Ferulic acid (FA) was purchased from Victory Biological Technology Co., Ltd. (Sichuan, China). Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Oil Red O staining test kits, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), 4′,6-diamidino-2-phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Solarbio (Beijing, China). Assay kits for total TG, cholesterol (TC), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies for PGC-1β and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from ProteinTech (Chicago, IL, USA). RNAiso Plus was purchased from Takara (Shiga, Japan). The TransScript First-Strand cDNA Synthesis SuperMix and TransStart Top Green quantitative reverse transcription polymerase chain reaction (qRT-PCR) SuperMix were purchased from TransGen (Beijing, China). TurboFect was purchased from Thermo Scientific (Waltham, MA, USA).

This animal experiment was approved by the Ethics Committee of Animal Experimentation of Shanxi University (SXULL2020046). A total of 20 male C57BL/6J mice (5 weeks old), weighing 20 ± 5 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in a specific pathogen-free room at 22 ± 2 °C under a 12:12-h light–dark cycle.

The mice were randomly divided into four groups: one control group and three treatment groups, with five mice per group. The control group was fed a normal diet, while the three treatment groups were fed a high-fat diet (HFD). Among the HFD groups, one was given an equal volume of water, one was given simvastatin (5 mg kg⁻¹ day⁻¹), and one was fed FA (30 mg kg⁻¹ day⁻¹). At the end of the study, all mice were anesthetized with ether, and their tissues were immediately collected, weighed, and frozen in liquid nitrogen for further analysis.

The PLCPRF5, Hepatocellular Carcinoma, Grade 2 (HepG2), and Beijing Institute of Liver Cancer (BEL-7402) BEL-7402 cell lines were obtained from the Chinese Type Culture Collection (Shanghai, China). The cells were cultured in DMEM supplemented with 10% FBS and antibiotics at 37 °C in a humidified atmosphere containing 5% CO₂. FFA (a mixture of oleic acid and palmitic acid at a ratio of 2:1) was used to treat hepatocytes to establish the MASLD model. For steatosis induction, HepG2 and PLCPRF5 cells were treated with 1 mM FFA for 24 h, whereas BEL-7402 cells were treated with 0.5 mM FFA for 24 h.

The solid-phase extraction assay was performed as described previously (16). PLCPRF5 or HepG2 cell lysates were incubated with FA crystallization at 4 °C overnight, and the supernatant was removed by centrifugation. The precipitated complex was washed with PBS to remove unbound proteins. The interaction between the PGC-1β protein and FA was then assessed.

The crystal structure of PGC-1β (ID: AF-Q86YN6-F1) was obtained from the protein database and downloaded in Protein Data Bank (PDB) format. The small-molecule structure of FA (Compound CID: 445858) was downloaded from the PubChem database. AutoDock Vina (17) was used to simulate the binding of FA with PGC-1β.

DARTS assays were performed according to a previously described protocol (18). PLCPRF5 cells were incubated in 10-cm dishes to 80% confluence and lysed in RIPA buffer containing a protease inhibitor. After centrifugation, the sediment was collected. The bicinchoninic acid (BCA) method was used to detect protein concentration. Each sample was divided into two aliquots, one for proteolysis with pronase and the other for mock proteolysis. Western blot analysis was performed after digestion. The concentration of FA was 40 μg/mL.

CETSA was performed according to a previously described protocol (19). The concentration of FA was 40 μg/mL.

To observe the protein degradation process, PLCPRF5 and HepG2 cells were treated with cycloheximide (CHX) (20 μg/mL). Total protein was extracted at the specified time points and analyzed using Western blotting.

The cells were treated with MG132 (20 μM) for 6 h before collection. Cell lysates were then prepared, immunoprecipitated using a PGC-1β antibody, eluted with 20 μL of lysis buffer, and denatured with SDS sample buffer. Ubiquitination modification of PGC-1β was assessed using Western blotting.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was used to measure mRNA levels. Total RNA was extracted from cells and tissues using Trizol reagent. RNA was reverse-transcribed using an all-in-one first-strand cDNA synthesis kit. mRNA levels were quantified by qRT-PCR. GAPDH mRNA was used as the control. Relative expression was calculated using the 2^−ΔΔCt method. Primer sequences are listed in Supplementary Table S1.

The data are presented as the mean ± SEM. Analysis of variance (ANOVA) with post-hoc Tukey’s testing was used to compare the means of ≥3 groups. Univariate comparison was performed using Student’s t-test. p-values of <0.05 were considered statistically significant.

To determine the effects of FA on the regulation of MASLD, an HFD-induced mouse model was used. It was found that FA significantly reduced body weight and liver weights compared to HFD diet-fed mice (Figures 1A–C; Supplementary Figure S2A). Food intake was not altered by FA (Supplementary Figure S2B). Meanwhile, the FA supplementation reduced the levels of serum total TG and TC, as well as the levels of serum ALT and AST (Figures 1D,E). Histologically, hematoxylin and eosin (H&E) and Oil Red O staining revealed the presence of hepatic steatosis and fat droplets in the hepatocytes of HFD diet-fed mice, while FA treatment ameliorated these changes (Figures 1F,G). Consistent with the above phenomenon, FA supplementation markedly reduced the content of TG in the liver (Figure 1H). Simvastatin, which can improve lipid metabolic disorders, was used as a control to evaluate the effect of FA. The results showed that FA attenuated lipid metabolism in a manner consistent with simvastatin. Collectively, these data suggest that FA intervention significantly ameliorated lipid accumulation in the livers of HFD-induced mice.

We then examined the effects of FA in hepatocytes undergoing lipid accumulation. The results of MTT assays revealed that no cytotoxicity was observed at concentrations below 60 μg/mL (Figure 2A). BODIPY and Oil Red O staining further demonstrated that lipid accumulation in PLCPRF5, HepG2, and BEL-7402 cells was significantly decreased by FA intervention (Figures 2B,C). The decreased triglyceride levels also suggested that lipid accumulation in hepatocytes was alleviated following FA treatment (Figure 2D). These results emphasize that FA effectively alleviates lipid accumulation in FFA-treated hepatocytes.

To further investigate the molecular mechanisms underlying FA effects on FFA-induced lipid accumulation, the target proteins of FA in FFA-induced PLCPRF5 were identified using a solid-phase extraction assay. Silver staining assay revealed that the FA group had specific binding proteins compared to the ethanol group (Figure 3A). These proteins were analyzed by mass spectrometry, and 322 FA-specific binding proteins were identified (Figure 3B). KEGG enrichment analysis revealed that the target proteins of FA were enriched in metabolic pathways, including the fatty acid metabolic pathway closely related to MASLD (Figure 3C). PPAR gamma-coactivator-1beta (PPARGC1β, also known as PGC-1β), the most enriched in the above pathway, is considered to be the target protein of FA (Figure 3D). To substantiate the interaction between FA and PGC-1β protein, a solid-phase extraction assay was used. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a distinct protein band of 100 kDa, which is consistent with the size of PGC-1β protein, was observed in the FA group compared to the ethanol group (Figure 3E). The results of Western blotting further confirmed the binding of FA to PGC-1β (Figure 3F). Interestingly, molecular docking analysis showed that PGC-1β possessed binding pockets for FA binding, with a binding score of −5.4 kcal/mol, and that FA forms hydrogen bonds with the K357 site of PGC-1β (Figure 3G). Moreover, PhosphoSitePlus prediction identified that K357 is the ubiquitination modification site of PGC-1β (Figure 3H). These results indicate that PGC-1β is a direct target of FA.

Based on the above results, we hypothesized that FA could regulate the PGC-1β protein through the ubiquitin–proteasome pathway. The results indicated that PGC-1β protein expression was significantly decreased after FA intervention (Figures 4A,B). Moreover, DARTS and CETSA assays showed that FA reduced the stability of the PGC-1β protein (Figures 4C,D). Meanwhile, the protein synthesis inhibitor CHX was used to evaluate the effect of FA on the degradation of PGC-1β and showed that the half-life of PGC-1β was shortened after FA intervention (Figure 4E). Furthermore, the proteasome inhibitor MG132 prevented PGC-1β downregulation following FA intervention (Figure 4F), suggesting that FA regulates the expression of PGC-1β through the ubiquitin–proteasome pathway. To further validate these data, we evaluated the effect of FA on PGC-1β ubiquitination using an in vitro ubiquitination assay. The results revealed that FA reduced the stability of PGC-1β by increasing the ubiquitous modification of PGC-1β (Figure 4G). The results indicate that FA inhibited the expression of PGC-1β protein through the ubiquitin–proteasome pathway.

To test whether PGC-1β is involved in the improvement effect of FA against fatty liver, we overexpressed PGC-1β protein before 40 μg/mL FA in FFA-induced hepatocytes. Oil Red O staining showed that overexpression of PGC-1β protein promoted FFA-induced lipid accumulation and blocked the improvement effect of FA on lipid accumulation (Figures 5A,B). Furthermore, FA treatment decreased TG content in FFA-induced hepatocytes, whereas the overexpression of PGC-1β protein partially counteracted the inhibitory effect of FA on TG content (Figures 5C–E). These results suggest that overexpression of PGC-1β protein eliminates the beneficial effect of FA treatment on fatty liver.

To investigate the molecular mechanism by which FA regulates non-alcoholic fatty liver through PGC-1β, proteins interacting with PGC-1β were predicted using the BioGRID Database and the IntAct Molecular Interaction Database. These target proteins were imported into Metascape for gene enrichment analysis. The results revealed that the proteins interacting with PGC-1β were enriched in pathways related to fatty liver disease (Figure 6A) and were involved in PPARA, thereby activating gene expression, the nuclear receptor transcription pathway, and the intracellular receptor signaling pathway (Figures 6B–D). The protein–protein interaction network and Molecular Complex Detection (MCODE) analysis indicated that PGC-1β targets proteins were involved in PPARA-activated gene expression, PPARA-mediated regulation of lipid metabolism, and transcriptional regulation of white adipocyte differentiation (Table 1, Figure 6E). PGC-1β is known to function as a transcriptional coactivator of SREBP-1, a key regulator of liver adipogenesis (7). As shown in Figure 6F, an interaction between PGC-1β and SREBP1 was observed. Furthermore, overexpression of PGC-1β protein promoted the expression of SREBP1 and blocked the inhibitory effect of FA on SREBP1 (Figure 6G). FA decreased the expression of FASN and SCD1 genes, the key enzymes of lipogenesis, while overexpression of PGC-1β protein partially counteracted the inhibitory effect of FA on FASN and SCD1 (Figures 6H,I). Based on this study, ferulic acid, through the PGC-1β/SREBP1 axis, inhibits lipogenesis to improve non-alcoholic fatty liver.