J Yeungnam Med Sci.  2024 Apr;41(2):103-112. 10.12701/jyms.2023.01389.

DA-6034 ameliorates hepatic steatosis and inflammation in high fat diet-induced obese mice

• Kim HM
• Kwon MH
• Lee ES
• Ha KB
• Chung CH

• KMID: 2554771
• DOI: http://doi.org/10.12701/jyms.2023.01389

Abstract

Background
Nonalcoholic fatty liver disease (NAFLD) is characterized by an increase in hepatic triglyceride content and increased inflammatory macrophage infiltration through the C-C motif chemokine receptor (CCR) 5 pathway in the liver. DA-6034 (7-carboxymethyloxy-3',4',5-trimethoxy flavone), is a synthetic derivative of eupatilin that exhibits anti-inflammatory activity in inflammatory bowel disease. However, the effect of DA-6034 on the inflammatory response in NAFLD is not well elucidated. Therefore, we aimed to determine the effect of DA-6034 on hepatic steatosis and inflammation.
Methods
Forty male C57BL/6J mice were divided into the following four groups: (1) regular diet (RD), (2) RD with DA-6034, (3) high fat diet (HFD), and (4) HFD with DA-6034. All mice were sacrificed 12 weeks after the start of the experiment. The effects of DA-6034 on macrophages were assessed using RAW264.7 cells.
Results
DA-6034 not only reduced hepatic triglyceride levels and lipid accumulation but also macrophage infiltration and proinflammatory cytokines in HFD-fed mice. According to fluorescence-activated cell sorter analysis, DA-6034 reduced the CD8+ T cell fraction in the liver of HFD-fed mice. DA-6034 also reduced CCR5 expression and the migration of liver macrophages in HFD-fed mice and inhibited CCR2 ligand and CCR4 ligand, which stimulated the migration of macrophages.
Conclusion
Overall, DA-6034 attenuates hepatic steatosis and inflammation in obesity by regulating CCR5 expression in macrophages.

Keyword

CCR5; DA-6034; Hepatic steatosis; Inflammation; Macrophages

Figure

• 

  Fig. 1. Cytotoxicity of eupatilin and DA-6034 in AML12 cells. (A) Chemical structures of eupatilin and DA-6034. (B) MTT cytotoxicity assay results of cell viability based on DA-6034 and eupatilin administered at different doses. Measurement of dissolved MTT in AML12 cells after DA-6034 and eupatilin treatment. *p<0.05, compared with 0 μM. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

• 

  Fig. 2. Weight gain and liver change owing to DA-6034 in HFD-fed mice. WT and DA-6034–treated mice were fed HFD or RD for 12 weeks. The change in (A) body weight, (B) food intake, and (C) liver weight. (D) Paraffin-embedded liver tissue was stained with hematoxylin and eosin. (E) Reduction of hepatic TG by DA-6034 in the HFD group. (F–J) Protein levels of (F, G) FAS, (F, H) SREBP-1, (F, I) phosphorylated NK-κB, and (F, J) IκB in the liver based on western blotting. Cropped membranes were used for western blotting. Data are presented as mean±standard error of the mean (n=10 animals per group). *p<0.05, compared with RD-fed mice. †p<0.05, compared with HFD-fed mice. RD, regular diet; HFD, high fat diet; WT, wild type; DA, DA-6034; TG, triglycerides; FAS, fatty acid synthesis; SREBP-1, sterol regulatory element-binding protein 1; IκB, inhibitor of kappa B; NF-κB, nuclear factor kappa B; p-NF-κB, phospho-NF-κB.

• 

  Fig. 3. DA-6034 effects on inflammation and macrophage migration. RAW 264.7 cells were treated with CCL2 (10 ng/mL) or CCL4 (10 ng/mL) in the presence and absence of DA-6034 (20 µM/mL) for 24 hours (n=4). (A, B) The number of transmigrated RAW 264.7 cells in the presence or absence of DA-6034 was measured using the migration assay. Original magnification, 200× (scale bar, 100 µm). (C) Macrophages infiltrating liver cells were detected via CD68 staining (C). (D–F) Protein levels of CCR2 and CCR5 in the liver based on western blotting. The protein levels of phosphorylated NK-κB in the RAW 264.7 cells (G, H) based on western blotting. Cropped membranes were used for western blotting. Data are presented as mean±standard error of the mean (n=10 animals per group). *p<0.05, compared with RD-fed mice or Veh. †p<0.05, compared with HFD-fed mice or LPS. CON, control; DA, DA-6034; CCL2, CCR2 ligand; CCL4, CCR4 ligand; Veh, vehicle; RD, regular diet; HFD, high fat diet; WT, wild type; CCR, C-C motif chemokine receptor; NF-κB, nuclear factor kappa B; p-NF-κB, phospho-NF-κB; LPS, lipopolysaccharides.

• 

  Fig. 4. DA-6034 reduces the level of proinflammatory immune cells in the liver. Flow cytometry analysis of immune cells from the liver of mice. (A) CD11c+/CD206– or CD11c–/CD206+ cell population was analyzed in the liver of RD-fed mice. (B) Percentages of M1 and M2 adipose tissue macrophages. (C) Fluorescence-activated cell sorting plots and gating of immune cells to detect the percentage of T cells (CD3+ cells) in the liver. Representative flow cytometric plots of the percentage of CD8+ T cells (CD3+/CD4–/CD8+) and CD4+ T cells (CD3+/CD4+/CD8–). (D) Percentages of CD8+ T cells and CD4+ T cells. Data are presented as mean±stanard error of the mean. *p<0.05 compared with RD-fed mice. †p<0.05 compared with HFD-fed mice. WT, wild type; DA, DA-6034; RD, regular diet; HFD, high fat diet.

Reference

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