were incubated with the corresponding secondary antibody HRP goat anti-rabbit lgG (H+L) (15015, Proteintech, Wuhan, China) or HRP goat anti-mouse lgG (H+L) (15014, Proteintech, Wuhan, China) for 1.5 h at room temperature. Detection was performed by using an ECL kit (Boster, Wuhan, China) and Chemiluminescence imager (Bio-Rad, Hercules, CA, USA). Finally, the images were analyzed using Image Lab software version 6.0. The results of relative phosphorylated protein expression were calculated by normalization to total protein, while others were expressed as the abundance of each target protein relative to β-actin.

All of the procedures involving animals and their care have been conducted strictly according to the Institutional Animal Care and Use Committee of Zhejiang University (AP code: ZJU20240807, 16 October 2024). Three-week-old specific pathogen-free male C57BL/6 mice were purchased from Slac Laboratory Animal (Slac, Shanghai, China) and equally randomized into four groups: (1) control group (Control), standard Zn diet (30 mg Zn/kg) and daily oral administration of 0.5% carboxymethyl cellulose sodium (CMC-Na; YuanYe, Shanghai, China); (2) fisetin group (Fis), standard Zn diet and daily oral administration of fisetin (200 mg/kg BW; suspended in 0.5% CMC-Na, S31422, purity ≥ 98%, YuanYe, Shanghai, China); (3) excess Zn group (Zn), excess-Zn diet (600 mg Zn/kg) and daily oral administration of 0.5% CMC-Na; (4) excess Zn + fisetin group (Zn + Fis), excess-Zn diet and daily oral administration of fisetin. The Zn source was zinc carbonate and the dosage of Zn referred to a previously published article [43]. Liver samples were collected after 16 days and snap-frozen in liquid nitrogen for future analysis.

Significant differences were analyzed by one-way ANOVA using the general linear model procedures of SPSS version 22 (SPSS Inc., Armonk, NY, USA). Data were presented as the mean ± SD. A value of p < 0.05 was considered statistically significant.