Inhibition of Acetylation Activity of p300/CBP Ameliorates Hepatic Steatosis Through Downregulation of ACSL4

ABSTRACT

Background & Aims

Metabolic dysfunction–associated steatotic liver disease (MASLD) is the most prevalent chronic liver disorder, with few optimal pharmacotherapies. Ongoing studies are providing evidence that histone acetylation contributes to the progression of MASLD. This study investigated the alterations of specific histone acetylation sites in MASLD tissues and the effects of B029‐2, a small‐molecule inhibitor targeting p300/CBP acetylation activity, in mouse models of progressive fatty liver disease.

Methods

The acetylation of H3K27/H3K18/H3K9 was assessed in liver samples from MASLD patients and mice. The effect of the B029‐2 in MASLD was assessed in MASLD models. A ChIP assay was conducted to investigate the epigenetic regulatory mechanism of ACSL4. Hepatic‐specific Acsl4‐knockout and overexpression mice were generated to validate the role of ACSL4 in MASLD.

Results

We observed a significant increase in p300 HAT activity, p300/CBP‐induced H3K18ac and H3K27ac levels in MASLD and B029‐2 markedly reduced hepatic steatosis in vitro and in vivo. Transcriptome analysis revealed that B029‐2 inhibited the expression of genes in the triglyceride biosynthetic process and long‐chain fatty‐acyl‐CoA biosynthetic process. ChIP assay showed that H3K18ac, H3K27ac and p300 bind to the promoter region of ACSL4, and their binding is reduced by B029‐2 treatment. AAV‐mediated hepatic‐specific overexpression of Acsl4 attenuated the effect of B029‐2 on MASLD in mice, while hepatic‐specific Acsl4‐deficient in mice blocked the effect of B029‐2.

Conclusions

p300/CBP‐mediated H3K18ac and H3K27ac regulate ACSL4‐dependent lipid biosynthesis in MASLD and might be a potential therapeutic target for the treatment of MASLD.

Lay Summary

Can blocking a specific protein activity help treat fatty liver disease? Cells use ‘tags’ such as acetylation to switch certain genes on or off. In this study, we found that in liver samples from patients with MASLD, there were unusually high levels of two particular tags (known as H3K18ac and H3K27ac). These tags are added by p300/CBP, which was overactive in diseased livers. We tested a small‐molecule inhibitor called B029‐2, which is targeting p300/CBP acetylation activity. Treatment with B029‐2 significantly reduced hepatic lipid accumulation in both cultured liver cells and mouse models of MASLD. To understand why this happened, we looked at which genes were affected. We found that B029‐2 turned down the activity of genes involved in making fats, particularly a gene called ACSL4. When we removed ACSL4 from the livers of mice, B029‐2 no longer had any effect. Conversely, when we artificially boosted ACSL4 activity, the benefits of B029‐2 were largely cancelled out. In short, our findings suggest that the p300/CBP enzymes play a major role in driving fat production in the liver by activating ACSL4. Blocking this process with a drug like B029‐2 could offer a new way to treat fatty liver disease, although much more research is needed.

Highlights

1. p300 HAT activity, H3K27ac and H3K18ac were significantly increased in the MASLD livers.

2. Targeting p300/CBP with B029‐2 attenuated hepatic steatosis by epigenetically regulating the expression of ACSL4.

3. B029‐2 ameliorated MASLD partially via inhibiting ACSL4‐dependent lipid biosynthesis.

Abbreviations

ACC

acetyl‐CoA carboxylase

ACSL4

Acyl‐CoA synthetase long chain family 4

ALT

alanine aminotransferase

AST

aspartate aminotransferase

CDAHFD

choline‐deficient, l‐amino acid‐defined, high‐fat diet

CPT2

carnitine palmitoyltransferase 2

CYP8B1

cytochrome P450 family 8 subfamily B member 1

DNL

de novo lipogenesis

FAO

fatty acid oxidation

FASN

fatty acid synthase

HATs

histone acetyltransferases

HDACs

histone deacetylases

HFD

high‐fat diet

MAFL

metabolic‐associated fatty liver

MASH

metabolic dysfunction–associated steatohepatitis

MASLD

Metabolic dysfunction–associated steatotic liver disease

MCD

methionine‐choline‐deficient diet

p300/CBP

E1A‐binding protein and its paralog CREB‐binding protein

PPARA

peroxisome proliferator‐activated receptor alpha

PUFAs

polyunsaturated fatty acids

TC

total cholesterol

TG

triglyceride

1. Introduction

Metabolic dysfunction–associated steatotic liver disease (MASLD) is the most widespread chronic liver disease, affecting about 30% of the global population [1, 2, 3]. MASLD would increase the risk for hepatocellular carcinoma, non‐liver cancer, cardiovascular disease, as well as increased risk for mortality, placing an enormous burden on individuals and health‐care systems [4]. Multifaceted lifestyle interventions are effective, but difficult to maintain. Recently, pharmacologic options have emerged: the thyroid hormone receptor‐β agonist resmetirom has achieved histological improvement in approximately 20% of MASH patients [5], and the GLP‐1 agonist semaglutide achieved both resolution of fatty liver and reduction of liver fibrosis in 32.7% of MASH patients [6]. Despite these advances, response rates remain modest and limited to a subset of patients. Thus, further exploration of additional mechanisms remains warranted to expand treatment options.

Ongoing studies are providing evidence that histone acetylation and epigenomic alteration contribute to the progression of MASLD [7, 8]. Histone acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Previous study showed that total HAT activity was correlated with histological steatosis and disease severity, and increased 2.07‐fold in livers from patients with MASLD [9]. The highly homologous enhancer factors, CBP and p300 (encoded by CREBBP and EP300, respectively), are representative members of HATs and catalyse lysines 18 and 27 of histone H3 (H3K18ac and H3K27ac) specifically, while H3K9ac was distinctly acetylated by the GCN5/PCAF subfamily of HATs [10]. Although the role of CBP in MASLD remains unclear, p300 has been reported as a key enzyme for the development of bisphenol S‐related MASLD [11]. It has been reported that p300 overexpression aggravated hepatic steatosis, insulin resistance and inflammation through promoting fatty acid synthesis [12]. All these suggest that inhibition of hepatic p300 activity may be beneficial for treating hepatic steatosis in fatty liver disease.

Acyl‐CoA synthetase long chain family 4 (ACSL4) is an important metabolic isoenzyme of polyunsaturated fatty acids (PUFAs) and a key positive contributor to ferroptosis [13]. The role of ACSL4 in MASLD is controversial. A study showed that suppressing ACSL4 expression promoted mitochondrial respiration, thereby increasing the capacity of hepatocytes to facilitate β‐oxidation of fatty acids and reducing lipid accumulation by upregulating PGC1α [14]. However, a recent study suggested that the loss of ACSL4 does not affect the progression of MASLD induced by CD‐HFD or the Western diet [15]. In addition, the epigenetic regulation during MASLD is still incompletely characterised.

B029‐2 was a highly selective, potent small molecule inhibitor of p300/CBP HAT, the pharmacokinetic property and inhibitory activity of which have been reported [16]. Our previous study found that B029‐2 could inhibit fatty acid biosynthesis in HCC cells [16], implying the therapeutic potential of B029‐2 in MASLD. In this study, we investigated the effect of B029‐2 on MASLD and found that inhibition of p300/CBP acetyltransferase activity by B029‐2 directly suppressed the transcriptional expression of ACSL4, thereby affecting the lipid biosynthetic process of the hepatocyte and alleviating hepatic steatosis in MASLD.

2. Material and Methods

2.1. Human Samples

Human liver samples were obtained from patients with suspected MASLD who underwent hepatic surgery due to liver hemangioma or hepatic cyst at the Eastern Hepatobiliary Surgery Hospital (Shanghai, China). Two blinded experienced pathologists independently used the Nonalcoholic Steatohepatitis Clinical Research Network (NASH–CRN) scoring system to diagnose MASLD and evaluate the NAFLD activity score (NAS). Samples with NAS of 0 were classified as non‐steatotic. Samples with a NAS of 1–3, a ballooning score of 0 and no fibrosis were allocated in the simple steatosis group (MAFL). Samples with NAS > 4 were assigned to the MASH group. The clinical characteristics of these subjects are provided in Table S1. Exclusion criteria for the study included excessive alcohol intake (> 140 g/week for men or > 70 g/week for women), drug abuse, autoimmune hepatitis or hepatitis C virus infection. All procedures involving human sample collection and application were approved by the ethics committee of Naval Medical University (no. 2018SL004) and adhered to the principles of the Declaration of Helsinki.

2.2. Animal Studies

Male C57BL/6J mice were obtained from GemPharmatech Co. Ltd. (Suzhou, China). Animal experiments were approved by the Institutional Animal Care and Use Committee of the Shanghai East Hospital, Tongji University (no. 2023083). The experiment was carried out in strict accordance with the guidelines. To establish fatty liver models, mice were maintained in environmentally controlled conditions under a 12 h light/dark cycle. Food and water were provided ad libitum. Mice were randomly assigned to different experimental groups. For the HFD‐induced fatty liver mice, male 6‐week‐old C57BL/6 mice were susceptible to an HFD diet (protein, 14.1%; fat, 60%; carbohydrates, 25.9%; TP2003, Trophic, Nantong, China) for 28 weeks. For the CDAHFD models, male 8‐week‐old C57BL/6 mice were fed a CDAHFD diet (choline‐deficient, l‐amino acid‐defined, high‐fat diet, Trophic, Nantong, China) for one week and then treated with B029‐2 (2.5 mg/kg) or DMSO (5%) via intraperitoneal injection every other day for an additional week, concurrent with the CDAHFD diet. For the MCD‐induced MASH, male 8‐week‐old C57BL/6J mice were fed an MCD diet (Methionine‐ and Choline‐Deficient Diet, TP3001, Trophic, Nantong, China) for 4 weeks, with B029‐2 (2.5 mg/kg) or DMSO treatment during the last week. For the HFF‐induced fatty liver mice, male 6‐week‐old C57BL/6 mice were fed a high‐fat and fructose diet (HFF, protein, 14%; fat, 42%; carbohydrates, 44%, 0.2% cholesterol; TP26304, Trophic, Nantong, China, in parallel a carbohydrate‐enriched drinking solution comprising 45% sucrose and 55% fructose) for 14 and 18 weeks and were administered with B029‐2 for the last two weeks. A normal chow diet (NCD) was used as the control.

Acsl4^f/f (flox/flox) and Acsl4^LKO (flox/flox, Alb‐cre) mice were obtained from Cyagen Biosciences Inc. (Suzhou, China). Male 8‐week‐old Acsl4^f/f and Acsl4^LKO mice were used for HFD‐ or CDAHFD‐induced MASLD models. The five‐week CDAHFD‐fed mice were treated with B029‐2 for the last two weeks. At the end of the experiments, mice were anaesthetised and euthanised after blood sampling from the ocular retro‐orbital space.

2.3. Virus

AAV8‐TBG‐Acsl4, adv‐Acsl4 and adv‐ACSL4 were purchased from WZ Biosciences Inc. AAV8‐TBG‐Acsl4 was constructed to comprise Acsl4 overexpression sequence in a hepatic‐specific adeno‐associated virus (AAV8), and AAV8‐TBG‐GFP for control. For hepatocyte‐specific ACSL4 overexpression in C57BL/6 mice, mice were injected via the tail vein with AAV8‐TBG‐Acsl4 or AAV8‐TBG‐GFP (5 × 10¹⁰ V.g.) two weeks before feeding the CDAHFD diet.

2.4. Reagents

The compound B029‐2 was designed and synthesised by the Drug Discovery and Design Center, Shanghai Institute of Materia Medica (Shanghai, China). Antibodies against H3K27ac (#8173T), H3K18ac (#13998T), H3K9ac (#9649T), H3 (#4499S), IgG (#3900S), p300 (#54062S), GAPDH (#2118T) and ACC (#3662S) were purchased from Cell Signalling Technology; antibody against GAPDH (KGAA002) was purchased from KeyGEN BioTECH; antibody against FACL4 (ab155282) was purchased from Abcam; antibody against F4/80 (GB11027) was purchased from Servicebio; antibody against Beta Actin (66009‐1‐Ig) was purchased from Proteintech.

2.5. Cell Lines and Cell Cultures

The mouse normal hepatocyte line AML.12 cells and HepG2 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). We routinely tested cell lines for Mycoplasma contamination using the MycoAlert Detection Kit (Lonza) and authenticated them by short‐tandem repeat analysis. HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Pricella, China) containing 10% heat‐inactivated fetal bovine serum (FBS; Gibco, NY, USA) at 37°C with 5% CO₂. AML.12 cells were cultured in DMEM with F‐12 (DMEM F‐12; Pricella, China), which was supplemented with 10% heat‐inactivated FBS (Gibco, NY, USA), 1% ITS liquid medium supplement (Gibco, NY, USA), 1% 100 μg/mL of penicillin and streptomycin (Sangon biotech, China) and 40 ng/mL of dexamethasone (Beyotime, China). All in vitro experiments were conducted in at least two independent experiments.

2.6. RNA Interference and Transfection

siRNAs were purchased from GenePharma (Shanghai GenePharma Co.) and were transfected using Lipofectamine 2000 (Invitrogen) reagent in 6‐well plates or 24‐well plates according to the manufacturer's protocols. The target sequences were listed in Table S2. After transfection for 48 or 72 h, the cells were collected and the specific silencing of ACSL4 expression was assessed using RT‐PCR and Western blotting.

2.7. Transcriptomic Analyses

The raw transcriptomic data associated with human MASLD samples are publicly accessible on Gene Expression Omnibus (GEO) under accession numbers GSE48452, GSE63067. The ATAC‐seq data and RNA‐seq data are accessible at NCBI‐BioProject under the accession number PRJNA613779. Mann–Whitney U‐test was performed to assess statistical significance. All calculations were performed using the R program (RStudio 4.2.3).

2.8. Rt‐PCR

tRNA was isolated from cells or tissues following the standard TRIzol (Takara) protocol. First‐strand cDNA of cells was synthesised from tRNA using RT Master Mix (Takara). First‐strand cDNA of tissues was synthesised from tRNA using the SuperScript IV First‐Strand Synthesis System (Invitrogen). The expression of various mRNAs was detected using SYBR Green–based RT‐PCR performed on the ABI StepOne Real‐time PCR Detection System (Life Technologies). The mRNA levels were normalised to those of β‐actin or GAPDH mRNA. At least three independent experiments were performed using each condition. Primer sequences are listed in Table S2.

2.9. Western Blot Analysis

Proteins were isolated from whole cells and tissues with lysis buffer (125 mmol/L Tris–HCl, pH 6.8, 25% glycerol, 5% SDS) supplemented with protease inhibitor (Roche), separated by SDS‐PAGE and then transferred onto nitrocellulose membranes (HAHY00010, Millipore). The membranes were blocked in PBST with 5% skim milk and then incubated with primary antibody overnight at 4°C or 2 h at 37°C. After incubation with a secondary antibody (donkey‐anti‐mouse or donkey‐anti‐rabbit, IRDye 700 or IRDye 800, respectively) for 1 h, signals were quantified using an Odyssey Infrared Imaging System (LI‐COR) at 700 or 800 nm.

2.10. Metabolic Studies in Mice

During the experiments, body weights were assessed at different time points. Blood glucose levels were measured using an ACCU‐CHEK Glucometer (Roche, Basel, Switzerland). Serum concentrations of triglycerides were determined using the triglyceride (TG) assay kit (ml076637, mlbio), and total cholesterol (TC) (ml076634, mlbio). Serum concentrations of Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) were determined using the Alanine aminotransferase Assay Kit and the Aspartate aminotransferase Assay Kit, respectively (Yuanju Bio, shanghai), according to the manufacturer's instructions.

2.11. Oil Red O Assay

HepG2 and AML.12 cells were plated in 6‐well or 24‐well plates. After culturing for 24 h in medium containing oleic acid (OA), cells were treated with/without B029‐2 for 48 h. For transfection experiments, cells plated in 6‐well plates were transfected with siRNA or adenovirus for 24 h, followed by culturing in OA‐containing medium for 24 h and subsequently treated with or without B029‐2. Differentiated cells prepared as described above were fixed in 10% formalin, rinsed in deionised water and then incubated in 60% isopropanol for 5 min. The cells were then stained with Oil Red O working solution containing 2 g/L Oil Red O, 60% isopropanol and 40% H₂O for 10 min at room temperature, washed three times in deionised water and incubated in 60% isopropanol. Then, the cells were photographed and counted to estimate cell density. Image analysis software (Image‐Pro Plus 6.0, Media Cybernetics) was used to measure the stained area. At least three independent experiments were performed for each condition.

2.12. Histological Analysis

Mice liver tissue samples were fixed in 4% paraformaldehyde solution (G1101‐500ML, Servicebio) for at least three days and embedded in paraffin. Tissue slides were stained with H&E to evaluate their morphologies for collagen‐specific staining using the Sirius red staining solution kit (BP‐DL029, Sbibio Life Sciences), according to the manufacturer's protocols. Oil Red O staining was used on frozen liver sections that were prepared in optimum cutting temperature (OCT) compound. The extent of MASLD was estimated by the NAFLD activity score (NAS). The intensity of steatosis, fibrosis, and infiltration of macrophages was calculated as the percentage of the positive area of Oil Red O staining, Sirius Red staining and F4/80 in the corresponding field of liver tissue using image analysis software (IMAGE‐PRO Plus 6.0; Media Cybernetics, Rockville, MD, United States).

2.13. Immunohistochemistry Analysis

Immunohistochemistry analysis (IHC) staining was performed on 4‐mm‐thick paraffin sections of tissues fixed in buffered formalin. Sections were deparaffinised in xylene and rehydrated in graded alcohols. After antigen retrieval, samples were placed in 3% H₂O₂ for 15 min to quench endogenous peroxide activity. Slides were blocked with 5% bovine serum albumin for 1 h, and incubated with primary antibodies against F4/80 (GB11027, Servicebio) at 4°C overnight. Then, the slides were incubated with HRP‐conjugated secondary antibodies for 1 h at room temperature, followed by DAB staining. Staining was developed using an EnVision Detection Rabbit/Mouse Kit (GK500710, GeneTech).

2.14. Chromatin Immunoprecipitation Assay

HepG2, AML.12 and Huh‐7 cells were harvested and cross‐linked in 1% formaldehyde for 10 min at room temperature. Then, cells were added with 125 mM glycine and incubated for 5 min at room temperature. Cells were washed with precooled PBS and centrifuged. Then, we discarded the supernatant and resuspended the cells in FA lysis buffer (50 mM HEPES‐KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, pH 8.0, 1% Triton‐X‐100, 0.1% SDS, protease inhibitor). The samples were sonicated, and 50 μL of the products were removed to assess the DNA fragment size. The remainder was stored at −80°C. Antibodies for control IgG (Cell Signalling Technology, #3900S), H3K27ac (Cell Signalling Technology, #8173T), H3K18ac (Cell Signalling Technology, #13998T) and H3K9ac (Cell Signalling Technology, #9649T) were used for immunoprecipitation. DNA extracted from 10 μL pre‐immunoprecipitated samples was used as input controls. Beads were washed with low‐salt ChIP wash buffer (0.1% SDS, 1% Triton‐X‐100, 2 mM EDTA, pH 8.0, 150 mM NaCl, 20 mM Tris–HCl, pH 8.0) 3 times and then with high‐salt ChIP wash buffer (0.1% SDS, 1% Triton‐X‐100, 2 mM EDTA, pH 8.0, 500 mM NaCl, 20 mM Tris–HCl, pH 8.0) once. The immune‐precipitated chromatins were eluted with ChIP elution buffer (1% SDS, 100 mM NaHCO₃) and incubated at 37°C for 30 min, followed by 6 h at 65°C to reverse the cross‐links. DNA was amplified by quantitative real‐time PCR and normalised to the input. DNA samples from immunoprecipitation with normal rabbit IgG were used as controls. The primer for a nonspecific site was used as the negative control. At least three independent experiments were performed. Primer sequences are shown in Table S2.

2.15. Statistical Analyses

Data analyses were performed with Prism 9 (GraphPad software). Two‐group datasets were analysed using Student's t‐tests for normally distributed data, while the Mann–Whitney U test was applied for non‐normally distributed data. One‐way ANOVA was used for the analysis of multiple groups involving a single variable. Two‐way ANOVA was employed to compare more than two groups with multiple variables. The results are presented as the mean ± SD. Statistical significance was denoted as *p < 0.05, **p < 0.01, ***p < 0.001 and ‘n.s.’ indicates no significant difference.

3. Results

3.1. H3K27 and H3K18 Acetylation in Hepatocytes Is Significantly Increased in MASLD

We first assessed the levels of histone acetylation in hepatocytes treated with OA and the livers from MASLD patients and mice. Western blotting showed that the levels of acetylated H3K27 and H3K18 in HepG2 and AML.12 cells were elevated by oleic acid (OA) stimulation, while no notable changes in H3K9ac levels were observed (Figure 1A). Similarly, the levels of H3K27ac and H3K18ac in fatty liver tissues of mice fed on HFD, CDAHFD, HFF and MCD were markedly higher than those in control liver tissues (Figure 1B–E, Figure S1A,B). Moreover, H3K27ac and H3K18ac in hepatic tissue of MASLD patients were also higher than those of healthy individuals (Figure 1F,G). In contrast, hepatic H3K9ac levels exhibited modest elevation in both MASLD patients and MCD‐fed mice compared to their respective controls, while no statistically significant alterations were observed in mice subjected to HFD, CDAHFD and HFF diet (Figure 1B–G, Figure S1A,B). Moreover, the HAT activity of p300 was significantly increased in the livers of MASLD patients and mice (Figure 1H). These findings indicate the enhanced activity of p300 in MASLD liver tissue and demonstrate p300/CBP as a promising candidate that warrants further functional investigation for therapeutic intervention.

FIGURE 1.

MAFLD exhibited hyperacetylation of H3K18 and H3K27 in hepatocytes. (A) Western blots showed the acetylation levels of H3K27 and H3K18 were significantly increased in OA‐induced HepG2 and AML.12 cells. (B, C) Western blots showed that the levels of H3K27Ac and H3K18Ac were significantly increased in hepatic tissues of HFD‐fed mice vs. control mice (B). Semiquantitative analysis was performed with Image Studio Ver 5.5 (C). H3 was used as an internal control for the semiquantitative analysis. (D, E) Western blots showed that the levels of H3K27Ac and H3K18Ac were significantly increased in hepatic tissues of CDAHFD‐fed mice compared to control mice (D). Semiquantitative analysis was performed with Imagine Studio Ver 5.5 (E). H3 was used as an internal control for the semiquantitative analysis. (F, G) The acetylation of H3K27/18/9 was detected by Western blots in hepatic tissues from patients without steatosis (no steatosis), with simple steatosis (MAFL) and with MASH (F). Semiquantitative analysis was performed with Image Studio Ver 5.5 (G). H3 was used as an internal control for the semiquantitative analysis. (H) The p300 HAT activity in the liver of MASLD patients (left) and HFF‐fed mice (right). All graphical data are presented as mean ± SD. The t‐test was performed to compare the differences described previously. *p < 0.05, **p < 0.01, ***p < 0.001. MAFL, metabolic dysfunction‐associated fatty liver; MASH, metabolic dysfunction‐associated steatohepatitis; OA, oleic acid.

3.2. Blocking p300/CBP Activity Reduced OA‐Induced Lipid Accumulation

Given that the initial pathological feature of MASLD is liver steatosis, driven predominantly by excessive caloric accumulation from carbohydrates and lipids. We specifically investigated the therapeutic potential of the p300 inhibitor, B029‐2, on hepatic lipid deposition. We first detected the IC50 of B029‐2 in HepG2 cells and AML.12 cells to avoid the toxicity of B029‐2 on hepatocytes, revealing IC50 values of 32.52 and 40.11 μM, respectively (Figure 2A). In addition, no significant alteration of the cell viability, ALT and AST levels was detected in AML.12 cells treated with 2 and 4 μM and in HepG2 cells treated with 1 and 2 μΜ (Figure S2A,B), while a significant decrease in p300 HAT activity was evident at a concentration of 1 μM in HepG2 cells (Figure S2C). Moreover, the levels of acetylated histones H3K27 and H3K18 in HepG2 and AML.12 cells were significantly decreased by B029‐2 at the concentrations well below IC50, whereas no significant alteration was detected in the acetylation of H3K9 (Figure 2B). To evaluate the anti‐steatotic potential of B029‐2, we established an in vitro model of hepatic lipid accumulation by priming HepG2 and AML.12 hepatocytes with OA. Subsequent pharmacological intervention with B029‐2 demonstrated dose‐dependent mitigation of OA‐induced lipid deposition as confirmed by Oil Red O staining (Figure 2C–F). Quantitative real‐time PCR revealed that B029‐2 abrogated the increased expression of genes involved in de novo lipogenesis (DNL) (ACC and FASN) in both OA‐treated HepG2 and AML.12 cells, while the genes related to bile acid biosynthesis (CYP7A1, CYP8B1) and fatty acid oxidation (CPT2) were upregulated (Figure 2G,H). Knockdown of p300 in HepG2 cells decreased the levels of H3K27ac and H3K18ac, attenuated OA‐induced lipid deposition and abolished the effects of B029‐2 (Figure S2D–H). Together, these data suggest that B029‐2 suppresses lipid accumulation in hepatocytes through inhibiting p300 activity.

FIGURE 2.

Blocking p300/CBP activity reduced OA‐induced lipid accumulation. (A) IC50 of B029‐2 in HepG2 and AML.12 cells. (B) B029‐2 treatment decreased the acetylation of H3K18 and H3K27 in HepG2 and AML.12 cells. The fold number indicates semiquantitative analysis of each target protein with Image Studio Ver 5.5. H3 was used as an internal control for the semiquantitative analysis. The vehicle control was adjusted to 1. (C, D) Oil Red O staining in control, OA‐induced and B029‐2 treated HepG2 cells (C) and the Oil Red O‐positive area was quantified using IMAGE‐PRO Plus (D). At least 3 independent experiments were performed. The 2‐way analysis of variance with multiple comparisons was applied to compare the differences described previously. (E, F) Oil Red O staining in control, OA‐induced and B029‐2‐treated AML.12 cells (E) and quantification of the Oil Red O‐positive area (F). At least 3 independent experiments were performed. The 2‐way analysis of variance with multiple comparisons was applied to compare the differences described previously. (G, H) Relative mRNA levels of genes related to DNL (ACC, FASN and SREBP1), bile acid biosynthesis (CYP7A1 and CYP8B1) and FAO (PPARα, CPT1 and CPT2) in OA‐induced HepG2 cells (G) and AML.12 cells (H) with or without B029‐2 treatment. Samples with DMSO‐treated were used as controls and normalised to 1; the t‐test was performed to compare the differences described previously. All statistical data represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

3.3. B029‐2 Attenuated MASLD in Mice

To investigate the effect of B029‐2 on MASLD models, we subjected mice to a CDAHFD diet for 2 weeks, within a one‐week course of treatment with B029‐2 (2.5 mg/kg) or DMSO via intraperitoneal injection (Figure 3A). As shown in Figure 3B–G, the liver‐to‐body weight ratio was decreased in B029‐2‐treated mice compared to that of control mice. Histological analyses, including haematoxylin–eosin and Oil Red O staining, revealed significant reductions in steatosis in B029‐2‐treated mice, accompanied by lower levels of serum triglyceride (TG) and total cholesterol (TC) (Figure 3H). Consistently, compared with liver tissue from CDAHFD‐fed control mice, liver tissue from B029‐2‐treated mice exhibited a marked downregulation of ACC and upregulation of CYP8B1 and CPT2 (Figure 3I,J). The population of F4/80‐positive cells was also significantly reduced in mice treated with B029‐2 (Figure 3B,G). In addition, serum alanine aminotransferases (ALT) and aspartate aminotransferase (AST) were notably lower in B029‐2‐treated mice compared with control mice (Figure 3K). To further investigate the impact of B029‐2 on MASH pathologies, we subjected mice to an MCD diet for 4 weeks, with B029‐2 (2.5 mg/kg) or DMSO treatment for the last week. The data further demonstrated that B029‐2 alleviates MCD‐induced hepatic steatosis, fibrosis and inflammation (Figure S3). The therapeutic effect of B029‐2 was also observed in an HFF‐induced MASLD model, as evidenced by reduced glucose intolerance, steatosis and dyslipidemia, as well as hepatic fibrosis and chronic inflammatory injury (Figure S4). These findings indicate that B029‐2 attenuated MASLD in mice.

FIGURE 3.

B029‐2 attenuated CDAHFD‐induced MAFLD. (A) Schematic illustration of the experimental protocol for CDAHFD‐induced MAFLD and B029‐2 intervention. Male 8‐week‐old C57BL/6J mice received a CDAHFD diet for 2 weeks, within a one‐week course of treatment with B029‐2 (2.5 mg/kg) or DMSO via intraperitoneal injection every other day (n = 6 mice per group). (B) Representative images of appearance, haematoxylin–eosin‐stained, Oil Red O‐stained, Sirius Red‐stained and IHC‐stained of F4/80 in liver sections. (C) Body weight and Ratio of liver weight to body weight. (D) Nonalcoholic fatty liver disease (NAFLD) activity scores. (E–G) Semiquantitative analysis of Oil Red O‐stained sections (E), Sirius Red‐stained sections (F) and F4/80 in IHC‐stained sections (G). (H) Serum levels of total cholesterol (TC) and triglycerides (TG) in B029‐2‐treated versus untreated CDAHFD‐fed mice. (I) Relative mRNA levels of genes related to DNL (Fasn, Srebp1 and Acc) in livers of B029‐2‐treated versus those in untreated CDAHFD‐fed mice. (J) Relative mRNA levels of genes related to bile acid biosynthesis (Cyp7a1 and Cyp8b1), and FAO (Pparα, Cpt1 and Cpt2) in livers of B029‐2‐treated versus those in untreated CDAHFD‐fed mice. (K) Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in B029‐2‐treated versus untreated CDAHFD‐fed mice. All graphical data are presented as mean ± SD. The t‐test was performed to compare the differences described previously. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.

3.4. B029‐2‐Mediated Epigenetic Regulation Alters the Expression of ACSL4 in Hepatocytes

To investigate the mechanistic basis of the therapeutic effects of B029‐2 on lipid accumulation, we analysed chromatin accessibility‐mediated gene expression changes in hepatocytes using our previous ATAC‐seq and RNA‐seq integrated datasets from B029‐2‐treated Huh‐7 cells [16]. Gene Ontology (GO) analysis of downregulated genes revealed eight lipid metabolism–associated pathways within the top 20 most significantly enriched biological processes following B029‐2 treatment. They were lipid metabolic process, triglyceride biosynthetic process, cellular lipid metabolic process, long‐chain fatty‐acyl‐CoA biosynthetic process, cholesterol metabolic process, steroid metabolic process, triglyceride metabolic process and bile acid metabolic process (Figure 4A). Considering the mechanistic linkage of transcriptional suppression to metabolic reprogramming, we focus on biosynthetic process, in which eight genes were involved in triglyceride biosynthetic process and long‐chain fatty‐acyl‐CoA biosynthetic process, including PNPLA3, ACC, FASN, GPAM, ACSL5, AGPAT2, ELOVL2 and ACSL4 (Figure 4B, Table S3).

FIGURE 4.

Transcriptomic profiling of hepatic metabolic dysregulation with B029‐2 intervention. (A) Lipid metabolic process in the top 20 biological process pathways on GO analysis of RNA‐seq and ATAC‐seq integrated datasets from B029‐2‐treated Huh‐7 cells. (B) Downregulated genes by B029‐2 in the triglyceride biosynthetic process and long‐chain fatty‐acyl‐CoA biosynthetic process from GO analysis of RNA‐seq and ATAC‐seq integrated datasets. (C) p300 occupancy at the promoters of FASN, ACC, ACSL5, GPAM, PNPLA3, AGPAT2, ACSL4 and ELOVL2 in HepG2 cells treated with/without OA. NC was used as a negative control. DNA samples from immunoprecipitation with normal rabbit IgG were used as controls and normalised to 1. (D) Linear correlation pattern showing a positive relationship between the mRNA levels of p300 and GPAM, and ACSL4 in MAFLD patients. (E) p300 occupancy at the promoters of GPAM and ACSL4 in HepG2 cells transfected with sip300. NC was used as a negative control. DNA samples from immunoprecipitation with normal rabbit IgG were used as controls and normalised to 1. (F) Relative ACSL4 and GPAM mRNA levels in p300‐deficient HepG2 cells. All graphical data are presented as mean ± SD. The 2‐way analysis of variance with multiple comparisons was applied to compare the differences described previously. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.

To further investigate the primary genes regulated by p300/CBP in MASLD models, we examined whether p300 occupied the promoter region of these candidate genes by using quantitative ChIP experiments. The results showed that p300 bound to the promoter region of FASN, ACC, GPAM and ACSL4, and the enrichment of p300 at the promoters of ACSL4 and GPAM was enhanced by OA stimulation (Figure 4C). ACSL4 catalyses long‐chain fatty acids (LCFAs) to acyl‐CoA for the synthesis of cellular lipids, and GPAM is the rate‐limiting enzyme in the conversion of FAs to storage triglycerides [17, 18]. RT‐PCR analysis also indicated a positive correlation between the mRNA levels of p300 and GPAM (R = 0.4708, p = 0.0033), ACSL4 (R = 0.3421, p = 0.0173) in MASLD patients' liver (Figure 4D). However, knockdown of p300 significantly reduced the enrichment of p300 at the promoter of ACSL4, but not at the promoters of GPAM (Figure 4E), suggesting that ACSL4 is predominantly regulated by p300. Moreover, p300 deficiency reduced ACSL4 expression (Figure 4F). Collectively, these findings indicated that B029‐2 may exert its anti‐steatotic effects through epigenetic modulation of ACSL4 in MASLD.

3.5. Epigenetic Regulation of ACSL4 in MASLD via the Histone Acetyltransferase Activity of p300/CBP

To further investigate the epigenetic regulation of ACSL4 in MASLD via the histone acetyltransferase activity of p300/CBP, we next searched the ChIP‐seq data from the Cistrome Data Browser. The data showed significant binding of H3K27Ac with the promoter of ACSL4 (Figure 5A). Moreover, the increased hepatic ACSL4 expression was further validated in MASLD patients and mice (Figure S5A–H). In addition, the upregulation of ACSL4 in OA‐treated HepG2 and AML.12 cells and hepatic ACSL4 expressions in CDAHFD and MCD fed mice were decreased by B029‐2 treatment (Figure 5B–D). Additionally, p300 knockdown abolished the effects of B029‐2 on ACSL4 expression (Figure S5I). The p300 HAT activity, H3K27ac and H3K18ac levels decreased in the livers of MASLD mice after B029‐2 treatment (Figure 5C,E). To further unravel the mechanism by which p300/CBP regulates ACSL4, we examined whether acetylated histone H3 occupied the promoter region of ACSL4. Quantitative ChIP experiments showed that H3K27Ac, H3K18Ac and H3K9Ac bound to the promoter region of ACSL4 and the enrichment of H3K18Ac and H3K27Ac at the promoters of this gene was enhanced by OA stimulation (Figure 5F). Moreover, the enrichment of H3K18Ac and H3K27Ac at the promoters of ACSL4 was reduced by treatment with B029‐2, while no reduction of H3K9Ac at these regions was detected (Figure 5F). Consistently, we found that the decreased occupancy of H3K18Ac and H3K27Ac was accompanied by the reduction of p300 occupancy at the promoters of ACSL4 (Figure 5G). These results indicated that p300 promoted the transcription of ACSL4 by regulating the binding of H3K18Ac and H3K27Ac to its promoters, and B029‐2 reduced the expression of ACSL4 through inhibiting the p300/CBP–induced histone acetylation.

FIGURE 5.

Epigenetic regulation of ACSL4 in MAFLD via the histone acetyltransferase activity of p300/CBP. (A) Genome‐browser view of the H3K27ac‐ChIP‐seq on the ACSL4 genes in Huh‐7 cells, HepG2 cells, hepatocytes and liver tissues of human and mice. (B) The expression levels of ACSL4 in OA‐induced HepG2 cells and AML.12 cells with/without B029‐2 treatment. (C) Western blots showed the expression levels of ACSL4 in liver tissues of CDAHFD‐fed mice with or without B029‐2 treatment, and MCD‐fed mice with or without B029‐2 treatment. (D) The expression levels of Acsl4 mRNA in liver tissues of CDAHFD‐fed mice with or without B029‐2 treatment (left), and MCD‐fed mice with or without B029‐2 treatment (right). (E) The HAT activity of p300 in liver tissues of CDAHFD‐fed mice after B029‐2 treatment. (F, G) ChIP assays were performed to analyse H3K27ac, H3K18ac, H3K9ac (F) and p300 (G) occupancy at the promoters of Acsl4 in AML.12 cells treated with/without B029‐2. NC was used as a negative control. DNA samples from immunoprecipitation with normal rabbit IgG were used as controls and normalised to 1. At least 3 independent experiments were performed. The data are presented as the mean ± SD. The analysis of variance with multiple comparisons and the t‐test were used to compare the differences mentioned previously. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.

3.6. B029‐2 Ameliorates MASLD Through Suppressing ACSL4 Expression

As the role of hepatocyte‐specific ACSL4 knockout in MASLD progression remained controversial [14, 15], we next sought to confirm the role of ACSL4 in MASLD. Oil Red O staining showed that ACSL4 knockdown significantly reduced lipid accumulation in OA‐induced HepG2 and AML.12 cells (Figure S6A–F). The expression levels of genes involved in DNL (ACC and FASN) were suppressed in ACSL4‐silenced cells (Figure S6G,H). We also confirmed that hepatic‐specific Acsl4‐deficient (Acsl4 ^LKO) ameliorates MASLD development in mice fed on a HFD or CDAHFD diet (Figures S7 and S8). Moreover, B029‐2 treatment produced no additional improvement in liver‐to‐body weight ratio, serum lipids, hepatic steatosis, fibrosis and liver injury in Acsl4 ^LKO mice with CDAHFD diet (Figure S8).

We then further investigate whether Acsl4 overexpression affects the inhibitory effect of B029‐2 on CDAHFD‐fed mice (Figure 6A). We found that Acsl4 overexpression did not alter body weight during the B029‐2 treatment, with only a slight reduction in food intake (Figure S9). Although hepatic Acsl4 overexpression did not significantly exacerbate MASLD, it remarkably increased the MASLD parameters, such as hepatic gross morphology (Figure 6B,C), serum TC levels (Figure 6D), ALT activities (Figure 6E), NAFLD activity score and Sirius red staining outcomes (Figure 6F–I), in mice treated with B029‐2. Moreover, the enhanced hepatic steatosis and increased F4/80‐positive cells were also observed in B029‐2‐treated mice with Acsl4 overexpression (Figure 6J–M). These findings revealed that hepatic Acsl4 overexpression significantly reversed the inhibitory effect of B029‐2 on hepatic steatosis, fibrosis and inflammation. Collectively, these data suggest that B029‐2 ameliorated hepatic steatosis partially through downregulation of Acsl4.

FIGURE 6.

ACSL4 overexpression reverses the effect of B029‐2 in vivo. (A) Schematic illustration of the experimental protocol for Acsl4 overexpression affecting the inhibitory effect of B029‐2 on CDAHFD‐fed mice. Male 6‐week‐old C57BL/6J mice were injected via the tail vein with AAV8‐TBG‐Acsl4 or AAV8‐TBG‐GFP (5 × 10¹⁰ Vg/mouse) two weeks before feeding the CDAHFD diet. Then they received a CDAHFD diet for 2 weeks, followed by B029‐2 (2.5 mg/kg) or DMSO treatment for the last week (n = 6 mice per group). (B) Representative images of liver appearance. (C) Ratio of liver weight to body weight. (D) Serum levels of total cholesterol (TC) and triglycerides (TG) in B029‐2‐treated versus untreated mice with/without Acsl4 overexpression and fed with/without CDAHFD diet. (E) Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in B029‐2‐treated versus untreated mice with/without Acsl4 overexpression and fed with/without CDAHFD diet. (F, G) Representative images of haematoxylin–eosin‐stained (F) and Sirius Red‐stained liver sections (G). (H) NAFLD activity scores. (I–K) Quantification of the positive area of Sirius Red‐stained liver sections (I), Oil Red O‐stained liver sections (J) and F4/80 in IHC‐stained liver sections (K) with IMAGE‐PRO Plus. (L, M) Representative images of Oil Red O‐stained liver sections (L) and F4/80 in IHC‐stained liver sections (M). The data are presented as the mean ± SD. The 2‐way analysis of variance with multiple comparisons was applied to compare the differences described previously. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.

4. Discussion

Histone acetylation is an epigenetic modification that can decondense, that is ‘open‐up’ chromatin to facilitate access to DNA for transcriptional or DNA repair proteins. Metabolism may exert complex influences on histone acetylation in the liver. Increased flux of acetylated histone forms and reduced HDACs was observed in the MASLD mouse liver [19, 20]. Chip‐seq analysis revealed that approximately 17% of differentially expressed genes exhibited H3K9ac peak changes within their promoters, indicating a strong correlation between H3K9ac dynamics and transcriptional dysregulation in HFD livers [21]. Another ChIP‐seq analysis mapping the genomic landscape of H3K27ac revealed that activated typical enhancers marked by this histone modification are enriched for lipid metabolism pathways in fatty liver [22, 23]. However, Carrer A, et al. did not observe the alterations of specific histone acetylation marks (H3K9ac, H3K14ac, H3K18ac, H3K23ac, H4K5ac, H4K8ac, H4K12ac, H4K16ac) in the liver of mice with a 4‐week HFD diet [24]. Here, we found that higher levels of acetylated H3K27 and H3K18 in fatty liver tissues of MASLD patients, as well as in the livers of MASLD models. In contrast, only a modest elevation of H3K9ac was observed in MASLD patients and MCD‐fed mice. H3K27ac and H3K18ac modification may be more important for MASLD progression.

H3K18Ac and H3K27Ac are two known histone acetylation types catalysed by p300/CBP. Although we use various mouse models of MASLD, they all recapitulate core pathological features of human MASLD/MASH, including hepatic steatosis, lipotoxicity and chronic inflammation. The similar epigenetic alteration of p300‐mediated histone acetylation in the MASLD models indicates that the activation of p300 may play an important role in MASLD. Studies revealed that adenovirus‐mediated overexpression of p300 promotes hepatic steatosis, and inhibition of p300 by siRNA reduces lipogenesis in human sebocytes [12, 25]. As a chemical tool that can efficiently inhibit the activity of p300/CBP, B029‐2 specifically decreases p300/CBP HAT activity, H3K18Ac and H3K27Ac and displays a significant antitumor effect in HCC [16]. But its role in MASLD remains unclear. Our study demonstrates that B029‐2‐mediated epigenetic modulation reprograms lipid metabolism genes in hepatocytes, effectively reducing lipid accumulation in both OA‐induced cells and MASLD models. These findings highlight the potential value of the p300/CBP inhibitor in the intervention of MASLD.

Previous studies have demonstrated an intriguing connection between elevated acetylation mediated by p300/CBP of transcription factors such as Foxa2, FXR, SREBP‐1c and ChREBP, and increased lipogenic gene expression [12, 26, 27, 28]. In our study, transcriptome analysis also revealed that B029‐2 downregulated the expression of lipogenic genes, primarily those involved in the triglyceride biosynthetic process and long‐chain fatty‐acyl‐CoA biosynthetic process. It has been well recognised that ACSL enzymes catalyse enzymatic reactions to esterify free FA to acyl‐CoA for entry into various metabolic pathways, including the cellular β‐oxidation system responsible for FA oxidation and anabolic pathways for the synthesis of phospholipids, cholesterol esters and triglycerides [29]. Within the ACSL family, ACSL4 has a marked substrate preference for PUFA. While there is debate regarding the role of ACSL4 in MASLD, most studies still suggest that ACSL4‐mediated positive regulation participates in the progression of toxin‐linked MASLD [30, 31, 32]. Herein, we further verified that hepatic ACSL4 deletion attenuated diet‐induced MASLD in mice. Moreover, the beneficial effect of B029‐2 on MASLD was partially reversed by ectopic expression of ACSL4, and hepatocyte‐specific ACSL4 knockout abolished the effect of B029‐2 on hepatic steatosis. In addition, a study by Sinha et al. had observed that increased ACSL4‐driven acetyl‐CoA promotes hyperacetylation of activating epigenetic marks such as H3K9 and H3K27 [33]. However, we revealed that p300/CBP promoted the transcription of ACSL4 by regulating H3K27Ac and H3K18Ac, providing a new understanding of the interrelationship between lipid metabolism and histone acetylation.

In summary, our study confirmed the epigenetic alterations of p300 HAT activity, H3K27Ac and H3K18Ac in MASLD, providing more evidence for HATs as a therapeutic target of MASLD. Our data also demonstrated that the therapeutic effect of the p300/CBP inhibitor B029‐2 on MASLD in vivo and in vitro by inhibiting the ACSL4 expression. Conceptually, our findings underscore the interplay between histone acetylation and lipid metabolism, and highlight the value of therapeutically targeting the HAT activity of p300/CBP in MASLD.

Author Contributions

All authors read and approved the final manuscript. L.‐Y.C. contributed to designing research studies, conducting experiments, acquiring data, analysing data and writing the manuscript. Y.‐P.G. and J.‐H.H. contributed to animal experiments and tissue collection, and acquisition and analysis of histological and biochemical data. C.‐Y.S. generated sequencing libraries and performed bioinformatics analysis. C.‐H.D. and X.Z. contributed to review and editing, W.‐F.X., X.Z. and X.Z. conceptualised and supervised the research, secured funding and interpreted the data.

Funding

This work was supported by the National Natural Science Foundation of China (Grants 82100608 to L.‐Y.C. and 82430022 to W.‐F.X.) and the National Key R&D Program of China (Grant 2023YFC2507500 to W.‐F.X.). This work was also funded by the New Quality Clinical Specialty Program of High‐End Medical Disciplinary Construction in Shanghai Pudong New Area (Grant 2025‐PWXZ‐04 to X.Z.).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: MASLD exhibited hyperacetylation of H3K18 and H3K27 in liver tissues.

Figure S2: siRNA‐mediated p300 knockdown reduced OA‐induced lipid accumulation.

Figure S3: B029‐2 attenuated MCD‐induced MASLD.

Figure S4: B029‐2 attenuated HFF‐induced MASLD.

Figure S5: ACSL4 is highly expressed in MASLD liver tissues.

Figure S6: Downregulation of ACSL4 reduced lipid accumulation in vitro.

Figure S7: Hepatic‐specific Acsl4 knockout improved HFD‐induced steatosis.

Figure S8: Hepatic‐specific Acsl4‐deficient blocks the effect of B029‐2 in vivo.

Figure S9: Body weight and food intake in mice with hepatic Acsl4 overexpression after one‐week B029‐2 treatment.

Table S1: The clinical characteristics of patients with normal liver (NL) or metabolic dysfunction‐associated fatty liver disease (MASLD).

Table S2: Primer and siRNA sequences.

Table S3: Gene ontology analysis of integrated ATAC‐seq and RNA‐seq datasets.

Cai L.‐Y., Guan Y.‐P., Hu J.‐H., et al., “Inhibition of Acetylation Activity of p300/CBP Ameliorates Hepatic Steatosis Through Downregulation of ACSL4 ,” Liver International 46, no. 10 (2026): e70846, 10.1111/liv.70846.

Data Availability Statement

The data that support the findings of this study are openly available in NCBI at https://www.ncbi.nlm.nih.gov/bioproject, reference number PRJNA613779.