Design, synthesis, and biological evaluation of (E)-3-(3-methoxy-4-substituted phenyl)-acrylic acid derivatives: Role of compound S17 in regulating lipid accumulation via AMPK activation

2.1.1 General procedure for the synthesis of compounds 2a-2i

To a stirred solution of substituted phenol 1a-1i (50.0 mmol) in acetone (30 mL), sodium hydroxide (10.00 g, 250.0 mmol) was added at room temperature. The reaction mixture was stirred under refluxing for 0.5 h, added chloroform (3.4 mL, 100.0 mmol) dropwise and refluxing for 3 h. Concentrate the resulting solution subsequently under vacuum, and the mixture was diluted with water (100 mL). The pH of mixture was adjusted to around 2 with 1 N HCl and then filtered. The filted cake was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:2, v/v) to obtain compounds 2a-2i. Their spectral data were provided in Supplementary Material.

2.1.2 General procedure for the synthesis of compounds 4a-4c

Vanillin 3 (3.05 g, 20.0 mmol), halohydrin (50.1 mmol), K₂CO₃ (5.54 g, 40.1 mmol), TBAI (1.48 g, 4.0 mmol), DMF (10 mL) were added into a dry 100 mL round bottom flask and heated to 50 °C under stirring for 5 h. After the reaction was completed, the mixture was poured into water (50 mL) and extracted with EtOAc (30 mL × 3). The combined organic phase was washed with saturated brine, dried over anhydrous MgSO₄, filtered and evaporated. The given residues were purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1, v/v) to obtain the compounds 4a-4c. Their spectral data were provided in Supplementary Material.

2.1.3 General procedure for the synthesis of compounds 5a-5u

To a solution of substituted phenyloxyisobutyric acid 2a-2i (2.5 mmol) in anhydrous dichloromethane (6 mL) was subsequently added 4a-4c (3.8 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (957 mg, 5.0 mmol), 4-dimethylaminopyridine (30 mg, 0.2 mmol) and triethylamine (0.9 mL, 6.2 mmol). The reaction mixture was stirred at room temperature for 5 h. Upon completion, the mixture was then poured into ice-water, extracted with dichloromethane (40 mL × 3). The combined extracts were then washed with saturated brine, dried over anhydrous MgSO₄ and concentrated. The crude products were purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1, v/v) to obtain the compounds 5a-5u. Their spectral data were provided in Supplementary Material.

2.1.4 General synthesis procedure for compounds S01-S21

A mixture of 5a-5u (2.0 mmol), malonic acid (416 mg, 4.0 mmol), piperidine (catalytic amount) and pyridine (5 mL) was added into a dry 25 mL round bottom flask. The reaction was heated to 90 °C under stirred for 8 h. The reaction solution was cooled to room temperature and poured into 20 mL of ice water. Then, the pH of mixture was adjusted to around 2 with 1 N HCl and extracted with CH₂Cl₂ (30 mL × 3). The combined organic layer was washed with water, saturated brine, dried over anhydrous MgSO₄ and evaporated to obtain a residue purified by silica gel column chromatography (CH₂Cl₂: Methanol = 20:1, v/v) to obtain the compounds S01-S21.

2.2.1 Cell line culture

HepG2 cells were maintained in DMEM (C11995500BT, Thermo Fisher) supplemented with 10 % FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. The cell culture was conducted at 37 °C in a humidified atmosphere with 5 % CO₂.

2.2.2 Antibodies and reagents

Ferulic acid (FA), 5-aminoimidazole-4-carboxamide-1-β-ribofuranoside (AICAR), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were acquired from MedChem Express (MCE). The BCA Protein Assay Kit (C503051-0500) and Oil Red O (A100684-0050) were obtained from Sangon Biotech Co., Ltd (Shanghai, China). Kits for the assays of triglyceride (TG, E1013-50), alanine aminotransferase (ALT, E2022-105), and aspartate aminotransferase (AST, E2024-105) were sourced from Pulilai Gene Technology Co., Ltd. (Beijing, China).

The following antibodies (at a dilution of 1:1000) were used: anti-β-actin (AC026) and anti-phospho ACC1 (AP0298) from ABclonal; anti-SREBP-1 (sc-13551) from Santa; anti-ACC1 (67373–1-Ig) and anti-AMPK (66536–1-Ig) from Proteintech; anti-phospho AMPKα (2535) from Cell Signaling Technology.

2.2.3 In vitro cytotoxicity assay

The cytotoxic effects of the compounds were evaluated in vitro using the MTT assay. HepG2 cells were enzymatically detached, resuspended, and plated in 96-well plates (2 × 10³ cells per well), followed by overnight incubation. The cells were subjected to the treatment of compounds under investigation, with O-methylferulic acid and AICAR serving as positive controls and DMSO as the negative control. After a 72 h incubation period, MTT reagent (5 mg/mL) was added to each well, and the plates were incubated for an additional 3 h. The optical density (OD) of the resulting formazan product was quantified at 490 nm (OD₄₉₀).

2.2.4 Preparation of free fatty acid (FFA) solutions

1. Oleic acid (OA) and palmitic acid (PA) solutions (0.1 mol/L)

Sodium oleate (141 mg) and sodium palmitate (128 mg) were weighed and placed into separate 15 mL centrifuge tubes. Each tube received 10 mL of PBS solution. The tubes were mixed and then heated at 80 °C until the fatty acids were completely dissolved.

1. Bovine serum albumin (BSA) solution (5 % w/v)

BSA (0.7 g) was weighed and added to a 50 mL centrifuge tube containing 14 mL of PBS. This mixture was heated at 80 °C while stirring until the BSA was fully dissolved.

1. OA and PA stock solutions (10 mmol/L)

For the OA stock solution, 1 mL of the 0.1 M OA solution was quickly transferred to a new 15 mL centrifuge tube containing 9 mL of the 5 % BSA solution and mixed well. Similarly, to prepare the PA stock solution, 0.5 mL of the 0.1 M PA solution was combined with 4.5 mL of the 5 % BSA solution.

1. Combined FFA solution (OA: PA = 2:1) at different concentrations

For experimental use, 2 mL of OA stock solution, 1 mL of PA stock solution, and 3 mL of DMEM with 20 % FBS were mixed to prepare a 5 mmol/L combined FFA solution (OA: PA = 2:1). The final concentration can be adjusted as required by diluting with DMEM containing 10 % FBS. FFA solutions should be freshly prepared prior to each experiment.

2.2.5 Development of an adipose accumulation model in HepG2 cells

HepG2 cells were plated in 6-well plates (1 × 10⁶/well) and cultured in DMEM (10 % FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin) at 37 °C in a 5 % CO₂ humidified atmosphere. To establish a model of fat accumulation, the cells were grown to 75 % confluence and then switched to serum-free DMEM overnight. Subsequently, they were treated with various concentrations of the target compounds dissolved in DMEM containing an FFA mixture (OA: PA = 2:1) for 24 h.

2.2.6 Oil Red O staining

After treatment with 1 mM FFAs and respective compounds or positive controls for 24 h, the HepG2 cells were prepared for lipid droplet visualization. Cells were washed twice with cold PBS and fixed with 10 % formaldehyde for 30 min. Following fixation, cells were washed thrice with water and stained with 0.5 % Oil Red O in a 60 % isopropanol solution for 20 min at room temperature. The stained cells were observed and imaged using an optical microscope (DMi1, Leica Microsystems Wetzlar GmbH) with a digital camera at 40 × magnification. Subsequently, the Oil Red O staining intensity was quantitatively analyzed at OD₄₉₀.

2.2.7 Measurement of intracellular TG levels

In vitro TG assays were conducted using the TG Assay Kit (E1013-50). The experimental procedures were carried out in accordance with the manufacturer's protocol, detailed information of which can be found at https://www.applygen.com/upload/download/20210713/20210713024524908.pdf.

HepG2 cells, treated with 1 mM FFAs along with the designated target compounds or positive controls for 24 h, were washed thrice with PBS. The cells were then lysed in lysis buffer for 30 min. Subsequently, the TG levels were determined following the protocols of the kit. Concurrently, protein concentrations were quantified using the BCA Protein Assay Kit (C503051-0500), adhering to the manufacturer's guidelines.

2.2.8 ALT and AST measurements

ALT and AST levels were measured in vitro using the ALT Assay Kit (E2022-105) and AST Assay Kit (E2024-105). The protocols followed were per the manufacturer's instructions, with more comprehensive details available at: https://www.applygen.com/upload/download/20220114/20220114102112664.pdf, and https://www.applygen.com/upload/download/20220114/20220114093422869.pdf.

Following 24 h exposure of HepG2 cells to 1 mM FFAs with the specified target compounds or positive controls, the cells were washed three times with PBS. A lysis buffer was used to lyse the cells for 30 min. The activities of ALT and AST were then assayed as per the instructions provided by the manufacturer. Protein levels were quantified concurrently using the BCA Protein Assay Kit (C503051-0500), following the manufacturer's protocol.

2.2.9 Western blot analysis

HepG2 cells (1 × 10⁶/well) were plated in 6-well plates. Following a 16-h incubation, the culture medium was replaced with fresh media containing varying concentrations of the target compounds or a control agent for a period of 24 h. For cell lysis, RIPA lysis buffer from Sangon Biotech, supplemented with a protease inhibitor cocktail from MCE, was employed for 30 min at ice-cold temperatures. Protein concentrations were determined using the BCA method. Equal amounts of proteins were resolved on 10 % SDS-PAGE and subsequently transferred onto polyvinylidene difluoride membranes. The membranes were then blocked with 5 % milk for 1–2 h. Primary antibodies were applied at a 1:1000 dilution, followed by incubation with HRP-conjugated secondary antibodies. Visualization of protein bands was conducted using ECL reagents from Bio-Rad (Hercules, CA). Quantification of the protein bands was achieved using ImageJ software, following normalization against the solvent control.

2.2.10 Statistical analysis

The data acquired from the experiments are expressed as the mean ± standard deviation (SD). To ensure the reliability of the results, each experimental assay was conducted at least thrice independently. For statistical analysis, GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA) was utilized. Tukey's post-test and student's t-test were utilized for multiple or single comparisons. The determination of statistical significance was conducted via t-tests and nonparametric tests, with p-value thresholds set at *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001.

3.2.1 Establishment of FFA-induced HepG2 cell model for steatosis

NAFLD is tightly associated with the hepatic manifestation of metabolic syndrome. In vitro models using FFA-challenged HepG2 cells are commonly employed to study NAFLD (Errafii et al., 2022, Guo et al., 2022, Ramos et al., 2022). Extensive research has highlighted that exposing HepG2 cells to 0.5–1 mM of FFA for 24 h results in significant lipid accumulation (Hassan et al., 2014, Tan et al., 2022, Yan et al., 2022).

In this study, we evaluated the impact of FFA at varying concentrations of FFA to identify the optimal concentration for inducing steatosis. MTT assay results (Fig. 2A) uncovered that FFA treatment up to 1 mM for 24 h did not render cytotoxic effects on HepG2 cells. The lipid droplet accumulation was assessed by Oil Red O staining, TG assay and ALT/AST enzyme assay. The result of Oil Red O staining unveiled that the number of lipid droplets correlated with the intense of Oil Red O in cells, which found to be concentration-responsive in FFA-treated cells (Fig. 2B&2F). The result of TG assay and ALT/AST assays also displayed a concentration-responsive increase in TG content and ALT/AST enzyme activities response to FFA treatment (Fig. 2C-2E).

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Fig. 2

Effect of FFA on cell viability and lipid accumulation in HepG2 cells. (A) Cell viability under varying FFA concentrations assessed by MTT assay. (B) Lipid droplet accumulation induced by FFA, as quantified by fluorescence absorption detection at OD₅₀₀ following Oil Red O staining. (C) Influence of different FFA concentrations on TG content in HepG2 cells post 24 h of treatment. (D, E) Effects of FFA at different concentrations on ALT (D) and AST (E) enzyme activity in HepG2 cells after 24 h of treatment. (F) Visualization of lipid droplet accumulation in HepG2 cells at different FFA concentrations (scale bar 50 μm, 40 × ); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus the control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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According to the MTT assay, the usage concentration of is in the range of 0.1 mM to 1 mM. Compared to 0.1 mM or 0.25 mM, the result of 0.5 mM or 1 mM shown more remarkable difference in above experiments, especially in Oil Red O staining and TG assay (Fig. 2B&2C). In ALT/AST assays, the result of 0.1 mM and 0.25 mM shown no remarkable difference (P > 0.05) or low remarkable difference (P < 0.05), but the result of 0.5 mM and 1 mM shown higher remarkable difference (P < 0.001, or P < 0.0001) (Fig. 2D&2E). Therefore, these results suggest that FFA concentrations of either 0.5 mM or 1 mM are optimal for establishing in vitro steatosis models in HepG2 cells.

3.2.2 Effects of target compounds on HepG2 cell proliferation

The influence of the target compounds on the viability of HepG2 cells was assessed using the MTT assay. To establish a reference for comparison, AICAR, an established AMPK activator that effectively augments AMPK phosphorylation in FFA-stimulated HepG2 cells, served as a positive control (Sanchez et al., 2013). The cell viability results, summarized in Table 1, were obtained from a 24 h exposure to the compounds using the MTT assay. These findings indicate that the viability of HepG2 cells was largely unaffected by the treatment with most of the compounds at concentrations up to 400 μM. Hence, the concentration of 400 μM for the target compounds was selected for subsequent experiments.

3.2.3 Screening of target compounds on lipid accumulation in FFA-induced HepG2 cells

The extent of cellular lipid accumulation was assessed using Oil Red O staining. This method was instrumental in clarifying the effects of target compounds on the FFA-induced steatosis model in HepG2 cells (Hassan et al., 2014, Tan et al., 2022, Yan et al., 2022). Notably, FFA treatment led to an increase in both the size and intensity of intracellular lipid droplets. As demonstrated in Fig. 3A, the lipid content in FFA (1 mM)-challenged cells escalated to 228.8 % compared to the control group. At a concentration of 100 μM, most compounds did not significantly diminish lipid droplet accumulation in FFA-challenged cells. However, two exceptions, compounds S10 and S17, noticeably reduced the intracellular lipid content to 178.4 % and 170.7 %. Further investigations, as indicated in Fig. 3B and Fig. S1, revealed that at the concentration of 400 μM, the majority of the target compounds prominently impeded lipid accumulation. Among these compounds, S10 and S17 were efficacious in reducing the size and number of intracellular lipid droplets (Fig. 3B&3C). The summary of the structure–activity relationship (SAR) of the synthesized compounds was depicted in Fig. 4.

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Fig. 3

Influence of compounds S01-S21 on lipid accumulation in FFA-challenged HepG2 cells. (A, B) Detection of lipid droplet accumulation via fluorescence absorption. The model group received 1 mM FFA (OA: PA = 2:1) and DEME for 24 h. The control group and various sample groups were subjected to treatment with or without 1 mM FFA (OA: PA = 2:1), DEME, and 500 μM of AICAR or the respective target compounds. Following Oil Red O staining, fluorescence absorption was measured at an OD of 500 nm. (C) Extent of lipid droplet accumulation in response to the target compounds in the absence or presence of FFA (1 mM) for 24 h (scale bar 50 μm, 40 × ). The data, derived from three independent experiments, are presented as mean ± SD. #### P < 0.0001 versus the control group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus the FFA (1 mM) group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4

Summary of the structure–activity relationships (SAR) in the target compounds.

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We conducted a SAR analysis for compounds S01-S21 to evaluate their inhibitory effect on lipid accumulation in FFA-challenged HepG2 cells. As depicted in Fig. 3, a notable observation was made regarding the influence of the linker length and the nature of substitution on the phenyl group. For compounds S01-S06, with R of a mono- or di-substituted phenyl group and a linker length of two carbon units (n = 2), compound S06 (R = 3,4-dichlorophenyl) demonstrated the most potent suppressive effect on lipid accumulation. Similarly, among the compounds S07-S13 (linker, n = 3) and S14-S21 (linker, n = 4), certain compounds, specifically S10 (R = 2-bromophenyl, n = 3), S13 (R = 3,4-dichlorophenyl, n = 3), S17 (R = 2-bromophenyl, n = 4), and S21 (R = 3,4-dichlorophenyl, n = 4), exhibited more potent inhibitory effects compared to their counterparts with different substituents on the phenyl ring. This pattern suggests a preference for electron-withdrawing groups (EWGs) on the benzene ring in enhancing the effect of the compound. In addition, among all compounds, those with a four-carbon linker (S14-S21) displayed a more pronounced inhibitory effect on lipid accumulation compared to those with shorter linkers (S01-S06, n = 2; and S07-S13, n = 3), especially compound S17. This observation implies that increasing the linker length enhances the ability of these compounds to curb lipid accumulation in FFA-treated HepG2 cells. Collectively, these results reveal that compounds S10 and S17 shown better activity than other compounds, and we choose the two compounds for the molecular biological experiments.

3.2.4 Impact of compounds S10 and S17 on HepG2 cell proliferation under FFA treatment

The effect of S10 and S17 treatments on the proliferation of HepG2 cells subjected to FFA was evaluated using the MTT assay. As presented in Table 1, cell viability remained unaffected at concentrations up to 400 μM for FA, AICAR, S10, and S17 under identical treatment conditions. In addition, the MTT assay was utilized to measure cell viability following 24 h of exposure to 1 mM FFA in conjunction with FA, AICAR, S10, or S17 (Fig. 5). It was noted that the co-treatment of FFAs and these compounds did not adversely affect the viability of HepG2 cells at the concentrations tested. Specifically, the proliferation rates (%) of HepG2 cells treated with S10 at 100, 200, 300, and 400 μM in the presence of 1 mM FFA were 99.44 %, 93.15 %, 95.75 %, and 90.61 %, respectively (Fig. 5C). Similarly, for S17 at the same concentrations, the proliferation rates were 98.25 %, 99.72 %, 97.25 %, and 92.45 % (Fig. 5D). Therefore, compounds S10 and S17 shown low cell cytotoxicity at the range of 100 μM to 400 μM on HepG2 cells, even in the presence of 1 mM FFA.

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Fig. 5

Effects of S10 and S17 on viability of FFA-stimulated HepG2 cells. Cells were exposed to FA with the indicated concentrations (A), AICAR (B), S10 (C), and S17 (D) in the absence or presence of FFA (1 mM, OA: PA = 2:1) for 24 h. Cell viability was assessed through the MTT assay. The data, derived from three independent experiments, are presented as mean ± SD. * P < 0.05 versus the control group.

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3.2.5 Suppressive effects of S10 and S17 on FFA-induced lipid accumulation in HepG2 cells

It was reported that FA inhibited adipogenesis and lipid accumulation in 3 T3-L1 cells, but lipid accumulation in cells declined after the treatment of 0.25 mM or 0.5 mM FA for 10 days (Ilavenil et al., 2017). To illustrating the impact of compounds S10 and S17 on FFA-induced lipid accumulation. HepG2 cells were exposed to these compounds alongside FFA, followed by analysis using Oil Red O staining to visualize lipid accumulation. Additionally, the intracellular TG levels were evaluated. The OD value of each well was determined to corroborate the microscopic observations. As displayed in Fig. 6A&6C, compounds S10 and S17 notably thwarted lipid accumulation and exhibited a more potent inhibitory effect than the positive control FA. Furthermore, the TG assay revealed that both S10 and S17 decreased intracellular TG content in a concentration-responsive manner (Fig. 6B). These results suggested that S10 and S17 exerted potent effect to reduced the intracellular lipid accumulation.

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Fig. 6

Impact of compounds S10 and S17 on lipid accumulation in HepG2 cells. (A) Detection of lipid droplet accumulation via fluorescence absorption. Cells were treated with specified concentrations of S10 or S17 in the absence or presence of FFA for 24 h. Following Oil Red O staining, fluorescence absorption was determined at an OD of 500 nm. (B) Quantification of intracellular TG levels was performed using the TG Assay Kit. (C) Visualization of the effects of S17 on lipid droplet accumulation in the absence or presence of FFA (1 mM) for 24 h (scale bar 50 μm, 40 × ). The data, derived from three independent experiments, are presented as mean ± SD. #### P < 0.0001 versus the control group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus the FFA (1 mM) group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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3.2.6 Suppressive effects of S10 and S17 on ALT and AST activities in FFA-challenged HepG2 cells

The enzyme activity of ALT and AST are key markers of NAFLD to indicate liver damage (Wang et al., 2012). Study has shown that the activity of ALT and AST in hepatic cells will significantly increased after treated by FFA or high fat diet, and it also can be reduced by nature products (Chen et al., 2012, Lee et al., 2015). As demonstrated in Fig. 2D&2E, FFA treatment at the concentrations of 0.5 mM and 1 mM resulted in increased ALT and AST activities.

In this study, we tested the ALT and AST activity in both culture medium and cell lysis to validate the inhibitory effect of S10 and S17 on lipid accumulation. The result of ALT and AST activity in culture medium revealed that comparison to the positive control FA, both S10 and S17 have significant effect on the enzyme activity (Fig. 7A & 7B). The assays in cell lysis perform approximately identically, S10 and S17 attenuated the FFA-induced increase in ALT and AST activities in HepG2 cells (Fig. S2). As a result, S10 and S17 showed pronounced capabilities in restricting the activities of ALT and AST in cell lysis.

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Fig. 7

Impact of S10 and S17 on FFA-Induced ALT and AST activity in HepG2 cells. (A) Measurement of ALT activity in culture medium via fluorescence absorption following treatment with designated concentrations of S10 or S17 in the absence or presence of 1 mM FFA for 24 h, with FA serving as a control. (B) Measurement of AST activity in culture medium via fluorescence absorption following treatment with designated concentrations of S10 or S17 in the absence or presence of 1 mM FFA for 24 h, with FA serving as a control. The data, derived from three independent experiments, are presented as mean ± SD. # P < 0.05, ## P < 0.01, ### P < 0.001versus the control group. * P < 0.05, ** P < 0.01 versus the FFA (1 mM) group.

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3.2.7 S17 activates AMPK/p-AMPK and represses the expression of SREBP-1 and its downstream proteins in HepG2 cells

SREBP-1 is a crucial transcription factor in lipid metabolism regulation, the hepatic intracellular lipid accumulation is mediated by the expression of the fatty-acid synthesis protein regulated by SREBP-1, like ACC1 (Flowers and Ntambi, 2008, An et al., 2020). In this study, we first measured two key regulators of hepatic lipogenesis SREBP-1 and ACC1, including the protein expression of SREBP-1 and ACC1, and the phosphorylated form of ACC1. Western blot assays were utilized to explore the mechanism of S17 about the lipid-lowering effects in FFA-treated cells.

As shown in Fig. 8A&8B, treatment of S17 (100, 200, 300, and 400 μM) in HepG2 cells for 24 h resulted in a dose-responsive decrease in SREBP-1 protein expression. The change was also observed in parallel assays using AICAR as a positive control. In addition, S17 downregulated the level of ACC1 and ACC1 phosphorylation, but the relative proportion of p-ACC1/ACC1 shown a corresponding increase (Fig. 8C). Hence, it is evident that S17 constrained the expression of SREBP-1 and its downstream proteins.

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Fig. 8

Inhibitory Effects of compound S17 on SREBP-1 in HepG2 cells. (A) Influence of varying concentrations of S17 on the protein levels of SREBP-1 and ACC1, as well as the phosphorylation level of ACC1. HepG2 cells were treated with or without S17 for 24 h. (B, C) Grayscale analysis of the Western Blot data of Figure A. The results were showed as means ± SD (n = 3) of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 compared with control group.

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Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) plays an important role in regulating hepatic lipid metabolism, study has demonstrated that AMPK represses the gene expression of SREBP-1, which in turn leads to a reduction in hepatic lipid accumulation (Li et al., 2011, Attal et al., 2022, Fang et al., 2022). Moreover, AMPK guide phosphorylation of ACC, blocking the synthesis of malonyl-CoA. Inhibition the expression or acticity of AMPK activited SREBP-1, then induced its downstream lipogenic proteins including ACC1.

As illustrated in Fig. 9A, treatment of HepG2 cells with S17 (100, 200, and 300 μM) in the presence of FFAs for 12 h notably enhanced the phosphorylation of AMPK and ACC1 in a dose-responsive manner, while concurrently reducing SREBP-1 protein levels. The grayscale analysis (Fig. 9B-9D) revealed that S17 decrease expression of SREBP-1, and activated the phosphorylation of ACC1. However, compared to AICAR, S17 prominently upregulated the protein level of AMPK (346.8 % of contrl), and the relative proportion of p-AMPK/AMPK was obviously lower than AICAR. Therefore, S17 regulating hepatic lipid accumulation by activating both AMPK and phosphorylation of AMPK.

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Fig. 9

Inhibitory Effects of compound S17 on SREBP-1 in HepG2 cells. (A) Influence of varying concentrations of S17 on the protein levels of AMPK, SREBP-1, and ACC1, along with the phosphorylation levels of AMPK and ACC1. HepG2 cells were treated with designated concentrations of S17 for 12 h. AICAR served as the positive control, and β-actin was used as a loading control. (B-D) Grayscale analysis of Western Blot of SREBP-1 (B), p-ACC1/ACC1 (C), and p-AMPK/AMPK (D) in Figure A. The results were showed as means ± SD (n = 3) of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 compared with control group.

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To further confirm that S17-induced decline in lipid accumulation is mediated through the AMPK signaling pathway, HepG2 cells were pre-treated with the AMPK inhibitor, compound C, prior to S17 treatment. Results from Oil Red O staining, presented in Fig. 10A&10B, compound C partially reversed the effect of S17, demonstrate that the lipid accumulation suppressive effect of S17 is dependent on AMPK signaling pathway and other factors. To evaluate the inhibitory effect of S17 in FFA-induced cells, western blots was used to measure the expression of APMK and other lipogenic proteins. The results shown that when cells incubated with S17 in the presence of FFAs for 12 h, S17 activated AMPK expression and ACC1 phosphorylation to impede lipid accumulation (Fig. 10C-10E), but the pretein level of SREBP-1 increased. Additionally, the phosphorylation level of AMPK exhibited no significant differences, which may according to the introduction of FFA. As a result, these compounds needs further modification to enhancing their activity on the inhibitory effect of lipid accumulation.

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Fig. 10

Effects of compound S17 on the AMPK pathway in FFA-stimulated HepG2 cells. (A) Measurement of lipid droplet accumulation using fluorescence absorption. Cells were treated with or without indicated concentrations of ferulic acid (FA), S17 or compound C in the absence or presence of FFA for 24 h. After staining with Oil Red O, fluorescence absorption was determined at an OD of 500 nm. (B) Visualization of lipid droplet accumulation influenced by ferulic acid (FA) and S17 in the absence or presence of FFA (1 mM) and compound C for 24 h (scale bar 50 μm, 40 × ). The results were showed as means ± SD (n = 3) of at least three independent experiments. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 versus the linked group. * P < 0.05, ** P < 0.01, *** P < 0.001, versus the FFA (1 mM) group. (C) Influence of S17 on protein levels of AMPK, SREBP-1, and ACC1, as well as the phosphorylation levels of AMPK and ACC1. HepG2 cells were treated with or without S17 in the absence or presence of FFA (1 mM) for 12 h. AICAR served as a positive control, with β-actin as a loading control. (D-F) Grayscale analysis of Western Blot of SREBP-1 (D), p-ACC1/ACC1 (E), and p-AMPK/AMPK (F) in Figure C. The results were showed as means ± SD (n = 3) of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 versus the control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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