Que (3,3,4,5,7-pentahydroxyflavone, purity ≥95%) was purchased from Solarbio (Beijing, China). Fetal bovine serum (FBS) and DMEM were obtained from Gibco (United States). The primary antibodies, including anti-glutathione peroxidase 4 (GPX4), anti-cyclooxygenases-2 (COX2), anti-ACSL4, anti-GAPDH, and IgG (H + L)-HRP secondary antibodies were purchased from Proteintech Bio, Inc. (Wuhan, China).

Six-week-old male C57BL/6J mice (18–22 g) were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Mice were maintained under specific pathogen-free conditions at 23 ± 2°C and a 12-h light/dark cycles and had free access to food and water. All animal protocols were approved by the Animal Care and Use Committee of the Xinxiang Medical University.

After 1  week of adaptive feeding, all mice were randomly divided into the following four groups (n = 10 per group): 1) the normal diet (ND) group, mice fed a ND (18% calories from fat; TROPHIC Animal Feed High-tech Co., Ltd., China); 2) the HFD group, mice fed a HFD (45% calories from fat; TROPHIC Animal Feed High-tech Co., Ltd., China) for 12 weeks when the experiment beginning; 3) the HFD-Que/L group, mice fed a HFD supplemented with 50 mg/kg body weight; and 4) the HFD-Que/H group, mice fed a HFD supplemented with 100 mg/kg body weight. After the experiment beginning, mice in the Que-treated groups were administered Que (50 and 100 mg/kgbw) via orally gavage daily for 12 weeks, and mice in the ND and HFD groups were administered deionized water at the same time via oral gavage. After treatment, the mice were sacrificed by intravenous pentobarbital injection (20 mg/kg). Blood samples were obtained by cardiac puncture to separate the serum. Liver tissues were quickly removed, weighed and then fixed in 4% formaldehyde solution for histopathologic examination or snap-frozen in liquid nitrogen and stored at −80°C until use.

L-02 cells were incubated at 37°C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Cell viability was assessed by the Cell Counting Kit-8 (CCK-8) assay (Beyotime Biotechnology, Shanghai, China). L-02 cells were treated with Que for 24 h, after which CCK-8 reagent was added to the cell wells, and the absorbance was determined at 450 nm.

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined using commercial enzyme kits (Nanjing Jiancheng Bioengineering, Inc., Nanjing, China). Hepatic total lipids were extracted as previously described (Loison et al., 2002), and the levels of total cholesterol (TC) and total triglycerides (TG) in livers were then measured using commercial assay kits (Sangon Biotech, Inc., Shanghai, China) according to the manufacturer’s protocols.

Iron content in liver tissue and cells was determined by a colorimetric assay kit according to the manufacturer’s instructions (MLBIO, Inc., Shanghai, China).

Frozen liver sections from OCT-embedded liver samples were stained with Oil Red O to assess fat accumulation. Liver samples were fixed in 4% paraformaldehyde for 24 h at room temperature and embedded in paraffin, and 5 μm-thick sections were then stained with hematoxylin-eosin (H&E) for histological assessment. A point-counting method was used for calculating hepatic steatosis as previously described (Catta-Preta et al., 2011; Xu et al., 2017).

To establish the MD, L-02 cells at 80% confluency were incubated with a 0.5 mmol/L free fatty acid (FFA) mixture (Kunchuang Biotechnology, Xi’an, China) for 24 h and then used for further experiments. Palmitic acid (PA) was coupled to palmitate-free bovine serum albumin (BSA) in a ratio of 2 mM PA: 3% BSA in a 40°C water bath to prepare the FFA mixture.

To evaluate intracellular TG content, L-02 cells were scraped. Then TG content was determined using a commercial assay kit (Sangon Biotech, Inc., Shanghai, China) according to the manufacturer’s protocols.

Before treatment with the FFA mixture, L-02 cells were pretreated with the ferroptosis specific inhibitor ferrostatin-1 (Fer-1, 10 μmol/L) (APEx Bio, United States) for 4 h.

The contents of reduced glutathione (GSH)/GSSG (oxidized glutathione) in the liver tissues of mice and cell lysates were measured by commercial assay kits (Nanjing Jiancheng Bioengineering, Inc., Nanjing, China) according to the manufacturers’ instructions. The level of 4-hydroxynonenal (4-HNE) in the liver tissues of mice and cell lysates was determined with a commercial ELISA kit (MLBIO, Inc., Shanghai, China).

To observe the lipid droplets in hepatocytes, L-02 cells were incubated in six-well plates, treated as described, washed with phosphate-buffered saline three times, fixed with 4% paraformaldehyde for 30 min, and stained with Oil red O for 15 min at room temperature. A microscope connected to a digital camera (Olympus, Tokyo, Japan) was used to observe and capture lipid droplets in L-02 cells.

The L-02 cells were pretreated with Que (50 μmol/L) or mito-TEMPO (60 μmol/L) (APEx Bio, United States) for 4 h and then incubated with the FFA mixture for 24 h to establish the steatotic hepatocyte model. After the experimental treatment, L-02 cells were incubated in 5 μmol/L MitoSox Red Mitochondrial Superoxide indicator (Thermo, United States) in phosphate buffered saline at 37°C for 30 min in the dark. The cells were washed with warm phosphate-buffered saline three times and then immediately analyzed by flow cytometry.

The L-02 cells were pretreated with Que (25 and 50 μmol/L) for 4 h and then incubated with the FFA mixture for 24 h to establish the steatotic hepatocyte model. Cells were incubated with the Molecular Probes BODIPY 581/591C11 (Invitrogen, United States) working solution (5 μmol/L) at 37°C for 30 min in the dark. The cells were washed with phosphate buffered saline three times and measured using flow cytometric analysis.

Total proteins were extracted from liver tissues and L-02 cells using RIPA lysis buffer protein extraction reagent (key GEN, Nanjing, China). The protein concentrations of the samples were measured using a BCA protein assay kit (DINGGUO, Bio Co., Ltd., Beijing, China). Protein samples were electrophoresed on polyacrylamide gels and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk in PBS and incubated with primary antibodies against GPX4, COX-2, ACSL4, and GAPDH overnight at 4°C, followed by incubation with the corresponding secondary antibody for 1 h. The protein bands were detected using an ECL detection system (Tanon, Shanghai, China). The relative intensities of the target bands were quantified by Image Studio Software (Li-Cor Biosciences) and normalized to the optical density of GAPDH.

Data are expressed as the mean ± standard deviation (SD). All data were analyzed with SPSS 25.0 (SPSS, Chicago, IL, United States). Comparisons of groups were analyzed using one-way ANOVA followed by post hoc analysis (Bonferroni posttest), and a p value less than 0.05 was considered to indicate statistical significance.

As shown in Figures 1A,B, HFD-fed mice showed increased body weight gains and the ratio of liver weight/body weight compared to those of mice in the ND group, and administration of Que significantly alleviated the increases in body weight gain caused by the HFD. To assess the lipid metabolism status after HFD feeding, the levels of TG and TC in the serum and liver were measured (Figures 1C–F). Compared to the ND group, the serum and hepatic levels of TG and TC were markedly elevated in the HFD group. Moreover, Que supplementation attenuated the TG and TC levels in HFD-fed mice in a dose-dependent manner. Liver sections stained with H&E and Oil Red O were used to evaluate the liver histopathological changes in the four groups. As shown by H&E staining, the occurrence of hepatic steatosis was significantly higher in the HFD-fed mice than in the mice of the ND group (Figure 2A). Consistent with the results of hepatic steatosis, mice in the HFD group showed more lipid droplet accumulation than those in the ND group (Figure 2B). Furthermore, the hepatic steatosis percentage and lipid droplets were much lower in the HFD-Que/L and HFD-Que/H group mice than in mice of the HFD group (Figure 2C). In addition, the serum levels of AST and ALT were increased in the mice of the HFD group compared with the ND group, and the elevated serum AST and ALT levels in the HFD-fed mice were alleviated by Que supplementation to varying degrees (Figures 2D,E).

To explore whether Que alleviated HFD-induced hepatic lipid peroxidation in vivo, we measured the levels of 4-HNE and GSH/GSSG (Figures 3A,B). Compared with those in the ND group, the concentration of 4-HNE was obviously upregulated and the ratio of GSH/GSSG was obviously downregulated in the HFD group. However, the concentrations of 4-HNE in the HFD-Que/L and HFD-Que/H groups were significantly lower than that in the HFD group. Moreover, the ratio of GSH/GSSG in the Que-treated mice was significantly higher than that in the HFD group. These data indicated that Que has a protective effect on HFD-induced hepatic lipid peroxidation.

To study whether Que has an inhibitory effect on hepatic ferroptosis in vivo, we determined the iron content and the protein expression of GPX4, COX-2, and ACSL4 in liver tissues (Figures 3C–G). Compared to the ND group, the liver iron content was markedly increased in the HFD group, and the elevated liver iron content in the HFD-fed mice were alleviated by Que supplementation (Figure 3C). Compared with that in the ND group, GPX4 expression was significantly downregulated in the HFD group, while COX-2 and ACSL4 expression was significantly higher in the HFD group than in the ND group (Figures 3D–G). Moreover, Que supplementation markedly increased GPX4 protein expression and obviously decreased the COX-2 and ACSL4 protein expression in liver tissues (Figures 3D–G). These data indicated that long-term HFD treatment induced hepatic ferroptosis in vivo, which was rescued by Que supplementation.

The viability of L-02 cells exposed to Que was evaluated by CCK-8 assays. L-02 cells were treated with different doses of Que (0–100 μmol/L) for 24 h. The viability of L-02 cells treated with Que showed a slight decline at the high dose of Que (100 μmol/L) (Figure 4A). The flow cytometry results showed that lipid ROS accumulated in steatotic L-02 cells, and Que treatment reduced the production of lipid ROS in steatotic L-02 cells (Figures 4B,C). As shown in Figures 4D,E, lipid accumulation in the MD group was significantly higher than that in the control group. Compared with the MD group, Que (50 μmol/L) decreased the lipid accumulation in steatotic L-02 cells (Figures 4D,E).

The content of 4-HNE was apparently increased and the ratio of GSH/GSSG was decreased in the MD group compared with the control group (Figures 5A,B). Moreover, the increase in 4-HNE and the decrease in the GSH/GSSG ratio in steatotic hepatocytes were reduced by Que treatment (Figures 5A,B). The cellular iron content in the MD group was higher than the control group, and elevated cellular iron content in steatotic hepatocytes was decreased by Que treatment (Figure 5C). As shown in Figures 5D–G, compared with those in the control group, the protein expression of GPX4 was downregulated and the protein expression of COX-2 and ACSL4 was upregulated in the MD group. Additionally, both the decreased GPX4 expression and the increased COX-2 and ACSL4 expression in the MD group were rescued by Que treatment. Taken together, these data suggest that Que inhibits ferroptosis in steatotic hepatocytes.

To explore the link between MtROS and ferroptosis in steatotic hepatocytes, we pretreated steatotic L-02 cells with Mito-TEMPO (the MtROS scavenger). Compared with the control group, the level of MtROS was significantly increased in the MD group (Figures 6A,B). As presented in Figures 6A,B, Que and Mito-TEMPO had similar effect on the reducing of MtROS in steatotic hepatocytes. To further explore the role of ferroptosis in lipid droplet accumulation and lipid peroxidation in steatotic hepatocytes, the ferroptosis specific inhibitor Fer-1 was used. As shown in Figures 6C,D, the lipid droplet accumulation and the TG content in steatotic L-02 cells was reduced by Que treatment and Fer-1. We next compared the effects of Que with Mito-TEMPO and Fer-1 on lipid peroxidation and ferroptosis in steatotic hepatocytes. We found that Que exerted similar effects to these of Mito-TEMPO and Fer-1 in reducing the level of lipid peroxidation and inhibiting ferroptosis in steatotic hepatocytes (Supplementary Figures S1, S2). Together, these data indicated that ferroptosis was involved in lipid accumulation and cellular ferroptosis in steatotic L-02 cells was mediated by MtROS, which was inhibited by Que treatment.