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Research Article
Nobiletin protects against alcohol-induced mitochondrial dysfunction and liver injury by regulating the hepatic NRF1-TFAM signaling pathway
REDOX REPORT
D. LU ET AL.
Lu Dan a
Huang Aiping b
Tong Xiaoqing c
Zhang Xiaoyan c
Li Songtao def
https://orcid.org/0000-0002-0734-3562
Yu Xiaolong c
a Department of Digestion, Zhejiang Hospital, Hangzhou, People’s Republic of China
b Department of Blood donation service, Blood Center of Zhejiang Province, Hangzhou, People’s Republic of China
c Department of Nutrition, Zhejiang Hospital, Hangzhou, People’s Republic of China
d School of Public Health, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China
e Institute of Nutrition and Health, School of Public Health, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China
f Academy of Chinese Medical Science, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China
CONTACT Xiaolong Yu rainboy2018@163.com Department of Nutrition, Zhejiang Hospital, Xihu district, Hangzhou 310013, Zhejiang, People’s Republic of China
Supplemental data for this article can be accessed online at https://doi.org/10.1080/13510002.2024.2395779.

2 9 2024
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https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Objectives

Alcohol and its metabolites, such as acetaldehyde, induced hepatic mitochondrial dysfunction play a pathological role in the development of alcohol-related liver disease (ALD).

Methods

In this study, we investigated the potential of nobiletin (NOB), a polymethoxylated flavone, to counter alcohol-induced mitochondrial dysfunction and liver injury.

Results

Our findings demonstrate that NOB administration markedly attenuated alcohol-induced hepatic steatosis, endoplasmic reticulum stress, inflammation, and tissue damage in mice. NOB reversed hepatic mitochondrial dysfunction and oxidative stress in both alcohol-fed mice and acetaldehyde-treated hepatocytes. Mechanistically, NOB restored the reduction of hepatic mitochondrial transcription factor A (TFAM) at both mRNA and protein levels. Notably, the protective effects of NOB against acetaldehyde-induced mitochondrial dysfunction and cell death were abolished in hepatocytes lacking Tfam. Furthermore, NOB administration reinstated the levels of hepatocellular NRF1, a key transcriptional regulator of TFAM, which were decreased by alcohol and acetaldehyde exposure. Consistent with these findings, hepatocyte-specific overexpression of Nrf1 protected against alcohol-induced hepatic Tfam reduction, mitochondrial dysfunction, oxidative stress, and liver injury.

Conclusions

Our study elucidates the involvement of the NRF1-TFAM signaling pathway in the protective mechanism of NOB against chronic-plus-binge alcohol consumption-induced mitochondrial dysfunction and liver injury, suggesting NOB supplementation as a potential therapeutic strategy for ALD.

KEYWORDS

Alcohol-related liver disease
flavonoids
nobiletin
hepatic steatosis
mitochondrial dysfunction
oxidative stress
NRF1
TFAM
Science and Technology Program of Zhejiang Province 10.13039/501100008990 2022KY488 Zhejiang Traditional Chinese Medicine Administration 10.13039/501100012175 2023ZL222 This work was supported by grants from the Science and Technology Program of Zhejiang Province (2022KY488) and Zhejiang Traditional Chinese Medicine Administration (2023ZL222).
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pmcIntroduction

The primary cause of alcohol-related liver disease (ALD) in the United States is chronic-plus-binge alcohol consumption, which accounts for nearly half of all liver cirrhosis deaths [1, 2]. Initially, the impact of alcohol manifests as simple fatty liver (steatosis), which can be reversed; however, prolonged and excessive alcohol consumption leads to increasingly severe liver damage, advancing through stages of hepatitis, fibrosis, and potentially leading to cirrhosis and hepatocellular carcinoma [3]. Although considerable progress has been made in understanding the disease's progression and underlying mechanisms, the intricate molecular pathways and corresponding cellular responses remain poorly understood, hindering the development of effective clinical interventions.

As the primary site for alcohol metabolism, the liver undertakes the conversion of alcohol into acetaldehyde through alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1) [4]. Following this, acetaldehyde undergoes further processing into acetic acid via acetaldehyde dehydrogenase (ALDH). Acetaldehyde, a major toxic metabolite, is one of the principal culprits mediating fibrogenic and mutagenic effects of alcohol in the liver [4]. Furthermore, these metabolic processes generate reactive oxygen species (ROS), which significantly contribute to alcohol-induced oxidative stress in the development of ALD [5]. The excessive production of ROS, coupled with compromised antioxidant defenses, initiates cellular oxidative stress, triggering mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and lipid accumulation in the liver [6]. Additionally, unresolved mitochondrial dysfunction can lead to heightened production of mitochondrial ROS (mtROS), exacerbating alcohol-induced oxidative stress, inflammation, and programed cell death, forming a vicious circle [7]. Despite efforts to address alcohol-induced mitochondrial dysfunction and oxidative stress in ALD treatment, clinically effective compounds specifically tailored to alcohol-related liver injury remain scarce.

Flavonoids, the most abundant class of naturally occurring polyphenolic compounds present in plants, are widely dispersed in plant-based foods and accumulate in relatively high levels [8]. Nobiletin (NOB), a polymethoxylated flavone sourced from citrus fruit peels, has exhibited therapeutic promise in various disease models encompassing neurological [9], inflammatory [10], cardiac [11], and metabolic ailments [12]. Renowned for its potent antioxidant properties, NOB has been observed to counteract oxidative stress and apoptosis in cardiomyocytes following hypoxic injury [13], as well as demonstrate neuroprotective effects against mitochondrial dysfunction [14]. In a murine model of non-alcoholic steatohepatitis, NOB supplementation has been shown to attenuate hepatocyte death, liver inflammation, and fibrosis by modulating hepatic oxidative stress and mitochondrial dysfunction [15]. The manifold health benefits of NOB, spanning anti-inflammatory, antioxidant, anti-aging, and endoplasmic reticulum (ER) stress-alleviating effects, have garnered increasing attention [9, 16, 17]. However, whether and how NOB administration may mitigate alcohol-induced liver damage remain largely unknown. This study aimed to evaluate the protective effects of NOB against alcohol-induced hepatic injury in both ALD mice and cultured hepatocytes. Our results demonstrated, for the first time, that NOB supplementation as a novel intervention effectively shielding against alcohol-induced mitochondrial dysfunction, oxidative stress, and liver injury by regulating the hepatic NRF1-TFAM signaling pathway. Consequently, alcohol-induced ER stress, inflammation, and hepatocellular apoptosis in ALD are all mitigated by NOB. These findings underscore the potential of NOB as a valuable candidate for alleviating hepatic mitochondrial dysfunction and oxidative damage in response to alcohol consumption.

Material & methods

Chronic-plus-binge-plus-binge alcohol feeding and treatments

Male C57BL/6N wild type (WT) mice were housed in ventilated cages maintained under specific pathogen-free conditions. All animal experiments strictly adhered to the approved protocol (SYXK2017-0013) by Hangzhou Medical College Animal Facility. Twelve weeks aged mice were subjected to a Lieber-DeCarli alcohol (alcohol-fed, AF) or an isocaloric maltose dextrin control (pair-fed, PF) liquid diet for a duration of eight weeks plus one binge as previously described with some modifications (Modified NIAAA model) [18, 19]. According to previous studies [18, 20], this model can lead to a relatively severe liver injury in mice. Half of the mice were supplemented with 100 mg/kg/d NOB (N, Sigma Aldrich) or vehicle (DMSO/PBS), respectively as previously described [21, 22], via intraperitoneal injection for the final four weeks, resulting in four experimental groups: PF/N, AF/N, PF/C, and AF/C (n = 16 each). The ethanol content (% w/v) in the diet initiated at 3.6 for the initial two weeks and incrementally increased by 0.3% every two weeks, peaking at 4.5% for the final two weeks. Daily food intake was recorded for all groups, with pair-fed mice receiving an equivalent amount of food consumed by alcohol-fed mice on the preceding day. NOB administration did not significantly affect the food intake in both PF and AF mice. Tissue collection occurred four hours post-gavage with ethanol (4 g/kg) or isocaloric dextrin, following which mice were anesthetized, and tissues were harvested for subsequent analysis.

For hepatocyte-specific overexpression of NRF1, 10-week-old C57/BL6 mice were injected via the retro-orbital sinus with recombinant AAV8 gene transfer vectors. Each mouse received 6 × 10^11 genome copies (GC) of the vector carrying mouse NRF1 complementary DNA (cDNA) under the control of a hepatocyte-specific promoter (TBG). Mice injected with AAV8-TBG-null vectors served as controls (AAV8-Null).

Liver immune cells isolation

Primary hepatocytes and liver immune cells were isolated following a previously described protocol with slight modifications [20]. Initially, livers were perfused with ice-cold sterile PBS via the portal vein and subsequently digested for 20 min at 37°C in a digestion buffer. Following digestion, dissociated cells were harvested and filtered through a 100 μm cell strainer (R&D Systems), followed by centrifugation at 50 g for 4 min at 4°C. Supernatants were collected to assess the proportion of neutrophils and monocytes by flow cytometry. The pellets were resuspended in 20 ml of 40% ice-cold Percoll (Sigma Aldrich) and centrifuged at 180 g for 5 min at 4°C. Following this step, viable hepatocytes settled at the bottom of the 50 ml tubes. Primary hepatocytes were then collected for subsequent analysis of mitochondrial function.

Cell culture and treatment

Alpha mouse liver 12 (AML-12) cells, a type of mouse liver cells, were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Aldrich), supplemented with 10% FBS and 100 U/ml penicillin/streptomycin, within cell culture flasks equipped with solid caps. Upon reaching 80% confluence overnight, the cells were subjected to treatment with alcohol toxicants, specifically 100 μmol/L acetaldehyde (Sigma-Aldrich), for 48 h, with or without prior pretreatment with 100 uM NOB for 16 h. Throughout the treatment period, the cell culture media containing acetaldehyde and NOB were refreshed every 24 h. For CRISPR transfection, a seeding density of 1.0 × 105–1.5 × 105 cells was maintained in six-well culture plates with antibiotic-free standard growth medium, 24 h prior to transfection. The cells were allowed to grow until reaching a confluency range of 40–60%. CRISPR/Cas9 knockdown or control plasmids sourced from Santa Cruz were transfected into the AML12 cells accordingly. Subsequently, stable cloned cells were chosen through a selection process. Following the selection, the efficacy of target gene knockdown in the cells was confirmed through RT-qPCR and Western blot analysis.

Real time-PCR

In the context of quantitative real-time PCR, the left lobe of the liver was chosen from each mouse. Total RNA extraction from liver tissues and hepatocytes employed the TRIzol method. Twenty milligrams of liver samples were homogenized with 1 mL of TRIzol (Thermo Fisher Scientific) in a homogenizer (for hepatocytes, approximately 1 × 107 cells/1 mL TRIzol), followed by the addition of 200 μL of chloroform. After vigorous shaking for 30 s, the mixture underwent centrifugation at 4 °C for 15 min at 12,000 rpm. The upper aqueous phase was transferred, and isopropanol (v/v 1:1) was gently added and mixed, followed by incubation at −20 °C for 30 min and subsequent centrifugation at 4 °C for 15 min at 12,000 rpm. The supernatant was discarded, and 1 mL of 75% ethanol (prepared with diethylpryrocarbonate (DEPC)-treated water) was added. The precipitate was washed three times, air-dried at room temperature, and then dissolved with 100 μL of DEPC water. Complementary DNA (cDNA) synthesis from total RNA (1 μg) utilized TaqMan Reverse Transcription Reagents (Thermo Fisher Scientific) following the manufacturer's recommendations. For amplification, 20 nanograms of cDNA were used, and quantitative PCR was conducted using the SYBR green PCR master mix (Qiagen). Samples were normalized to the general housekeeping gene Rn18s, and calculations were performed using the 2−ΔΔCt method. The primer details are provided in Supplementary Table 1.

Western blot

For Western blot analysis, the middle lobe of the liver was chosen from each mouse. Protein lysates were extracted using lysis buffer supplemented with the protease inhibitor and phosphatase inhibitor (Sigma-Aldrich). Aliquots containing 50 μg of proteins were loaded onto an 8–12% SDS-PAGE, trans-blotted onto a PVDF membrane, blocked with 5% skimmed milk in Tris-buffered saline solution with 0.1% Tween-20 for 30 min at room temperature, and incubated with primary antibodies (Supplementary Table 2). Membranes were washed and incubated with secondary antibodies. Bound complexes were detected via enhanced chemiluminescence. Bands intensity was quantified by ImageJ (NIH), and the ratio to β-actin was calculated and given as fold changes, setting the values of control groups at 1.

Flow cytometry

To prepare for flow cytometry analysis, 150 million cells were suspended in 200 microliters of staining buffer kept at a cold temperature. The buffer included murine Fc-block (CD16/32) antibody (Biolegend) and incubated for 5 min to minimize nonspecific binding. Neutrophils and monocytes in the liver were identified by adding antibodies such as Ly6c, Ly6 g, CD45, and CD11b (Biolegend) to the cell suspension in flow cytometry staining buffer (Biolegend). The mixture was then incubated on ice in the dark for 35 min. Subsequently, 7-aminoactinomycin D (7-AAD) antibody (Biolegend) was added and incubated for the last 5 min at room temperature to complete the staining process. Mitochondrial superoxide indicator MitoSOX Red at a concentration of 1 micromolar (Thermo Fisher Scientific) was utilized to detect mitochondrial ROS (mtROS) production. The mitochondrial membrane potential (MMP) was assessed using Tetramethylrhodamine, ethyl ester (TMRE) from Thermo Fisher Scientific. The expression levels of MTCO1 and SDHA in the liver and hepatocytes were measured using the MitoBiogenesisTM Flow Cytometry Kit (Abcam) following the manufacturer’s instructions. For the detection of reactive oxygen species (ROS) content in primary mouse hepatocytes, H2DCFDA at a concentration of 10 micromolar (Thermo Fisher Scientific) was introduced into the medium. Hepatocellular apoptosis was examined using an Annexin V probe (Thermo Fisher Scientific) as per the manufacturer’s protocol. Samples underwent analysis using a flow cytometer (BD Bioscience), and the data were analyzed using FlowJo software from TreeStar. The supplementary Table 3 contains the antibody information for flow cytometry analysis.

Histological and immunohistochemistry analysis

Immunohistochemistry

Liver tissue paraffin sections were incubated with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidases and with normal serum (from the same species producing the secondary antibody) for 20 min. Then the tissue sections were incubated with primary antibodies (Supplementary Table 2) at 4 °C overnight, followed by incubation with the corresponding Dako EnVision + System HRP Labelled Polymer Anti-Rabbit or Anti-Mouse secondary antibody (Agilant) at room temperature for 30 min.

Histopathology

Hematoxylin and eosin (H&E) staining was conducted with formalin-fixed liver tissue paraffin sections for observation of pathological changes. The ALD activity score were valued as previous described [23]. The histological sections of ALD were scored based on the pathological criteria of the nonalcoholic fatty liver disease activity score (NAS). This ALD activity score includes the evaluation of steatosis, lobular inflammation, and hepatocyte ballooning.

Biochemistry analysis

The right lateral lobe of the liver was specifically selected for biochemical analysis. Mitochondrial complex I activity was assessed using commercial kits obtained from Abcam, following the manufacturer’s instructions and as previously outlined. Hepatic NAD+ levels and the NAD+/NADH ratio were determined using a commercial kit from Biovision, following the manufacturer’s instructions and as previously detailed. Triglyceride and free fatty acid levels in the liver were quantified utilizing the Triglyceride Assay Kit and Free Fatty Acid Assay Kit from Biovision, respectively, following the manufacturer’s instructions. Serum ALT and AST activities were measured calorimetrically using Infinity kits from Thermo Scientific, following the manufacturer’s instructions. TBARS levels were detected by following the manufacturer’s instructions. ATP levels were quantified using an ATP Determination Kit from Abcam (ab83355). Liver tissues (at a ratio of 1:10 w/v) or cells (at a concentration of 106 cells/ml) were resuspended in a reaction buffer provided by the kit, containing 1 mM dithiothreitol, 0.5 mM luciferin, and 12.5 μg/ml luciferase. Intensity readings were obtained for the mixtures using an automatic microplate reader, and ATP concentrations were calculated utilizing an ATP standard curve. The generated nicotinamide adenine dinucleotide (NADH) subsequently reduced a colorless probe to a colored product with strong absorbance at 450 nm. GSH and GSSG concentrations in the liver were measured using a commercial kit from BioVision. Liver triglycerides (TG) and free fatty acids (FFA) were measured by homogenizing 40 mg of liver samples with 400 μL of chloroform – Triton X-100 solution (1% Triton X-100 in pure chloroform). Following homogenization, the extract was centrifuged at 3000 rpm for 10 min, and the organic phase (lower phase) was carefully collected. The collected organic phase was then air-dried at 50 °C to eliminate chloroform, followed by vacuum drying for 30 min to remove any residual trace of chloroform. The dried lipids (in Triton X-100) were reconstituted in 200 μL of fatty acid assay buffer and vigorously vortexed for 5 min. Subsequently, the contents of free fatty acids and triglycerides were determined in accordance with the manufacturer’s instructions. The triglyceride assay kit from Biovision offers a sensitivity ranging between 2 and 10,000 μM, while the FFA assay kit from Biovision has an upper limit of 2 μM. Total cholesterol (TC) measurement involved the extraction of 10 mg of tissue with 200 μL of chloroform/isopropanol/NP-40 solution (7:11:0.1) in a homogenizer. The resulting extract underwent centrifugation at 12,000 rpm for 5–10 min, and all liquid (organic phase, excluding the pellet) was carefully transferred to a new tube. The transferred liquid was then subjected to air-drying at 50 °C to remove chloroform, followed by vacuum drying for 30 min to eliminate any residual organic solvents. The dried lipids were dissolved in 200 μL of cholesterol assay buffer by sonicating or vortexing until homogeneity was achieved. Finally, the total cholesterol content was determined as per the manufacturer’s instructions (ThermoFisher Scientific). The lactate dehydrogenase (LDH) assay involved collecting the culture medium and analyzing it using an LDH assay kit from Thermo Scientific, following the manufacturer’s instructions. This kit offers a detection range spanning from 1 to 10 mU/mL. Acetaldehyde levels were measured by using a commercial assay kit (LSbio) following the manufacturer’s instructions. Aldehyde dehydrogenase (ALDH) activity was assessed using a commercial kit (BioVision, Milpitas, CA) according to the manufacturer's protocol. In brief, ALDH present in liver homogenates, ileum mucosa, or liver subcellular fractions oxidized acetaldehyde. The resulting nicotinamide adenine dinucleotide (NADH) subsequently reduced a colorless probe to produce a colored product with significant absorbance at 450 nm. ALDH activity was quantified as mmol NADH/min/mg protein.

Statistical analyses

The analyses were performed using SPSS 19.0 software (SPSS, IL, USA). Data are expressed as the mean ± standard deviation (SD). Results were analyzed using the one-way analysis of variance (ANOVA) with Tukey’s post hoc test or two-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, where it was appropriate. In all tests, P values less than 0.05 were considered statistically significant.

Results

Nobiletin administration ameliorates chronic-plus-binge-induced liver injury in mice

To evaluate the role of NOB in the development of ALD, C57BL/6N wild-type mice were subjected to the Lieber-DeCarli control or alcohol diet for 8 weeks plus one binge (4 g/kg). As shown in Figure 1A, chronic-plus-binge-induced histopathological change was significantly reversed by NOB administration. After alcohol intoxication, serum levels of both ALT and AST were both significantly increased in AF mice compared with PF controls (Figure 1B); with NOB supplementation, mice displayed significantly lowered values (Figure 1B). Alcohol-increased hepatic TG levels were considerably ameliorated by NOB (Figure 1C). Furthermore, alcohol-increased hepatic free fatty acids (FFAs) (Figure 1D) and cholesterol levels (Figure 1E) were all reversed by NOB administration. Chronic-plus-binge alcohol consumption-induced ER stress is a hallmark for liver injury which play an essential for alcohol-induced apoptosis. In this study, the ER stress marker, CCAAT-enhancer-binding protein homologous protein (CHOP), was significantly activated after chronic-plus-binge alcohol feeding (Figure 1F–G). This effect was significantly reversed by NOB supplementation (Figure 1F–G). Consistently, alcohol-increased protein levels of hepatic cleaved-caspase3 (cCASP3) were ameliorated by NOB (Figure 1G). Notably, NOB itself did not cause any harmful effect on the liver based on the tested indexes (Figure 1A–G). Figure 1. Nobiletin administration ameliorated alcohol-induced liver injury in mice. C57BL/6 male mice were pair-fed or alcohol-fed for 8 weeks, with or without Nobiletin (NOB) at a dose of 100 mg/kg/day. This regimen was followed by a single binge of alcohol (4 g/kg) administered 4 h before tissue collection. (A) H&E staining (n = 6). Scale bars: 50 μm. Star: lipid droplets. (B) Serum ALT and AST levels (n = 7). (C) Hepatic TG levels (n = 6). (D) Hepatic FFA levels (n = 8). (E) Hepatic total cholesterol levels (n = 5). (F) Immunohistochemistry of CHOP in the liver. Images were captured by light microscope. Scale bars: 50 μm. (G) Western blot analysis of hepatic CHOP and cCASP3 (n = 3). Statistical comparisons were made using two-way ANOVA with Tukey’s post hoc test. The bars with different characters differ significantly (P < 0.05). PF, pair-fed; AF, alcohol-fed; C, control group; N, nobiletin supplementation group.

Nobiletin supplementation alleviates hepatic inflammatory cell infiltration in response to chronic-plus-binge alcohol consumption

Inflammatory cell infiltration in the liver, a significant hallmark of alcohol-induced steatohepatitis, was analyzed by flow cytometry. As shown in Figure 2A, alcohol-induced CD45+/CD11b+/Ly6c+ monocyte infiltration into the liver was markedly ameliorated by NOB administration (Figure 2A). After chronic-plus-binge alcohol consumption, CD11b + CD11b+/Ly6g+ neutrophils are also significantly increased in the mouse liver, however, this effect was reversed by NOB administration (Figure 2A). Consistently, compared with AF/C mice, AF/N mice displayed remarkably lower mRNA levels of C–C Motif Chemokine Ligand 2 (Ccl2), C-X-C motif chemokine ligand 1 (Cxcl1), and Tumour Necrosis Factor alpha (Tnf-α) in the liver (Figure 2B). Figure 2. Alcohol-induced hepatic inflammation was reversed by nobiletin administration. C57BL/6 male mice were pair-fed or alcohol-fed for 8 weeks, with or without Nobiletin (NOB) at a dose of 100 mg/kg/day. This regimen was followed by a single binge of alcohol (4 g/kg) administered 4 h before tissue collection. (A) Representative dot plots and gating strategy for 7-AAD-CD45 + CD11b + Ly6chi monocytes and CD45 + CD11b + Ly6chi neutrophils (n = 8). (B) Relative hepatic Ccl2, Cxcl1, and Tnf-α levels (n = 8). Statistical comparisons were made using two-way ANOVA with Tukey’s post hoc test. The bars with different characters differ significantly (P < 0.05). PF, pair-fed; AF, alcohol-fed; C, control group; N, nobiletin supplementation group.

Alcohol-induced hepatic oxidative stress is significantly ameliorated by nobiletin supplementation

Oxidative stress plays a critically pathological role in the development of ALD. In line with previous reports, the data from this study showed that chronic-plus-binge alcohol consumption led to apparent hepatic oxidative stress manifested by the elevations of 4-hydroxynonenal (4-HNE) protein adduct formation (Figure 3A) and Thiobarbituric acid reactive substances (TBARS) contents (Figure 3B). As shown in Figure 3A, alcohol-increased 4-HNE protein adducts in the liver were remarkably attenuated by NOB administration. Consistently, NOB administration also reversed the increase of TBAR levels in the liver in response to chronic-plus-binge alcohol-fed mice (Figure 3B). Figure 3. Alcohol-induced hepatic oxidative stress was ameliorated by nobiletin administration. C57BL/6 male mice were pair-fed or alcohol-fed for 8 weeks, with or without Nobiletin (NOB) at a dose of 100 mg/kg/day. This regimen was followed by a single binge of alcohol (4 g/kg) administered 4 h before tissue collection. (A) Immunohistochemistry of hepatic 4-HNE staining (n = 6). Scale bars: 50 μm. (B) Hepatic TBAR levels (n = 6). (C) Hepatic GSH levels and GSH/GSSG ratio (n = 6). (D) Hepatic NAD+ levels and NAD+/NADH ratio (n = 6). (E) Flow cytometry analysis of total hepatic ROS using an H2DCFDA probe (n = 6). Data are the summary of the mean fluorescence intensity (MFI). Data are the summary of the mean fluorescence intensity (MFI). Statistical comparisons were made using two-way ANOVA with Tukey’s post hoc test. The bars with different characters differ significantly (P < 0.05). PF, pair-fed; AF, alcohol-fed; C, control group; N, nobiletin supplementation group.

chronic-plus-binge alcohol consumption-decreased hepatic Glutathione (GSH) levels and GSH/GSSG ratio was also reversed by NOB administration (Figure 3C). Hepatic NAD+ levels and NAD+/NADH ratio were dramatically decreased by alcohol feeding, whereas this effect was ameliorated by NOB administration (Figure 3D). Moreover, flow cytometry analysis indicated that alcohol-increased hepatic total ROS levels were also significantly reversed by NOB supplementation (Figure 3E).

Ethanol and acetaldehyde metabolism in the liver consumes NAD+ and generates ROS. However, in this study, both the serum alcohol and acetaldehyde levels were not affected by NOB administration (Supplementary Figure 1A). The protein levels of ADH, CYP2E1, and ALDH2 were also comparable between AF/C mice and AF/N mice (Supplementary Figure 1B). The hepatic total ALDH activity were also not affected by NOB administration (Supplementary Figure 1B).

Western blot results showed that NOB administration did not affect the protein levels of glutathione peroxidase 1 (GPX1) and superoxide dismutase type 2 (SOD2) in the mouse liver (Supplementary Figure 2).

Nobiletin administration markedly reversed alcohol-induced hepatic mitochondrial dsyfunction in mice

Due to hepatic mitochondrial dysfunction play a detrimental role in alochol-induced oxidative stress and liver injury. We further determined if NOB can improve alcohol-induced mitochondrial dysfunction. As show in the Figure 4A, alcohol-decreased the hepatic adenosine triphosphate (ATP) levels was remarkably reversed by NOB administration. Primary hepatocytes isolated from AF/C mice exhibited significantly lower mitochondrial membrane potential than those from PF/C mice, whereas this effect was ameliorated in AF/N mice (Figure 4B). The enzymatic activity of mitochondria respiratory complex I, which control cellular NAD+/NADH redox balance, was significantly decreased by chronic-plus-binge alcohol feeding (Figure 4C). Whereas, NOB supplementation restored alcohol-reduced mitochondria respiratory complex I activity (Figure 4C). Figure 4. Nobiletin administration protects alcohol-induced hepatic mitochondrial dysfunction in mice. C57BL/6 male mice were pair-fed or alcohol-fed for 8 weeks, with or without Nobiletin (NOB) at a dose of 100 mg/kg/day. This regimen was followed by a single binge of alcohol (4 g/kg) administered 4 h before tissue collection. (A) Hepatic ATP levels (n = 6). (B) FACS analysis of mitochondrial membrane potential (MMP) using a TMRE probe in the mouse liver (n = 6). (C) Hepatic complex I acitvity (n = 6). (D) Immunohistochemistry of OXPHOS in the liver. Images were captured by light microscope. Scale bars: 50 μm. (E) mtDNA levels (MTND1) relative to nuclear DNA (SDHA) in mouse liver (n = 6). (F) The protein levels of MTCO1/SDH ratio by flow cytometry. (G) The mRNA levels of mtDNA-encoded complexes subunits (n = 6). (H) Western blot analysis of hepatic MTCO3 and MTATP6 (n = 3). (I) Hepatic mtROS levels analysis (n = 8). Statistical comparisons were made using two-way ANOVA with Tukey’s post hoc test. The bars with different characters differ significantly (P < 0.05). PF, pair-fed; AF, alcohol-fed; C, control group; N, nobiletin supplementation group.

We next analyzed the effects of alcohol and NOB on hepatic mitochondrial respiratory chain complexes, the proper function of which are essential for maintaining mitochondrial oxidative phosphorylation (OXPHOS). As shown in Figure 4D, protein levels of hepatic OXPHOS (NDUFB8/SDHB/MTCO1/UQCRC2/ATP5A antibody cocktail) were significantly decreased after alcohol consumption. However, this effect was significantly reversed by NOB administration (Figure 4D). Interestingly, alcohol-decreased hepatic relative mtDNA contents (MTND1 relative to SDHA) were significantly reversed by NOB administration (Figure 4E). Consistently, the ratio of mtDNA-encoded mitochondrial complexes subunits, cytochrome c oxidase subunit 1 (MTCO1) to nuclear DNA encoded complex (SDH) was also remarkably higher in NOB/AF than that in WT/AF (Figure 4F). Alcohol-decreased mRNA and protein levels of mtDNA-encoded gene (Mtco3, and Mtatp6) were all reversed by NOB administration (Figure 4G–H). We next measured the levels of mitochondrial ROS (mtROS) in isolated primary hepatocytes by flow cytometry. Compared with WT/PF mice, WT/AF mice displayed remarkably higher levels of mtROS, whereas this effect was dramatically ameliorated by NOB supplementation (Figure 4I).

The restoration of TFAM is involved in the beneficial role of nobiletin on acetaldehyde-induced hepatocyte mitochondrial dysfunction, oxidative stress, and cell death

To understand the mechanisms underlying NOB-restored hepatic mtDNA contents in ALD, major regulators involved in mtDNA replication, transcription, and maintenance were examined. Intriguingly, AF/N mice displayed higher hepatic mitochondrial transcription factor A (TFAM) mRNA and protein levels compared with AF/C control mice, as evaluated by RT–PCR (Figure 5A), IHC staining (Figure 3B), and western blot (Figure 5C). However, the mRNA levels of DNA polymerase subunit gamma (Plog), DNA-directed RNA polymerase, mitochondrial (Polrmt), and twinkle mtDNA helicase (Twinkle) were comparable between AF/C mice and AF/B mice (Figure 5A). Figure 5. Nobiletin protect against acetaldehyde-induced mitochondrial dysfunction and apoptosis via regulating TFAM in hepatocyte. (A-C) C57BL/6 male mice were pair-fed or alcohol-fed for 8 weeks, with or without Nobiletin (NOB) at a dose of 100 mg/kg/day. This regimen was followed by a single binge of alcohol (4 g/kg) administered 4 h before tissue collection. (A) The mRNA levels of hepatic Tfam, Twinkle, and Polrmt (n = 6). (B) IHC of hepaitc TFAM (n = 5). (C) Western blot of hepatic TFAM (n = 3). (E-J) Tfam knockdown and Tfam overexpression AML-12 cells were generated and treated with acetaldehyde or NOB for 48 h. (D) The protein levels of TFAM in AML-12 cells (n = 3). The concentration of acetaldehyde is 25, 50, and 100 μM, respectively. (E) Relative mtDNA levels (n = 6). (F) Hepatocellular ATP levels (n = 6). (G) Relative mitochondrial ROS levels (n = 6). Data are the summary of the mean fluorescence intensity (MFI). (H) Relative total ROS levels (n = 6). (I) Released LDH levels (n = 6). Data are the summary of the mean fluorescence intensity (MFI). (J) Annexin V positive cells (n = 6). For panel E-J, the concentration of acetaldehyde is 100 μM. Statistical comparisons were made using two-way ANOVA with Tukey’s post hoc test. The bars with different characters differ significantly (P < 0.05). PF, pair-fed; AF, alcohol-fed; C, control group; N/NOB, nobiletin supplementation group; ACH, acetaldehyde.

Furthermore, we found that acetaldehyde exposure reduces the protein (Figure 5D) and mRNA levels (Supplementary Figure 3A) of TFAM in AML-12 hepatocytes in a dose and time dependent manner. However, 100 mM ethanol administration did not affect the protein levels of TFAM in AML-12 cells (Supplementary Figure 3E). NOB administration significantly ameliorated acetaldehyde-reduced mRNA and protein levels of TFAM in AML-12 cells (Supplementary Figure 3C–D). To evaluate the effect of TFAM reduction in acetaldehyde-mediated mitochondrial dysfunction, Tfam knockdown alpha mouse liver 12 (AML-12) hepatocytes were generated by using CRISPR Cas9 approach. As shown in the Supplementary Figure 3B, Tfam mRNA and protein levels were efficiently decreased after Tfam KD. Whereas, Tfam KD alone is efficently lead to mtDNA reduction in AML12 cells (Figure 5E). Interestingly, lack of Tfam in AML-12 cells significantly exacerbated acetaldehyde-induced mtDNA reduction (Figure 5E), ATP depletion (Figure 5F), mtROS overgeneration (Figure 5G), oxidative stress (Figure 5H), and cell death (Figure 5I–J). Moreover, Tfam overexpression significantly increased mtDNA levels (Figure 5E) and protect against acetaldehyde-induced mitochondrial dysfunction (Figure 5F–H) and cell death (Figure 5I–J), suggesting NOB-mediated TFAM induction is involved in the protective role of acetaldehyde-induced mitochondrial dysfunction and cell death in hepatocyte.

Nobiletin administration restores acetaldehyde-induced hepatic TFAM reduction via restoring NRF1 expression

To explore the possible mechanisms by which NOB regulates TFAM expression, major signaling molecules related to mitochondrial biogenesis were measured. Interestingly, the protein (Figure 6A) and mRNA levels (Supplementary Figure 4A) of nuclear respiratory factor 1 (NRF1) but not nuclear factor-erythroid 2-related factor 2 (NRF2) (Supplementary Figure 4B) were increased in AF/N mice compared with AF/C mice. Share the same trend of TFAM, acetaldehyde exposure also significantly decreased the expression of NRF1 in AML-12 hepatocyte in a time and dose dependent manner (Figure 6B). In AML-12 cells, NOB supplementation also significantly protects against acetaldehyde-reduced mRNA and protein levels of NRF1 (Supplementary Figure 4C–D). Figure 6. Nobiletin ameliorated acetaldehyde-decreased Tfam expression via restoring Nrf1 in hepatocyte. (A) C57BL/6 male mice were pair-fed or alcohol-fed for 8 weeks, with or without Nobiletin (NOB) at a dose of 100 mg/kg/day. This regimen was followed by a single binge of alcohol (4 g/kg) administered 4 h before tissue collection. The protein levels of hepatic NRF1 (n = 3). (B) AML-12 cells are treated with acetaldehyde at 25, 50, or 100 μM for 12, 24, or 48 h, respectively. NRF1 protein levels in AML-12 cells (n = 3). The concentration of acetaldehyde is 25, 50, and 100 μM, respectively. (C) AML-12 hepatocytes were transfected with either NRF1 overexpression or NRF1 knockdown CRISPR plasmids. Hepatic NRF1 and TFAM protein levels after Nrf1 OE or KD (n = 3). (D-E) NRF1 KD or NRF1 OE cells were treated with 100 μmol/L acetaldehyde (Sigma-Aldrich), for 48 h, with or without prior pretreatment with 100 uM NOB for 16 h. (D) Relative hepatocellular mtDNA levels (n = 6). (E) Relative mitochondrial ROS levels (n = 6). Data are the summary of the mean fluorescence intensity (MFI). (F) Relative total ROS levels (n = 6). (G) Released LDH levels (n = 6). For panel D-G, the concentration of acetaldehyde is 100 μM. Statistical comparisons were made using two-way ANOVA with Tukey’s post hoc test. The bars with different characters differ significantly (P < 0.05). PF, pair-fed; AF, alcohol-fed; C, control group; N/NOB, nobiletin supplementation group; ACH, acetaldehyde.

By using Nrf1 KD cells, we identified that knockdown of Nrf1 in AML-12 significantly cause TFAM reduction (Figure 6C) and mtDNA depletion (Figure 6D). In contrast, Nrf1 OE can significantly increased the mRNA and protein levels of TFAM (Figure 6C). Furthermore, Nrf1 OE alone can also increased the mtDNA levels (Figure 6D). Next, we treated Nrf1 KD and Nrf1 OE hepatocytes with acetaldehyde for 48 h respectively. Interestingly, acetaldehyde-induced mitochondrial dysfunction was significantly ameliorated by Nrf1 OE (Figure 6D–E and Supplementary Figure 5A) but exacerbated by Nrf1 KD (Figure 6D–F and Supplementary Figure 5A). Furthermore, the protective effect of NOB on acetaldehyde-induced mitochondrial dysfunction were abolished in Nrf1 KD hepatocytes, suggesting Nrf1 is involved in the protective role of NOB in acetaldehye-treated hepatocyte (Figure 6D–F). Acetaldehyde-induced hepatocellular apoptosis was also ameliorated by Nrf1 OE but exacerbated by Nrf1 KD as evaluated by LDH levels and Annexin V positive cells (Figure 6G and Supplementary Figure 5B).

Hepatocyte-specific nrf1 overexpression ameliorates alcohol-induced hepatic mitochondrial dysfunction and oxidative stress in mice

To further validate the protective of NRF1 in alcohol-induced hepatic mitochondrial dysfunction in mice, hepatocyte-specific Nrf1 overexpressing (AAV8-Nrf1) mice as well as control mice (AAV8-null) were generated. The mRNA (Supplementary Figure 6A) and protein levels (Figure 7A) of NRF1 in the liver of Nrf1 overexpressing mice were increased nearly 5-fold and 6-fold, respectively, at the time of starting alcohol feeding. The Nrf1 induction was only observed in the mouse liver but not in gut, adipose tissue, heart or brain (Supplementary Figure 7). After eight weeks of alcohol feeding, hepatic TFAM protein and mRNA levels in the AAV8-Nrf1AF mice were also higher than the AAV8-null/AF mice (Figure 7A and Supplementary Figure 6B). Figure 7. Hepatocyte-specific Nrf1 overexpression protects alcohol-induced mitochondrial dysfunction and oxidative stress. Hepatocyte-specific Nrf1 overexpression mice were generated by injected in the retrial orbital sinus with recombinant adeno-associated viral (AAV) serotype 8 gene transfer vectors bearing a liver-specific promoter combination (TBG) with mouse Nrf1 sequence. Mice injected with null-vector are served as control. These mice were fed with alcohol for 8 weeks plus a single binge of alcohol (4 g/kg) 4 h before tissue collection. (A) Protein levels of NRF1 and TFAM in the liver (n = 3). (B) Hepatic mtDNA contents (MTND1 relative to SDHA). (C-D) mtDNA-encoded genes expression (n = 6). (E) ATP contents in the liver (n = 6). (F) FACS analysis of mitochondrial membrane potential using TMRE probe (n = 6). Data are the summary of the mean fluorescence intensity (MFI). (G) Hepatic mitochondrial complex I activity (n = 5). (H) Immunohistochemistry of MTCO1 in the liver. Images were captured by light microscope. Scale bars: 50 μm. (I) Hepatic mtROS levels analysis (n = 6). (J) Immunohistochemistry of 4-HNE in the liver. Images were captured by light microscope. Scale bars: 50 μm. (K) Hepatic GSH levels and GSH/GSSG ratio (n = 6) and NAD+ levels (n = 6). Statistical comparisons were made using two-way ANOVA with Tukey’s post hoc test. The bars with different characters differ significantly (P < 0.05). AF, alcohol-fed.

Hepatic mtDNA contents and the mRNA levels of mtDNA-encoded mitochondrial complexes subunits were remarkably increased by Nrf1 overexpression (Figure 7B–D). Accordingly, hepatic protein levels of MTCO1 were higher in AAV8-Nrf1/AF mice than that in AAV8-null/AF mice (Figure 7H). Alcohol-decreased hepatic ATP contents were reversed by Nrf1 overexpression (Figure 5E). Overexpression of Nrf1 in the liver also ameliorated chronic-plus-binge alcohol feeding-perturbed mitochondrial membrane potential (Figure 5F) and mitochondrial respiratory complex I activity (Figure 5G).

Subsequent flow cytometry analysis showed that AAV8-Nrf1/AF mice displayed lower levels of mtROS and total ROS levels in the liver than that in AAV8-null/AF mice (Figure 5I). Consistently, alcohol-induced hepatic 4-HNE adducts formation, GSH reduction, and NAD+ reduction were all ameliorated by Nrf1 overexpression (Figure 7J–K).

Hepatocyte-specific nrf1 overexpression protects against alcohol-induced liver injury in mice

We found that AAV8-Nrf1/AF mice displayed markedly fewer hepatic lipid droplets in the liver compared with AAV8-null/AF mice (Figure 8A). Lower levels of serum ALT and AST was observed in AAV8-Nrf1/AF mice than that in AAV8-null/AF mice (Figure 8B–C). Chronic-plus-binge-increased liver TG, FFA, and cholesterol levels were ameliorated by Nrf1 overexpression (Figure 8D–F). Hepatocyte-specific Nrf1 overexpression ameliorated alcohol-mediated monocytes and neutrophils infiltration in the liver (Figure 8G). The mRNA levels of hepatic Ccl2 and Cxcl1 were also lower in AAV8-Nrf1/AF compared with AAV8-null/AF mice (Figure 8H). Furthermore, hepatic protein levels of CHOP as well as cCASP3 were all lower in AAV8-NRF1/AF mice (Figure 8I and Supplementary Figure 8). Overall, the results suggesting hepatocyte NRF1 protect against alcohol-induced mitochondrial dysfunction and liver injury. Figure 8. Hepatocyte-specific Nrf1 overexpression protects alcohol-induced liver injury. Hepatocyte-specific Nrf1 overexpression mice were generated by injected in the retrial orbital sinus with recombinant adeno-associated viral (AAV) serotype 8 gene transfer vectors bearing a liver-specific promoter combination (TBG) with mouse Nrf1 sequence. Mice injected with null-vector are served as control. These mice were fed with alcohol for 8 weeks plus a single binge of alcohol (4 g/kg) 4 h before tissue collection. (A) H&E staining (n = 6). Scale bars: 50 μm. Star: lipid droplets. (B-C) Serum ALT and AST levels (n = 7). (D) Hepatic TG levels (n = 6). (E) Hepatic FFA levels (n = 8). (F) Hepatic total cholesterol levels (n = 5). (G) Representative dot plots and gating strategy for 7-AAD-CD45 + CD11b + Ly6chi monocytes and CD45 + CD11b + Ly6chi neutrophils (n = 8). (H) Relative hepatic Ccl2, Cxcl1, and Tnf-α levels (n = 8). (I) Immunohistochemistry of CHOP in the liver. Images were captured by light microscope. Scale bars: 50 μm. (J) Mechanical graphic. Statistical comparisons were made using two-way ANOVA with Tukey’s post hoc test. The bars with different characters differ significantly (P < 0.05). AF, alcohol-fed.

Discussion

This study reveals, for the first time, that Nobiletin (NOB) supplementation protects against alcohol-related liver disease by mitigating acetaldehyde-induced hepatic mitochondrial dysfunction. Utilizing chronic-plus-binge alcohol-fed mice and AML-12 hepatocytes, we observed that NOB administration significantly reversed the detrimental effects of alcohol/acetaldehyde on mitochondrial function, oxidative stress, ER stress, and subsequent hepatocellular damage. The mechanism underlying these effects involves the restoration of hepatic Tfam via regulating Nrf1 activation, thereby contributing to the amelioration of hepatocellular injury following NOB administration (Figure 8J).

Hepatocyte is densely packed with mitochondria, which is the primary site of ATP production [20, 24]. By regulating pivotal anabolic and catabolic pathways, mitochondria orchestrate cell metabolism, notably through oxidative phosphorylation [25]. It is composed of five multisubunit complexes encoded by both nuclear and mitochondrial DNA, and its balance is vital for cellular equilibrium and influences cellular fate [26]. Studies in both humans and mice indicate that alcohol-induced liver damage, including hepatic mitochondrial DNA loss/mutation and decreased respiratory chain complex activity, is closely linked to liver damage during the initiation and progression of ALD [20, 27, 28]. These alcohol-induced changes in mitochondrial structure and function contribute to oxidative stress, steatosis, and pro-inflammatory response, exacerbating hepatic mitochondrial dysfunction in a self-perpetuating cycle [29–31]. Targeting alcohol-induced mitochondrial dysfunction presents a promising avenue for therapeutic intervention of ALD [32, 33]. Previous study has shown that flavonoids display protective properties against mitochondria-mediated cell death [34, 35]. Epidemiological data also suggest that certain flavonoids alleviate diseases linked to amelioration of oxidative stress and mitochondrial dysfunction [36, 37]. NOB, known for its ability to enhance mitochondrial function emerges as a potential therapeutic candidate [38]. However, the role of NOB in the development of ALD is still unclear. Here, our results showed that NOB administration remarkably protects against alcohol-induced mitochondrial dysfunction and liver injury in mice. NOB supplementation in mouse liver demonstrates mitigation of ATP depletion, maintenance of membrane potential, prevention of mtDNA depletion, and reduction of mitochondrial ROS production in alcohol-fed mice. Moreover, in AML-12 hepatocytes, NOB treatment alleviates acetaldehyde-mediated mitochondrial dysfunction, oxidative stress, and apoptosis. Consistently, in animal model, our study reveals that NOB supplementation effectively counteracts alcohol-induced steatosis, oxidative stress, ER stress, hepatic inflammation, and apoptosis. These observations highlight the potential of NOB as a therapeutic agent for alcohol-induced liver injury by regulating mitochondrial function. Previous studies have demonstrated that a single alcohol binge is sufficient to cause hepatic mitochondrial dysfunction and oxidative stress in experimental animals [39, 40]. Given that our model incorporates both chronic and binge alcohol exposure, the administration of NOB may play a beneficial role in mitigating liver damage induced by chronic alcohol consumption, and also, at least partially, in counteracting the hepatic mitochondrial dysfunction and oxidative stress caused by acute alcohol binges. These findings suggest a potential therapeutic effect of NOB on both chronic and acute alcohol-induced hepatic damage. Therefore, future studies are warranted to specifically investigate the effects of NOB on hepatic mitochondrial dysfunction and oxidative stress resulting from a single alcohol binge.

Alcohol metabolism within the liver produces acetaldehyde, a highly reactive and toxic compound known to induce various harmful effects, including mitochondrial dysfunction, oxidative stress, ER stress, proinflammatory responses, and apoptosis [41]. Acetaldehyde interact directly with DNA and lead to point mutations and chromosomal damage, as well as form adducts with proteins, disrupting liver function and structure [42, 43]. Previous study have underscored acetaldehyde as a key pathological factor in ALD development, suggesting that enhancing its clearance could ameliorate alcohol-induced liver injury [4]. Given NOB's established protective effects against diverse forms of liver injury, we hypothesized its potential in safeguarding against acetaldehyde-induced hepatocellular injury during ALD progression. Indeed, our findings demonstrate that NOB remarkably mitigates acetaldehyde-induced mitochondrial dysfunction and oxidative stress. Furthermore, we observed that NOB effectively reduces acetaldehyde-induced ER stress by activating the hepatocellular CHOP, thereby diminishing subsequent cellular apoptosis. Previous study demonstrated that chronic-plus-binge alcohol consumption-mediated oxidative stress can disrupt the activity of hepatic ALDH, which in turn, leads to acetaldehyede accumulation in the liver [4]. However, in this study, both serum acetaldehyde levels and hepatic ALDH activity were not affected by NOB, suggesting acetaldehyde metabolism did not involved in the beneficial role of NOB in alcohol-induced liver injury.

TFAM stands out as one of the most abundant DNA-binding proteins localized within mitochondria, playing a crucial role in regulating mtDNA replication, transcription, and packaging [44]. Complete deletion of Tfam throughout the body results in severe mtDNA depletion [45], while tissue-specific Tfam deficiency disrupts OXPHOS in various cell types [46–48]. Previous studies have indicated that alcohol-induced reduction of hepatic TFAM levels contributes to the pathology of ALD [20]. Notably, hepatocyte-specific overexpression of TFAM markedly mitigates alcohol-induced mitochondrial dysfunction and cellular injury [20]. In this study, we observed that administration of Nobiletin (NOB) effectively reversed alcohol/acetaldehyde-induced reduction of TFAM in hepatocytes. Our findings suggest that the restoration of TFAM signaling is integral to the protective role of NOB against alcohol-induced mitochondrial dysfunction and liver injury. Several lines of evidence support this notion. Firstly, NOB administration restored acetaldehyde-induced Tfam reduction both in vitro and in vivo. Secondly, Tfam overexpression in AML-12 cells notably attenuated acetaldehyde-induced mitochondrial dysfunction, oxidative stress, and cell death. Finally, the protective effects of NOB against acetaldehyde-induced mitochondrial dysfunction, oxidative stress, and cell death were all abolished in Tfam knockdown (KD) cells. Taken together, these results suggest that the restoration of TFAM function mechanistically underpins the protective effects of NOB against acetaldehyde-induced mitochondrial dysfunction and hepatocellular injury.

NRF1 serves as a crucial nuclear transcription factor primarily responsible for positively regulating genes related to mitochondrial biogenesis, including TFAM [49]. Its specific and direct binding to the TFAM promoter has been demonstrated through chromatin immunoprecipitation assays [50]. Silencing of NRF1 results in a corresponding knockdown of TFAM expression and a decrease in mitochondrial DNA (mtDNA) content [50]. Similar to TFAM, genetic ablation of NRF1 also leads to embryonic lethality [51], highlighting its role as a master regulator in TFAM-mediated mtDNA homeostasis. Previous studies have shown that alcohol-induced reduction of hepatic NRF1 levels correlates with ER stress-mediated TFAM reduction and mitochondrial dysfunction in mice [20]. NRF1 overexpression has been found to reverse ER stress-mediated TFAM inhibition and mtDNA depletion, indicating that NRF1 reduction is a key contributing factor to alcohol-induced mitochondrial dysfunction and liver injury [20]. In our present study, we observed that both the protein and mRNA levels of NRF1 were restored by NOB administration. Liver-specific overexpression of Nrf1 markedly attenuated alcohol-induced TFAM reduction, mitochondrial dysfunction, oxidative stress, and liver injury. In vitro experiments revealed that the protective effects of NOB against acetaldehyde-induced TFAM reduction, mitochondrial dysfunction, and cell death were abolished in Nrf1 knockdown cells. Conversely, Nrf1 over-expression alone was sufficient to increase TFAM expression and protect against acetaldehyde-mediated hepatocellular injury. These findings collectively suggest that NRF1 induction is involved in the NOB-mediated protection against mitochondrial dysfunction and liver injury in our ALD mouse model. While PGC-1α has been identified as a major regulator of NRF1-TFAM signaling in maintaining mitochondrial homeostasis [52], further studies are needed to evalute whether NOB control hepatic NRF1-TFAM signaling pathway via PGC-1α. Additionally, previous research has indicated that activating transcription factor 4 (ATF4) directly suppresses NRF1 transcription by binding to the CRE site in the NRF1 promoter [20]. Therefore, we cannot rule out the involvement of other pathological factors in NOB-mediated hepatic NRF1 recovery in ALD.

In summary, our study presents novel evidence showcasing the protective effects of Nobiletin administration against acetaldehyde-induced liver injury in ALD. Our findings reveal that Nobiletin supplementation effectively mitigates chronic-plus-binge alcohol-induced steatosis, inflammation, ER stress, and oxidative stress by alleviating mitochondrial dysfunction. Mechanistically, Nobiletin restores the decreased expression of Tfam induced by acetaldehyde through the activation of Nrf1 in hepatocytes. These results suggest that Nobiletin holds promise as a potential therapeutic option for the treatment of ALD.

Supplementary Material

July_revised_NOB supplementary cleaned.doc

Abbreviations

4-HNE, 4-Hydroxynonenal; 7-AAD, 7-Aminoactinomycin D; AAV8, Adeno-Associated Virus Serotype 8; ADH, alcohol dehydrogenases; AF, alcohol-fed; ALD, alcohol-related liver disease; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ATP, Adenosine triphosphate; CCL2, C–C Motif Chemokine Ligand 2; cCASP3, cleaved caspase-3; CHOP, C/EBP homologous protein; CXCL1, chemokine (C-X-C motif) ligand 1; CYP2E1, cytochrome P450 2E1; FFAs, free fatty acids; GSH, glutathione; GSSG, Glutathione disulfide; H&E, hematoxylin and eosin; KD, knockdown; LDH, Lactate dehydrogenase; Ly6c, lymphocyte antigen 6 family member C; Ly6 g, lymphocyte antigen 6 family member G; MFI, mean fluorescence intensity; MTATP6, mitochondrial encoded ATP synthase membrane subunit 6; mtDNA, mitochondrial DNA; MTCO1, cytochrome c oxidase subunit 1; MTCO3, cytochrome c oxidase subunit 3; mtROS, mitochondrial reactive oxygen species; NAD, nicotinamide adenine dinucleotide; NOB, Nobiletin; NRF1, nuclear respiratory factor 1; OE, overexpression; OXPHOS, oxidative phosphorylation; PF, pair-fed; ROS, reactive oxygen species; SDH, succinate dehydrogenase; TFAM, mitochondrial transcription factor A; TG, triglycerides; TMRE, tetramethylrhodamine, ethyl ester; TNF-α, tumor necrosis factor alpha.

Author contributions

Y.X and L.D. conceived and designed research. All the authors performed experiments and data analysis. All the authors participated in manuscript preparation.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data available statement

All data is available upon reasonable request.
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