Melatonin Prevents the Progression of MASLD via Inhibiting FFAs-Induced Ferroptosis through KEAP1/NRF2/HO-1 Pathway

Abstract

The accumulation of free fatty acids (FFAs) in hepatocytes is a key characteristic of metabolic dysfunction-associated steatotic liver disease (MASLD), which leads to lipid peroxidation and ultimately results in ferroptosis. Currently, there is an absence of efficacious therapeutic options available for the management of MASLD. Consequently, an in-depth exploration of the roles of FFAs and ferroptosis in the progression of MASLD may reveal hitherto unidentified therapeutic targets. In the study, we established an early lesion model of MASLD, namely NAFL, and comprehensive analyses of lipid metabolism, hepatocellular injury, iron homeostasis, and ferroptosis were performed. The HFD and FFAs treatment significantly elevated the expression of enzymes associated with lipid synthesis, including ACC1 and FASN, leading to enhanced lipid accumulation in hepatocytes. Additionally, HFD and FFAs resulted in increased iron loading and a reduction in the levels of the antioxidant enzyme GPX4, which ultimately triggers ferroptosis. In contrast, the administration of melatonin effectively inhibited the activity of lipid synthesis-related enzymes, decreased hepatic lipid deposition, alleviated free fatty acid-induced iron dysregulation, and mitigated liver damage. Mechanistically, melatonin has been shown to attenuate hepatocyte ferroptosis by modulating the KEAP1/NRF2/HO-1 pathway, which in turn diminishes free fatty acids-induced oxidative stress. In conclusion, melatonin alleviates MASLD progression by curbing FFAs-induced oxidative stress and ferroptosis. These findings provide valuable insights into the mechanisms underlying MASLD progression and highlight melatonin as a potential therapeutic agent for the management of MASLD.

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a worldwide chronic liver disease, its predecessor being non-alcoholic fatty liver disease (NAFLD), characterized primarily by lipid accumulation in hepatocytes (Fan et al., 2024). It affects more than a third of the world’s adult population, with an even higher incidence observed in Asia (Tacke et al., 2024). MASLD encompasses a spectrum of liver diseases ranging from simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH), which may progress to cirrhosis and hepatocellular carcinoma (Wang et al., 2024). Several factors, including apoptosis, necrosis, and autophagy, have been shown to exacerbate its pathogenesis(Shojaie et al., 2020). Notwithstanding the exhaustive research that has been conducted, effective treatments for MASLD remain unavailable.

In the initial stages of MASLD, there is an imbalance in the body’s lipid metabolism or an excessive intake of free fatty acids (FFAs), which leads to an accumulation of fatty acids in hepatocytes, resulting in hepatic steatosis (Ipsen et al., 2018). FFAs, a major metabolic by-product released from adipose tissue and other cell types, have been observed to accumulate in the liver, thereby promoting the progression of MASLD (Anwar et al., 2023). Fatty acid synthase (FAS) catalyzes the production of long-chain fatty acids, either directly or indirectly, serving as a source of FFAs (Heeren and Scheja, 2021). The expression of FAS is subject to stringent regulation by various gene transcription factors that respond to intracellular and extracellular signals. It is evident that these factors modulate the expression of FAS and influence the metabolism of FFAs, which ultimately impacts the rate of hepatic lipid synthesis (Kimura et al., 2020; Paul et al., 2022). A hallmark pathological feature of MASLD is the accumulation of lipids, n-6 polyunsaturated fatty acids (PUFAs), such as linoleic acid within hepatocytes(Lands, 2014). In the liver, PUFAs are susceptible to peroxidation in iron-dependent reactions, resulting in the production of lipid peroxides (Jiang et al., 2021). This oxidative process has been demonstrated to cause damage to cell membranes and to trigger ferroptosis, a form of regulated cell death (Videla and Valenzuela, 2022). It is hypothesized that the modulation of lipid-metabolizing enzymes is critical for cell ferroptosis and may provide a promising approach to address MASLD.

Ferroptosis is a newly identified form of iron-dependent cell death distinguished by lipid peroxidation and intracellular iron accumulation (Pope and Dixon, 2023). FFAs serve as essential precursors for lipid peroxidation, while iron catalyzes the peroxidation of PUFAs via the Fenton reaction, which produces highly reactive lipid peroxides that ultimately contribute to cellular death (He et al., 2022). Studies have indicated that patients suffering from MASLD frequently present with hepatic iron overload, which is characterized by an excess of oxidative stress products generated through the Fenton reaction, and has been demonstrated to result in cellular damage (Tong et al., 2022). Dysregulated hepatic iron metabolism contributes to ferroptosis, thereby worsening liver injury and facilitating the progression of MASLD to MASH. This process is initiated by the activation of hepatic stellate cells, which in turn leads to an increase in liver fibrogenesis (Gao et al., 2022). Consequently, strategies focused on modulating iron metabolism, reducing oxidative stress, or inhibiting lipid peroxidation hold promise as effective approaches to mitigate the progression of MASLD.

Melatonin, a hormone secreted by the pineal gland, plays diverse physiological roles, including the regulation of the sleep-wake cycle, antioxidant activity, immunomodulation, and anti-tumor effects (Bhattacharya et al., 2019; Minich et al., 2022). A growing body of evidence highlights a close relationship between melatonin and lipid metabolism. For instance, melatonin has been demonstrated to regulate the hypothalamic-pituitary-adrenal (HPA) axis through the activation of melatonin receptors (MT1 and MT2, human G-protein-coupled receptors), influencing lipolysis and fat synthesis in adipocytes (Wang et al., 2019; Jin et al., 2022). Under dark conditions, melatonin inhibits insulin activity, reduces fat synthesis in adipocytes, promotes lipolysis, and elevates FFAs levels in the blood (Small et al., 2023). The accumulation of lipids within the liver can initiate a series of intracellular processes, including lipid peroxidation and an increase in iron loading. Notably, melatonin has been shown to regulate hepatic lipid metabolism, thereby impeding the accumulation of excessive lipid (Ku et al., 2023). As a potent antioxidant, melatonin has been shown to inhibit ferroptosis and liver inflammation by reducing lipid peroxidation and oxidative stress, thereby alleviating liver injury (Chitimus et al., 2020). However, it remains unclear whether melatonin directly affects hepatocytes ferroptosis by modulating lipid synthesis during the progression of MASLD.

In this study, we employed an in vitro and in vivo model of MASLD induced by FFAs and a high-fat diet to explore the role of melatonin in the progression of MASLD. Our findings demonstrated that melatonin effectively inhibited fatty acid synthesis, reduced lipid accumulation in hepatocytes, diminished intracellular iron loading, and suppressed hepatocyte ferroptosis through its antioxidant properties, which ultimately alleviated liver injury. These results suggest that melatonin may serve as a potential therapeutic agent for decreasing hepatic lipid accumulation and inhibiting ferroptosis in MASLD.

MATERIALS AND METHODS

Reagents

Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Nanjing Wisent Biotechnology Co. Ltd. (Nanjing, China). Free fatty acid solvent kit (No. KC006) was acquired from Xi’an Kunchuang Science and Technology Co. Ltd. (Xi’an, China). Melatonin (No. PHR1767) was procured from Merck KGaA (Darmstadt, Germany). The Cell counting kit-8 (CCK-8; No. GK10001) was obtained from GIPBio (CA, USA). The modified oil Red O kit (No. C0158S), the lipid oxidation (MDA) detection kit (No. S0131S), and the BCA kit (No. P0009) were purchased from Shanghai Beyotime Technology Co. Ltd. (Shanghai, China). The superoxide dismutase (SOD) kit (No. BC5165), the reduced glutathione (GSH) kit (No. BC1175), the Hoechst 33342 staining solution (No.C0030), and the DAPI solution (No.C0065) were obtained from Beijing Solarbio Technology Co. Ltd. (Beijing, China). The triglyceride kit (TG; No. A110-1-1) and total cholesterol kit (T-CHO; No. A111-1-1) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Primary antibodies, such as anti-NRF2 (No.380773), anti-GPX4 (No.381958), anti-HO-1 (No. R24541) and anti-TFR1 (No. R25971) were acquired from Chengdu Zenbio Technology Co. Ltd. (Chengdu, China), anti-FASN (No.C20G5) from Cell Signaling (MA, USA), anti-SREBP1 (No. ab313881) from Abcam (Cambridge, UK). Additionally, primary antibodies, including anti-ACC1 (No. 29119-1-AP), anti-SCD1 (No.28678-1-AP), anti-DMT1 (No.81609-1-RR), anti-iNOS (No.18985-1-AP), and anti-β-Actin (No.66009-1-Ig) were from Wuhan Proteintech Biotechnology Co. Ltd. (Wuhan, China). The first-strand cDNA synthesis kit (No. 11171ES03) and AceQ qPCR SYBR Green Master Mix (No. 11201ES03) were purchased from Shanghai Yesen Biotechnology Co. Ltd. (Shanghai, China).

Cell culture

The human hepatocellular carcinoma cell line HepG2 was obtained from Wuhan Prosperity Life Sciences Co., Ltd. (Wuhan, China). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂. For subsequent experiments, cells in the logarithmic growth phase were utilized.

Cell counting kit-8 (CCK-8) assay

Free fatty acids (FFAs) were prepared at a 2:1 molar ratio of oleic acid (OA) to palmitic acid (PA) dissolved in fatty-acid-free BSA. HepG2 cells were seeded into 96-well plates at a density of approximately 1×10⁴ cells per well and allowed to adhere for 6 h. Subsequently, the medium was replaced with a basal medium containing varying concentrations of FFAs (0, 0.1, 0.25, 0.5, 1.0, and 2.0 mM, calculated as OA equivalents) and/or melatonin (0, 0.5, 1, 2.5, 5, and 10 µM). Following a 24-h incubation period, 10 µL of CCK-8 was added to each well, followed by an additional 2-h incubation at 37°C. The absorbance was measured at a wavelength of 450nm using a fully automated microplate reader (BioTek; VT, USA).

Divalent iron (Fe²⁺) detection

The staining working solution was prepared by mixing 5 µL of probe solution with 10 mL of cell staining buffer, yielding a final concentration of 1 µM. The solution was directly applied to the cells for staining. Following a thorough cleansing with cell staining buffer, 1 mL of the staining solution was added to each 1×10⁶ cells, followed by an incubation at 37°C in a humidified atmosphere containing 5% CO₂ for 1 h. Subsequently, the nuclei of live cells were stained with the Hoechst solution for a duration of 10 min. The cells were then washed with phosphate-buffered saline (PBS) and visualized using a laser scanning confocal microscope (Carl Zeiss; BW, Germany).

Immunofluorescence (IF) assay

HepG2 cells were washed with PBS and then fixed in 4% paraformaldehyde at room temperature. The cells were further permeabilized with 0.2% Triton X-100 and blocked with 5% BSA to prevent nonspecific binding. Subsequently, the slides were subjected to an overnight incubation at 4°C with primary antibodies against ACC1 or NRF2. After washing with PBS, the slides were co-incubated with AF488-conjugated streptavidin at room temperature in the dark. Finally, the nuclei were stained with DAPI for 15 min, and the cells were imaged using a confocal microscope.

Animal modeling

Three-week-old male Balb/c mice were purchased from the Laboratory Animal Center of Anhui Medical University (Ethics Certificate No. LLSC20221000). Following a one-week acclimatization period, the mice were randomly assigned to two groups, namely the normal chow diet (NCD) group and the high-fat diet (HFD) group. The specific food composition of the HFD is presented in Supplementary Table 1. After six weeks, half of the mice in the HFD group were subjected to a further treatment with melatonin at a dose of 108 mg/kg for an additional six weeks via gavage, and this subgroup was designated as the HFD+Melatonin (HFD+M) group. The melatonin dosage was selected based on previously published studies with minor modifications (Sun et al., 2023).

Lipid detection

HepG2 cells were seeded into 60 mm culture dishes at a density of approximately 1×10⁵ cells per dish and allowed to adhere for 12 h. The culture medium was then replaced with basal medium containing FFAs and/or melatonin, and the cells were cultured for an additional 24 or 48 h.

In the context of animal experiments, the serum levels of triglycerides (TG) and total cholesterol (T-CHO) were measured in mice subjected to a high-fat diet (HFD) and those additionally treated with melatonin (HFD+M) using a commercial kit. Absorbance was recorded at a wavelength of 500 nm using a fully automated microplate reader.

Oil red O staining

HepG2 cells were seeded into six-well plates at a density of 5×10³ cells per well. Upon reaching approximately 50% confluence, the culture medium was replaced with a medium containing 0.5 mM FFAs and/or melatonin, and the cells were incubated for an additional 24 or 48 h. Following incubation, 1 mL of Oil Red O staining solution was added to each well for 30 min. Then the solution was discarded, and the cells were thoroughly washed with distilled water. The stained cells were subsequently observed and imaged using a fluorescence microscope (Olympus; Tokyo, Japan).

For the purpose of staining liver tissues, a staining wash solution was applied to cover the tissue, followed by immersion in Oil Red O working solution for staining. The sections were then subjected to a brief treatment with the staining wash solution and subsequently immersed in distilled water while being gently agitated on a shaker. The sections were mounted and sealed with neutral resin and imaged under a Leica microscope (Leica; Wetzlar, Germany).

Determination of lipid peroxidation and antioxidant activity

Liver samples were homogenized and subjected to a centrifugation process at 4°C to obtain clarified lysates. The protein concentrations in the lysates were determined using a BCA assay kit. The levels of malondialdehyde (MDA), reduced glutathione (GSH) and the activity of superoxide dismutase (SOD) were measured according to the manufacturer’s instructions.

Prussian blue staining

Paraffin-embedded tissue sections were deparaffinized and washed using distilled water for 2 min. The sections were then stained with Prussian blue staining solution for a period of 15-30 min, after which they were rinsed in distilled water for a further 2-3 min. Subsequently, the sections were counterstained with nuclear fast red solution for 5 to 10 min and then washed again in distilled water for a duration of 2 min. After dehydration with xylene, the sections were sealed using neutral gum. The stained sections were photographed and documented under a microscope to evaluate the presence of iron deposits.

Hematoxylin and eosin (H&E) staining

The deparaffinization and washing steps were carried out according to standard protocols, with the samples being immersed in distilled water for a duration of 2 min. The sections were then immersed in hematoxylin solution for 5 min, followed by rinsing and brief incubation in 1% hydrochloric acid for 45 s. Following a 30-s soak in a dilute lithium carbonate solution, the sections were dehydrated in 80% ethanol and stained with eosin for a further 30 s. To optimize the color contrast, the sections were further soaked in 95% ethanol, followed by dehydration in anhydrous ethanol. Following the process of rendering the sections transparent in xylene, they were mounted with a drop of neutral gum and photographed under the microscope.

Western blotting

HepG2 cells or mouse liver tissues were collected following treatment with FFAs and/or melatonin, and then lysed using a cell lysis buffer (RIPA: PMSF=100:1). The lysates were centrifuged to obtain the supernatant and then mixed with a loading buffer. The proteins were separated by SDS-PAGE gel and transferred to a PVDF membrane. The membranes were then blocked and incubated overnight at 4°C with various primary antibodies, such as SREBP1, FASN, ACC1, and SCD1. Then the membranes were washed and incubated with appropriate secondary antibodies. Finally, the protein bands were visualized using a gel imager (Tanon; Shanghai, China), and the intensity was semi-quantified using Image J software (National Institutes of Health; MD, USA).

qRT-PCR

HepG2 cells or mouse liver tissues were collected following treatment with FFAs and/or melatonin, and total RNA was extracted using standard protocols. cDNA was generated by reverse transcription of total mRNA using a first-strand cDNA synthesis kit, and then amplified by qRT-PCR using an AceQ qPCR SYBR Green master mix according to the manufacturer’s instructions. The qRT-PCR amplification was performed on a real-time fluorescence quantitative PCR instrument (Roche; Basel, Switzerland). The sequences of the primers are listed in Supplementary Table 2.

Statistics

Statistical analyses were performed by SPSS 23.0 software (IBM; NY, USA). Data are presented as the mean ± standard deviation (SD). Comparisons between two groups were conducted using an independent t-test, while comparisons among more than two groups were analyzed using one-way analysis of variance (ANOVA). A p-value less than 0.05 was considered to indicate statistical significance.

RESULTS

FFAs promote lipid deposition in hepatocytes

To investigate the role of FFAs in the progression of MASLD, HepG2 cells were treated with varying concentrations of FFAs (0, 0.1, 0.25, 0.5, 1.0, and 2.0 mM concentration of OA) for 24 h. The effect of FFAs on cell proliferation was assessed by CCK-8 assay, which revealed a dose-dependent reduction in cell viability along with increasing concentrations of FFAs (Fig. 1A). As illustrated in Fig. 1B, the presence of oil-red lipid droplets surrounding HepG2 cells was observed to significantly increase in a concentration-dependent manner when the concentration of FFAs was greater than or equal to 0.25 mM. The half maximal inhibitory concentration (IC50) of FFAs treatment in HepG2 cells was approximately 0.60 mM. (Fig. 1C), therefore, 0.5 mM of FFAs were used in the ensuing experiments. Subsequently, HepG2 cells were co-incubated with 0.5 mM of FFAs for 24 and 48 h, and it was found that cell viability decreased in a time-dependent manner. Additionally, an increased number of lipid droplets were observed in the vicinity of HepG2 cells treated with FFAs for 48 h in comparison to those treated for 24 h (Fig. 1D). Interestingly, FFAs also increased the levels of triglyceride (TG) and total cholesterol (T-CHO) in a time-dependent manner (Fig. 1E, 1F). Based on these findings, it was determined that 0.5 mM of FFAs for 24 and 48 h would be used to mimic the high-fat diet in the subsequent in vitro experiments.

Fig. 1.

FFAs promote lipid deposition in hepatocytes. (A) Cell viability of HepG2 cells treated with various concentrations of FFAs for 24 h (% of control). (B) Lipid deposition in hepatocytes stimulated by different concentrations of FFAs, and assessed by Oil Red O staining. (C) IC50 values of FFAs. (D) Oil Red O staining of HepG2 cells following 24 h and 48 h of 0.5 mM FFAs treatment. (E, F) The levels of TG and T-CHO in HepG2 cells incubated with 0.5 mM FFAs for 24 h and 48 h. Data are expressed as the mean ± SD of three independent experiments (n=3). Scale bar=20 μm, *p<0.05, **p<0.01, and ***p<0.001, compared to untreated HepG2 cells (con).

High-fat diet promotes hepatic lipid deposition in mice

In this study, an NAFL model was induced by a 12-week HFD protocol, aiming to simulate the lipid metabolic disorder process in the early lesion stage of MASLD. It was found that HFD feeding significantly increased the serum levels of TG and T-CHO compared to the normal diet (NCD) group (Fig. 2A, 2B). And liver steatosis was clearly observed in HFD-fed mice in comparison to the NCD group (Fig. 2C) as assessed by the Oil Red O staining assay. Moreover, the expression of transcription factor SREBP1 as well as lipogenesis-associated enzymes, such as ACC1, FASN, and SCD1, was significantly up-regulated in the HFD group relative to mice in the NCD group (Fig. 2D, 2E). Similar results were obtained through qRT-PCR analysis (Fig. 2F-2I). Collectively, these findings suggest that a high-fat diet elevated the expression of lipid metabolism-related enzymes and promoted lipid deposition in the liver.

Fig. 2.

High-fat diet promotes hepatic lipid deposition in mice. (A, B) The serum levels of TG and T-CHO in mice. (C) Histological analysis of hepatic lipid deposition via Oil Red O staining (scale bar=50 μm). (D) Western-blotting analysis the expression levels of FASN, ACC1, SREBP1 and SCD1 in liver tissues, (E) and the bands were semi-quantitatively measured. Relative mRNA levels of SREBP1 (F), ACC1 (G), FASN (H), and SCD1 (I) in liver tissues. Data are expressed as mean ± SD of three independent experiments (n=3). *p<0.05, **p<0.01, and ***p<0.001, compared to mice in the NCD group. NCD=Normal Chow Diet, HFD=High Fat Diet.

FFAs disrupt the balance between iron loading and antioxidant systems in the liver

In patients with MASLD, elevated levels of FFAs are frequently observed in the liver. FFAs have been demonstrated to induce the excessive production of reactive oxygen species (ROS) and to disrupt iron homeostasis. This imbalance exacerbates cellular iron overload, which further amplifies FFAs-induced lipid peroxidation and subsequent results in cell damage (Sun et al., 2021; Rochette et al., 2023). The findings of our present study revealed that co-culturing with FFAs significantly reduced the activity of the antioxidant enzyme GPX4 (Fig. 3A, 3B), while simultaneously increasing hepatocyte iron loading, as evidenced by a substantial rise in the iron transporter protein TFR1 (Fig. 3C, 3D). In addition, qRT-PCR assays also confirmed that FFAs inhibited the expression of GPX4 and increased the level of TFR1 (Fig. 3E, 3F). Similar results were observed in the livers of HFD-fed mice in vivo, where the levels of GPX4 were significantly decreased and TFR1 were increased (Fig. 3G, 3H), with qRT-PCR assay further validating these observations (Fig. 3I, 3J). In conclusion, the accumulation of FFAs enhances lipid peroxidation and intracellular iron loading in hepatocytes, thus leading to an imbalance in iron homeostasis in the liver.

Fig. 3.

FFAs disrupt the balance between iron loading and antioxidant systems in the liver. (A) Western-blotting analysis the expression of GPX4 in HepG2 cells. (B) and the bands were semi-quantitatively analyzed. (C) The expression of TFR1 in HepG2 cells was measured by Western-blotting assay, (D) and the bands were semi-quantitatively analyzed. (E-F) Relative mRNA levels of GPX4 and TFR1 in HepG2 cells. (G-H) Relative protein levels of GPX4 and TFR1 in liver tissues. (I-J) Relative mRNA levels of GPX4 and TFR1 in liver tissues. Data are expressed as the mean ± SD of three independent experiments (n=3). *p<0.05, **p<0.01, and ***p<0.001, compared to untreated HepG2 cells (con) or the NCD group. NCD=Normal Chow Diet, HFD=High Fat Diet.

Melatonin alleviates free fatty acid-induced lipid synthesis in hepatocytes

MASLD is a chronic liver condition closely associated with lipid metabolism disorders. Melatonin, an endogenous hormone, exhibits a wide range of biological functions, including antioxidant, anti-inflammatory, and the regulation of lipid metabolism. Recent studies have highlighted that melatonin has the capacity to inhibit the production of desaturated fatty acids and mitigate lipid metabolism disorders by modulating key enzymes involved in lipid synthesis (Ou et al., 2019; Hsu and Chien, 2023). In order to investigate the potential of melatonin in alleviating FFAs-related MASLD progression, we first co-incubated FFAs-pretreated HepG2 cells with various concentrations of melatonin (0, 0.5, 1.0, 2.5, 5.0, and 10.0 μM), and found that melatonin could reverse FFAs-induced cytotoxicity to a certain extent. Specifically, 1.0 μM and 2.5 μM of melatonin improved cell viability more significantly; however, higher concentrations of melatonin led to a decrease in cell viability (Fig. 4A). Therefore, melatonin at a concentration of 1.0 μM was selected for the subsequent study.

Fig. 4.

Melatonin alleviates free fatty acid-induced lipid synthesis in hepatocytes. (A) Cell viability at different concentrations of melatonin in HepG2 cells (% of control). (B) Oil Red O staining analysis of the effect of melatonin in HepG2 cells. (C, D) The effect of melatonin on FASN, ACC1, SREBP1, and SCD1 in HepG2 cells at both protein and mRNA levels. (E) Immunofluorescence analysis of ACC1 in HepG2 cells. (F) Relative mRNA levels of PPAR-γ, C/EBP-β, Caveolin-1, and Perilipin-1 in HepG2 cells. Data are presented as the mean ± SD of three independent experiments (n=3). scale bar=25 μm. *p<0.05, **p<0.01, and ***p<0.001, compared to HepG2 cells treated with FFAs. FFAs+M=Free Fatty Acids+Melatonin.

Subsequently, we explored the impact of melatonin on lipid metabolism in hepatocytes. Oil red O staining revealed that a significant accumulation of lipid droplets around the HepG2 cells following a 48-h exposure to FFAs, however, co-administration of melatonin significantly reduced the number of lipid droplets in the hepatocytes (Fig. 4B). Meanwhile, Western blotting and immunofluorescence (IF) analyses showed that the nuclear expression level of SREBP1 was significantly increased after FFAs upregulation, and significantly decreased after melatonin intervention (Supplementary Fig. 1A-1C). And then, western-blotting analysis showed that FFAs up-regulated the expression of SREBP1 and its downstream lipid synthases, including ACC1, FASN, and SCD1, while melatonin exhibited an opposite effect (Fig. 4C). Furthermore, qRT-PCR also showed that melatonin reduced the mRNA levels of lipid synthesis-related enzymes, which had been elevated by FFAs (Fig. 4D). Moreover, immunofluorescence assays also confirmed that melatonin was able to reverse FFAs-induced upregulation of ACC1 (Fig. 4E). Interestingly, it is found that melatonin not only diminished FFAs-induced up-regulation of lipogenesis factors, such as transcription factors peroxisome proliferator-activated receptor (PPAR-γ) and CCAAT/enhancer-binding protein β (C/EBP-β), but also reduced the expression of lipid droplet-producing factors, for example, perilipin-1 and caveolin-1 (Fig. 4F). Taken together, it was evident that melatonin could inhibit lipid synthesis and alleviate lipid droplet accumulation in hepatocytes in vitro.

Melatonin inhibits lipid synthesis and alleviates hepatic lipid deposition in vivo

To further elucidate the effect of melatonin on lipid metabolism in vivo, we first constructed a MASLD model via feeding mice with a high-fat diet over a period of twelve weeks, and mice in the melatonin treatment group received 108 mg/kg of melatonin at the beginning of the seventh week for six weeks. The administration of HFD feeding resulted in an increase in the weight of the body, the liver, the epididymal white adipose tissue (Epi-WAT), visceral white adipose tissue (Vis-WAT), subcutaneous white adipose tissue (Sub-WAT), and brown adipose tissue (BAT), while supplementation of melatonin significantly decreased these indices compared to the HFD group, except for BAT (Fig. 5A-5F). Additionally, melatonin treatment effectively reversed HFD-induced hepatic steatosis, and significantly reduced hepatic lipid droplet accumulation (Fig. 5G). Serological tests also showed that melatonin treatment led to a significant reduction in the blood TG (Fig. 5H) and T-CHO (Fig. 5I) levels, as evidenced by a comparison with the HFD group. In accordance with the results obtained in vitro, the application of melatonin led to a significant down-regulation of the expression of enzymes related to lipid synthesis in the liver, including FASN, ACC1, SREBP1, and SCD1, as compared to the HFD group (Fig. 5J, 5K). Furthermore, qRT-PCR assay also revealed that melatonin significantly reduced the mRNA levels of enzymes involved in lipid synthesis elevated by the HFD feeding (Fig. 5L). These results indicate that melatonin has the potential to impede lipid synthesis, and ameliorate HFD-induced lipid metabolism disruption.

Fig. 5.

Melatonin inhibits lipid synthesis and alleviates hepatic lipid deposition in vivo. The impact of melatonin on (A) Body weight, (B) Liver weight, (C) Epi-WAT, (D) Vis-WAT, (E) Sub-WAT, and (F) BAT. (G) Oil Red O staining the liver tissues (scale bar=50 μm). (H-I) The serum levels of TG and T-CHO in melatonin-treated mice. (J) The protein levels of FASN, ACC1, SREBP1, and SCD1 in liver tissues (K) and the bands were semi-quantitatively analyzed. (L) The relative mRNA levels of FASN, ACC1, SREBP1, and SCD1 in liver tissues. Data are expressed as mean ± SD of independent experiments (n=6 for A-F; n=3 for G-L). *p<0.05, **p<0.01, and ***p<0.001, compared to HFD group. HFD+M=High Fat Diet+Melatonin.

Melatonin mitigates FFAs-induced iron overload in hepatocytes

Lipid deposition has been demonstrated to impede the capacity of ferritin to store iron, resulting in an increase in free iron ions that subsequently drive iron-mediated peroxidation (Gao et al., 2022). Melatonin has been demonstrated to mitigate this effect by reducing hepatic lipid deposition, thereby lowering lipid peroxidation and oxidative stress (Saha et al., 2023). However, the specific mechanism through which melatonin regulates cellular iron imbalance remain unclear. To test the hypothesis that melatonin alleviates HFD-induced liver injury via inhibiting lipid synthesis and ferroptosis in hepatocytes. We treated HepG2 cells with Liproxstatin-1, a ferroptosis inhibitor, and found that Liproxstatin-1(Lip-1) significantly reversed FFAs-induced upregulation of transferrin receptor (TFR1) and divalent metal ion transporter (DMT1), and melatonin demonstrated similar efficacy by effectively restoring intracellular iron metabolism (Fig. 6A, 6B). Additionally, Fe²⁺ staining revealed that FFAs significantly altered intracellular iron accumulation in HepG2 cells, while treatment with either melatonin or Liproxstatin-1 substantially reduced intracellular iron accumulation, indicating their protective roles in mitigating FFAs-induced disturbances in iron homeostasis (Fig. 6C, 6D). To further investigate the effect of melatonin on iron metabolism in vivo, Prussian blue staining was carried out and revealed that HFD feeding significantly increased intracellular iron accumulation in the liver, as indicated by prominent brown-positive area, while melatonin intervention was able to reduce iron accumulation in the liver of HFD fed mice (Fig. 6E). Consistent with the findings in vitro, the application of melatonin resulted in a significant decrease in the levels of TFR1 and DMT1, as well as a partial restoration of iron homeostasis in the liver of HFD-fed mice (Fig. 6F, 6G). In conclusion, melatonin regulates intracellular iron metabolism, thereby improving intracellular iron overload and mitigating ferroptosis in hepatocytes.

Fig. 6.

Melatonin mitigates FFAs-induced iron overload in hepatocytes. (A) The protein levels of FTH1 and TFR1 in HepG2 cells were measured by Western-blotting assay, (B) and the bands were semi-quantitatively analyzed. (C) Ferrous iron (Fe²⁺) levels were detected using a fluorometric assay, (D) and semi-quantitative (scale bar=20 μm). (E) Prussian blue staining detected iron accumulation in liver tissues (scale bar=100 μm). (F-G) The relative expression levels of FTH1 and TFR1 in liver tissues. Data are presented as mean ± SD from three independent experiments (n=3). *p<0.05, **p<0.01, and ***p<0.001, compared with FFAs or HFD group. FFAs+L=Free Fatty Acids+Liproxstatin-1, FFAs+M=Free Fatty Acids+Melatonin, HFD+M=High Fat Diet+Melatonin.

Melatonin alleviates hepatocyte ferroptosis via modulating the KEAP1/NRF2/HO-1 pathway

Melatonin has been demonstrated to effectively mitigate ferroptosis by decreasing lipid peroxidation and oxidative stress (Galano and Reiter, 2018; Xu et al., 2020). However, the specific mechanisms through which melatonin alleviates ferroptosis remain unclear. Lipid peroxidation is a hallmark of ferroptosis, and glutathione peroxidase (GPX4), a key regulator of antioxidant stress, plays a pivotal role in modulating ferroptosis. It is noteworthy that melatonin exhibited minimal impact on the cystine/glutamate reverse transporter protein (SLC7A11), which is the direct upstream regulator of GPX4 and a critical component of the Xc-system (Supplementary Fig. 2A, 2B). Interestingly, nuclear factor erythroid 2-related factor 2 (NRF2), an upstream transcription factor of SLC7A11, was elevated following the intervention of melatonin. Therefore, it is hypothesized that melatonin may indirectly regulate GPX4 by modulating other factors downstream of NRF2.

To elucidate the effects of melatonin on FFAs-induced lipid peroxidation in HepG2 cells, we found that GPX4 was significantly downregulated following FFAs stimulation, but melatonin could reverse the effect of FFAs. Western blot analysis revealed that melatonin was able to activate the NRF2-regulated antioxidant gene Heme oxygenase-1 (HO-1), thereby enhancing the expression of GPX4 and alleviating lipid peroxidative damage in hepatocytes (Fig. 7A). HO-1 is a key enzyme in the cellular antioxidant defense system, with the capacity to impede lipid peroxidation and regulate ferroptosis through its effects on heme and free iron metabolism, and both this factor and NQO1 are directly regulated by NRF2. Furthermore, the regulation of upstream signals can also promote NRF2-mediated activation of HO-1. KEAP1, a negative regulator of NRF2, is subject to alteration in a high-fat environment, which potentially leads to the onset of fatty acid peroxidation and imbalance in cellular antioxidant responses. Using qRT-PCR assay, we confirmed that melatonin restored FFAs-induced downregulation of GPX4 expression via the KEAP1/NRF2/HO-1 pathway (Fig. 7B). Furthermore, immunofluorescence assays have demonstrated that the intervention of melatonin or Lip-1 can effectively reverse the detrimental effects of FFAs on GPX4 (Supplementary Fig. 2C). Moreover, immunofluorescence also corroborated the hypothesis that the expression of NRF2 in hepatocytes was significantly down-regulated following FFAs treatment; however, melatonin significantly increased NRF2 expression in hepatocytes (Fig. 7C). And consistent results were obtained in the co-localization quantitative analysis of NRF2 nuclear translocation (Supplementary Fig. 3A-3C). Additionally, to determine whether melatonin restores FFAs-induced GPX4 downregulation via the KEAP1/NRF2/HO-1 pathway in a ROS-dependent manner, we conducted mechanistic validation using the antioxidant N-acetylcysteine (NAC). Cells were pretreated with NAC (10 mM) for 2 h, followed by sequential addition of FFAs and melatonin. Western blotting results showed no statistically significant difference in GPX4 expression between the FFA+M group and the NAC+FFA+M group, with a slight downward trend observed in the latter (Supplementary Fig. 4A, 4B). These findings indicate that NAC pretreatment partially suppresses melatonin-mediated GPX4 upregulation, suggesting that melatonin may restore GPX4 expression by scavenging FFA-induced reactive ROS, thereby alleviating KEAP1-mediated inhibition of NRF2.

Fig. 7.

Melatonin alleviates hepatocyte ferroptosis via modulating the KEAP1/NRF2/HO-1 pathway. (A) Relative protein levels of NRF2, HO-1, and GPX4 in HepG2 cells. (B) Relative mRNA levels of KEAP1, NRF2, HO-1, and GPX4 in HepG2 cells. (C) Immunofluorescence analysis of NRF2 localization in HepG2 cells (scale bar=20 μm). (D-F) The levels of MDA, GSH, and SOD in liver tissues. (G) Histological analysis of liver sections stained with H&E (scale bar=50 μm). The levels of iNOS, NRF2, HO-1, and GPX4 in liver tissues were measured by Western-blotting assay (H) and qRT-PCR assay (I). (J) Melatonin prevents the progression of MASLD via inhibiting FFAs-induced ferroptosis through KEAP1/NRF2/HO-1 pathway. Data are presented as mean ± SD from three independent experiments (n=3). *p<0.05, **p<0.01, and ***p<0.001, compared to FFAs or HFD group. FFAs+M=Free Fatty Acids+Melatonin, HFD+M=High Fat Diet+Melatonin.

Subsequently, we assessed hepatic redox products and related enzymes in mice, and found that malondialdehyde (MDA) was significantly elevated in the HFD group, while the levels of antioxidant enzyme, such as superoxide dismutase (SOD) and reduced glutathione (GSH), were markedly decreased, and the intervention of melatonin was found to effectively reverse these changes (Fig. 7D-7F). Notably, exogenous melatonin supplementation significantly alleviated HFD-induced liver injury in mice (Fig. 7G). Consistent with our in vitro findings, melatonin treatment significantly up-regulated the expression of GPX4, HO-1, and NRF2, whilst concomitantly leading to a decrease in the expression of KEAP1 at both protein and mRNA levels (Fig. 7H, 7I). In conclusion, melatonin inhibits free fatty acids-induced lipid peroxidation in hepatocytes via the KEAP1/NRF2/HO-1 pathway, thereby mitigating ferroptosis-associated liver injury and the progression of MASLD (Fig. 7J).

DISCUSSION

MASLD is a prevalent chronic liver disease, primarily characterized by the accumulation of lipids and hepatic steatosis. The accumulation of lipids in hepatocytes, particularly free fatty acids, is a pivotal mechanism that leads to hepatocyte damage. Therefore, inhibiting lipid accumulation could be a potential therapeutic strategy to reduce hepatocellular injury and mitigate the progression of MASLD. Unfortunately, there are currently no effective pharmacological treatments available for the management of MASLD.

It has been demonstrated that excessive dietary fat intake, obesity, and insulin resistance have the capacity to elevate circulating levels of FFAs. Elevated FFAs not only induce lipid accumulation but also exacerbate insulin resistance, which further generates significant amounts of reactive oxygen species (ROS) in hepatocytes (Zhang et al., 2019). Furthermore, this has been observed to activate inflammatory signaling pathways and promote hepatocellular damage and fibrosis, thereby accelerating the progression of MASLD (Wang et al., 2022b). For instance, Li et al. reported that ATL III, an antioxidant compound, could activate the hepatic AdipoR1-mediated AMPK/SIRT1 signaling pathway to alleviate lipid deposition and oxidative stress, reduce liver injury, and prevent the development of liver fibrosis (Li et al., 2022). In the present study, we found that melatonin reduced FFAs-induced hepatic lipid accumulation and inhibited lipid peroxidation.

Ferroptosis is a novel form of iron-dependent cell death characterized by intracellular lipid peroxidation, iron ion overload, and impaired antioxidant systems (Rochette et al., 2023). The progression of MASLD is closely related to ferroptosis; however, the precise role and underlying mechanisms are not fully understood (Wang et al., 2022a; Wu et al., 2022). Recent studies have highlighted a strong association between FFAs and ferroptosis, especially polyunsaturated fatty acids. For instance, Krümmel B et al. found that FFAs induce the generation of ROS, increase intracellular iron levels, and cause mitochondrial dysfunction (Huimin et al., 2023; Kruemmel et al., 2022). During ferroptosis, intracellular Fe²⁺ catalyze the oxidation of FFAs, generating lipid peroxides that inflict damage to cell membranes and consequently lead to cell death. FFAs have also been demonstrated to elevate intracellular iron levels by regulating the expression of iron transporter proteins and promoting the degradation of iron storage proteins, thereby facilitating lipid peroxidation and ferroptosis (Ajoolabady et al., 2021; Rochette et al., 2023). Moreover, FFAs could also activate cellular signaling pathways to promote ferroptosis (Lee et al., 2021). For example, FFAs could activate the nuclear factor κB (NF-κB) signaling pathway, inducing the production of inflammatory factors, which exacerbate oxidative stress and ferroptosis (Zhao et al., 2023). In the current study, we found that FFAs induced a reduction in the antioxidant enzyme GPX4 and a concomitant increase in the iron transport protein TFR1, resulting in an imbalance in the cellular antioxidant system and promoting intracellular iron accumulation.

Melatonin, an indoleamine hormone primarily produced by the pineal gland, is a potent endogenous antioxidant with significant potential for neuroprotection, delaying aging, and preventing age-related cataracts (Bocheva et al., 2024; Mi et al., 2023). In the context of MASLD, previous studies have explored the role of melatonin. Bahrami et al. conducted a double-blind placebo-controlled clinical trial involving 55 patients diagnosed with NAFLD and found that oral melatonin significantly improved multiple indices (such as body weight and waist circumference), liver enzymes, and fatty liver grade in patients compared with the placebo group, suggesting a positive effect of melatonin on NAFLD (Bahrami et al., 2020). Zhang et al. confirmed in animal experiments that melatonin effectively alleviates liver inflammation, relieves endoplasmic reticulum (ER) stress and mitochondrial autophagy, improves fatty acid oxidation, and mitigates microbial rhythm and fat metabolic disorders in mice (Zhang et al., 2022). However, the effects of melatonin on lipid metabolism in the context of MASLD and the underlying mechanisms remain unclear. Several studies have reported that melatonin has the capacity to impede fat synthesis by regulating genes involved in fatty acid biosynthesis. For example, melatonin has been shown to downregulate the expression of key enzymes such as FAS and acetyl-CoA carboxylase (ACC), thereby reducing the de novo fatty acid synthesis (Ku et al., 2023; Saha et al., 2023). Our study confirmed that melatonin can directly regulate transcription factor SREBP1, thereby influencing the expression of FASN and ACC1. Additionally, melatonin activates proteins like hormone-sensitive lipase (HSL) and perilipin in adipocytes, promoting triglyceride hydrolysis (Park et al., 2020). In the present project, we observed that FFAs significantly elevated the expression of key regulators of adipocyte differentiation, such as C/EBP-β and PPAR-γ, and proteins involved in the regulation of lipid droplet size, morphology, and function, such as perilipin-1 and caveolin-1, however, melatonin effectively inhibited the expression of these proteins. These findings suggested that melatonin has the capacity to impede lipid synthesis via multiple pathways, potentially offering therapeutic value for lipid metabolism-related diseases. However, further clinical studies are needed to confirm the efficacy and safety of melatonin for treating lipid metabolism-related diseases.

Melatonin is a powerful antioxidant capable of directly scavenging ROS related to lipid peroxidation, thereby inhibiting the onset of ferroptosis. Numerous in vitro and in vivo studies have demonstrated that melatonin treatment significantly reduces the expression of several indicators of ferroptosis, such as malondialdehyde (MDA), a byproduct of lipid peroxidation, and promotes cell survival (Guan et al., 2023). Our findings showed that melatonin intervention led to a decrease in MDA levels, while boosting the expression of antioxidant markers, including GSH and SOD. Moreover, melatonin has also been reported to decrease cellular iron uptake and promote iron excretion, consequently reducing intracellular iron ion levels and mitigating iron-related damage (Rui et al., 2021). Consistently, we confirmed that melatonin downregulated the expression of TFR1 and DMT1, exhibiting a similar effect to ferroptosis inhibitors. Melatonin helps maintain the activity of GPX4 and enhances cellular antioxidant defenses by inhibiting lipid peroxidation. In a neurodegenerative model, melatonin has been shown to reduce neuronal ferroptosis via the activation of GPX4, and ultimately protect neurological function (Chen et al., 2024). In the present study, we revealed that melatonin effectively reversed FFAs-induced reduction of GPX4 levels. Additionally, we also found that melatonin exerted an indirect regulatory influence on GPX4 by modulating HO-1, a key factor in the downstream of NRF2. In the current study, taking the dual influence of iron ion metabolism and oxidative stress on ferroptosis as the main line, we reveal the pathway by which melatonin regulates ferroptosis in the development of MASLD. However, our study demonstrated that melatonin did not exert an effect on SLC7A11, it may be associated with differences in cell types and experimental conditions, including dosage and treatment duration (Kim et al., 2020). These findings suggest that melatonin mitigates FFAs-induced ferroptosis by regulating lipid metabolism and enhancing antioxidant defenses.

In summary, a high-fat diet exacerbates the accumulation of lipids in hepatocytes, which further leads to an imbalance in iron homeostasis and lipid peroxidation, thereby promoting ferroptosis. Melatonin not only reduces free fatty acid-induced lipid accumulation in hepatocytes by inhibiting enzymes involved in lipid synthesis but also inhibits lipid peroxidation and restores intracellular iron balance by regulating the KEAP1/NRF2/HO-1 pathway. These actions help attenuate ferroptosis-associated liver injury and mitigate the progression of MASLD. However, the precise mechanisms by which melatonin regulates lipid peroxidation and mitigates ferroptosis need in-depth investigation. Our results provide a new viewpoint for early intervention in MASLD lesions by synchronously analyzing the dynamic correlations among lipid metabolism, iron ion metabolism, and oxidative stress, and highlight the therapeutic potential of melatonin in managing MASLD.