2,5-Dihydroxybenzoic Acid Ameliorates Metabolic Dysfunction-Associated Steatotic Liver Disease by Targeting the CCL2-CCR2 Axis to Reduce Lipid Accumulation

Abstract

Background/Objectives: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease worldwide, contributing to metabolic dysfunction and increased healthcare costs. The green Mediterranean diet reduces intrahepatic fat and elevates the plasma levels of 2,5-dihydroxybenzoic acid (2,5-DHBA), suggesting a mechanistic role for 2,5-DHBA in hepatic lipid metabolism. This study aimed to evaluate the therapeutic potential of 2,5-DHBA in MASLD and elucidate its molecular mechanism. Methods: Lipid accumulation was assessed in oleic acid-treated HepG2 cells and a high-fat diet (HFD)-induced MASLD mouse model. RNA sequencing, molecular docking, and immunohistochemical staining were performed to investigate the molecular mechanisms, focusing on the chemokine (C-C motif) ligand 2 (CCL2)-CCL2 receptor (CCR2) axis. Results: 2,5-DHBA significantly reduced hepatic lipid accumulation in both HepG2 cells and HFD-fed mice in a dose-dependent manner. RNA sequencing revealed the marked downregulation of CCL2, a key proinflammatory mediator in MASLD pathogenesis. Molecular docking predicted that 2,5-DHBA competed with CCL2 for binding at the CCR2 axis. Immunohistochemistry further confirmed that 2,5-DHBA treatment lowered hepatic CCL2 expression, suppressed nuclear factor-κB activation, and reduced inflammatory cell infiltration. These findings suggest that 2,5-DHBA exerted anti-steatotic effects by modulating the CCL2-CCR2 signaling pathway. Conclusions: This is the first study to demonstrate that 2,5-DHBA attenuates hepatic steatosis via targeting the CCL2-CCR2 axis. These findings highlight its potential as a novel nutraceutical strategy for MASLD treatment.

Keywords: 2,5-dihydroxybenzoic acid; chemokine (C-C motif) ligand 2; chemokine (C-C motif) receptor 2; metabolic dysfunction-associated steatotic liver disease.

Figure 1

Effects of 2,5-DHBA on OA-induced lipid accumulation in HepG2 cells. (A) Dose response of OA. HepG2 cells were treated without or with various concentrations of OA for 24 h. (B) Dose response of 2,5-DHBA. HepG2 cells were treated with different concentrations of 2,5-DHBA in the presence of 0.5 mM OA for 24 h. Simvastatin (10 μM) was used as a positive control. Cells were stained with Oil Red O and imaged using a camera and optical microscope at 200× magnification (top panel). Scale bar = 50 μm. Representative images are shown. Quantitative analysis of lipid accumulation was performed by measuring Oil Red O content at 520 nm (bottom panel). Cell viability was assessed using CCK-8 assay (line graph). Values are mean ± standard error (n = 3). ### p < 0.001, compared to mock. ** p < 0.01 and *** p < 0.001, compared to OA.

Figure 1

Effects of 2,5-DHBA on OA-induced lipid accumulation in HepG2 cells. (A) Dose response of OA. HepG2 cells were treated without or with various concentrations of OA for 24 h. (B) Dose response of 2,5-DHBA. HepG2 cells were treated with different concentrations of 2,5-DHBA in the presence of 0.5 mM OA for 24 h. Simvastatin (10 μM) was used as a positive control. Cells were stained with Oil Red O and imaged using a camera and optical microscope at 200× magnification (top panel). Scale bar = 50 μm. Representative images are shown. Quantitative analysis of lipid accumulation was performed by measuring Oil Red O content at 520 nm (bottom panel). Cell viability was assessed using CCK-8 assay (line graph). Values are mean ± standard error (n = 3). ### p < 0.001, compared to mock. ** p < 0.01 and *** p < 0.001, compared to OA.

Figure 2

Effects of 2,5-DHBA on HFD-induced hepatic lipid accumulation in mice. (A) Short-term experiment. Mice were fed an HFD and orally administered various dosages of 2,5-DHBA for 7 days. Mice in the mock group were fed a normal diet. On day 7, mice were sacrificed, liver tissue was stained with Oil Red O, and lipid content was quantified at 520 nm. Values are mean ± standard error (n = 5). ### p < 0.001, compared to mock. ** p < 0.01, compared to HFD. (B) MASLD experiment. Mice were fed an HFD for 4 months. During the fourth month, mice received oral administration of 2,5-DHBA (100 mg/kg) for 4 weeks. Mice in the mock group were fed a normal diet. Liver sections were stained with H&E (left panel) and representative images are shown. Quantification of fat droplet area is presented in the right panel. Values are mean ± standard error (n = 10). ### p < 0.001, compared to mock. *** p < 0.001, compared to HFD.

Figure 3

Volcano plot of the expression of genes in the HFD (A) and 2,5-DHBA groups (B). Red dots indicate genes with a fold change ≥2 and statistically significant differences (adjusted p < 0.05). Blue dots represent DEGs with a fold change ≤−2. Gray dots indicate genes without statistically significant changes. The Y-axis shows the − Log₁₀ p-value, and the X-axis shows the Log₂ fold change. Dotted horizontal and vertical lines indicate significance thresholds. IL-1β, TNF-α, Ccl2, and Ccr2 are labeled on the plot.

Figure 4

KEGG pathway maps of MASLD affected by HFD and 2,5-DHBA. DEGs affected by HFD (A) and 2,5-DHBA (B) are shown. Red frames represent upregulated DEGs, and green frames represent downregulated DEGs. Fold changes are indicated by the color scale (top).

Figure 5

Interaction between 2,5-DHBA and CCR2. (A) Structure of CCR2. (B) Docking structure of 2,5-DHBA bound to CCR2. The structure of CCR2 is shown as a ribbon. The CCL2-binding region and 2,5-DHBA are shown as red and orange sticks, respectively. Enlarged images highlight key amino acid residues involved in the 2,5-DHBA/CCR2 interaction. Hydrogen bonds between 2,5-DHBA and CCR2 are represented by green lines.

Figure 6

IHC staining analysis of CCL2 expression, p65 activation, and inflammatory cell infiltration in response to 2,5-DHBA treatment. Mice were fed an HFD for 4 months. During the fourth month, mice received 2,5-DHBA (100 mg/kg) orally for 4 weeks. Mice in the mock group were fed a normal diet. Liver sections were stained with antibodies against CCL2, p65, and CD11b. Original magnification: 100×. Scale bar = 50 μm. Representative images are shown. Quantification of the stained areas or cells percentages is shown in the right panel. Values are mean ± standard error (n = 10). ## p < 0.01, ### p < 0.001, compared to mock. ** p < 0.01, *** p < 0.001, compared to HFD.