3,3'-Diindolylmethane Ameliorates Metabolism Dysfunction-Associated Fatty Liver Disease via AhR/p38 MAPK Signaling

Abstract

Background/Objectives: Metabolic dysfunction-associated fatty liver disease (MAFLD) is a chronic hepatic condition marked by lipid buildup, lipotoxicity, and inflammation. Prior research indicates that 3,3'-Diindolemethane (DIM), a natural indole-type phytochemical that is abundant in brassicaceae vegetables, has been reported to reduce body weight and improve lipid metabolism in mice subjected to a high-fat diet (HFD). The aryl hydrocarbon receptor (AhR), a nuclear receptor implicated in lipid metabolism and immune regulation, serves as a functional receptor for DIM. However, the underlying signaling pathways that regulate MAFLD remain elusive. Our objective is to ascertain the beneficial impact of DIM on MAFLD and the associated mechanisms. Methods: Hematoxylin and eosin staining, together with Oil Red O staining, were utilized to assess the pathological changes and lipid deposition in the liver. Biochemical analysis was employed to measure levels of triglyceride (TG), total cholesterol (TC), free fatty acid (FFA), aspartate transaminase (AST), alanine transaminase (ALT), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). The cell survival rate of HepG2 cells treated with palmitic acid (PA) and DIM was assessed using the CCK-8 assay. Flow cytometry was employed to measure the fluorescence intensity emitted by lipid droplets within cells. Western blotting analysis was performed to assess AhR pathway and fatty acid transporter expression levels in hepatic tissue. Results: Our results showed that DIM significantly attenuated body weight gain and hepatic injury brought on by HFD, decreased lipid droplet accumulation in HepG2 cells, and effectively suppressed the phosphorylation of p38 MAPK and the protein expression levels of fatty acid transporters CD36 and FATP4. Conclusions: DIM reduced lipid accumulation by activating AhR and suppressing p38 MAPK phosphorylation, thereby inhibiting fatty acid transport and inflammatory responses. These findings suggest that DIM may represent a promising therapeutic candidate for MAFLD, warranting further exploration for clinical applications.

Keywords: 3,3′-Diindolylmethane; CD36; MAFLD; aryl hydrocarbon receptor; p38 MAPK.

Figure 1

DIM attenuated body weight and alleviated liver injury in mice. (A) Trends in mouse body weight. (B) Differences in live weight. (C) Differences in live index. (D) The H&E and ORO images clearly and precisely illustrate the percentage of hepatocyte steatosis; (E) Quantitative analysis of ORO images. (F) Differences in serum AST. (G) Differences in serum ALT. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NCD group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. HFD group and % p < 0.05 indicates differences between groups at various time points.

Figure 2

DIM attenuated droplet accumulation in HepG2 cells. (A,B) The CCK-8 assay was used to evaluate the impact of PA and DIM on the cytotoxicity of HepG2 cells. (C) Lipid droplets in HepG2 cells were visualized using ORO staining. (D) Analysis of HepG2 cells’ lipid droplets stained with oil red O statistically. (E,F) DIM’s effect on the AST and ALT levels in the HepG2 cell culture medium. * p < 0.05, *** p < 0.001 vs. CON group, ### p < 0.001 vs. PA group, $ p < 0.05, $$$ p < 0.001 vs. PA + DIM group, and &&& p < 0.001 vs. PA + DIM + CH223191 group.

Figure 3

DIM attenuated the phosphorylation of p38MAPK in mice and cells. (A–D) Western blotting analysis of AhR, p-p38 MAPK, p38 MAPK and NF-kB protein levels in mice and densitometric quantification of them, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NCD group, # p < 0.05, ## p < 0.01 vs. HFD group. (E–H) Western blotting analysis of AhR, p-p38 MAPK, p38 MAPK and NF-kB protein levels in HepG2 cells and densitometric quantification of them, * p < 0.05, ** p < 0.01 vs. CON group, ## p < 0.01, ### p < 0.001 vs. PA group, $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. PA + DIM group, and & p < 0.05, && p < 0.01, vs. PA + DIM + CH223191 group.

Figure 4

DIM attenuated fatty acid transport in mice and cells. (A–E) Western blotting analysis of CD36, PPARγ, FATP4 and FABP1 protein levels in mice and densitometric quantification of them, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NCD group, ## p < 0.01, ### p < 0.001 vs. HFD group. (F–J) Western blotting analysis of CD36, PPARγ, FATP4 and FABP1 protein levels in HepG2 cells and densitometric quantification of them; (K) DIM’s effect on the FFA level in the HepG2 cell culture medium. (L,M) Lipid droplets in HepG2 cells were stained with Bodipy 493/503 for visualization. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CON group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. PA group, $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. PA + DIM group, & p < 0.05, && p < 0.01, &&& p < 0.001 vs. PA + DIM + CH223191 group.