Schisanhenol ameliorates non-alcoholic fatty liver disease via inhibiting miR-802 activation of AMPK-mediated modulation of hepatic lipid metabolism - PubMed (original) (raw)
Schisanhenol ameliorates non-alcoholic fatty liver disease via inhibiting miR-802 activation of AMPK-mediated modulation of hepatic lipid metabolism
Bin Li et al. Acta Pharm Sin B. 2024 Sep.
Abstract
Non-alcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis, is a common metabolic liver disease worldwide. Currently, satisfactory drugs for NAFLD treatment remain lacking. Obesity and diabetes are the leading causes of NAFLD, and compounds with anti-obesity and anti-diabetic activities are considered suitable candidates for treating NAFLD. In this study, biochemical and histological assays revealed that a natural lignan schisanhenol (SAL) effectively decreased lipid accumulation and improved hepatic steatosis in free fatty acid (FFA)-treated HepG2 cells and high-fat diet (HFD)-induced NAFLD mice. Further, molecular analyses, microRNA (miRNA)-seq, and bioinformatics analyses revealed that SAL may improve NAFLD by targeting the miR-802/adenosine monophosphate-activated protein kinase (AMPK) pathway. Liver-specific overexpression of miR-802 in NAFLD mice significantly impaired SAL-mediated liver protection and decreased the protein levels of phosphorylated (p)-AMPK and PRKAB1. Dual-luciferase assay analysis further confirmed that miR-802 inhibits hepatic AMPK expression by binding to the 3' untranslated region of mouse Prkab1 or human PRKAA1. Additionally, genetic silencing of PRKAA1 blocked SAL-induced AMPK pathway activation in FFA-treated HepG2 cells. The results demonstrate that SAL is an effective drug candidate for treating NAFLD through regulating miR-802/AMPK-mediated lipid metabolism.
Keywords: AMPK; Hepatic steatosis; Lipid metabolism; Non-alcoholic fatty liver disease; PPARα; SREBP-1c; Schisanhenol; miR-802.
© 2024 The Authors.
Conflict of interest statement
The authors declare no conflicts of interest.
Figures
Graphical abstract
Figure 1
Effect of SAL on lipid accumulation in FFA-treated HepG2 cells. (A) Effects of SAL (5, 10, and 20 μmol/L) on HepG2 cell viability with or without FFA. (B) Lipid droplet deposition in FFA-treated HepG2 cells was assessed using Oil red O staining. Effects of SAL (5, 10, and 20 μmol/L) on TG (C) and TC (D). The data are presented as the mean ± SD (n = 3). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group.
Figure 2
Effect of SAL on lipid accumulation through the AMPK pathway in FFA-treated HepG2 cells. (A) Representative Western blotting results and densitometric analysis of SREBP-1c and PPAR_α_. (B) qRT-PCR analysis of ACC, SCD1, FAS, DGAT1, DGAT2, and CPT-1 gene expression. (C) Representative immunofluorescence images of p-AMPK (green) expression and Western blotting analysis of AMPK. Phalloidin (red) and 4ʹ,6-diamidino-2-phenylindole (DAPI, blue) were used as cell skeleton and nuclear counterstaining probes, respectively. (D) Representative Western blotting results and densitometric analysis of p-ACC. The results are expressed as the mean ± SD (n = 3). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group.
Figure 3
SAL ameliorates hepatic steatosis in HFD-induced NAFLD mice. (A) Representative images of H&E, Oil red O, and Nile red staining of liver sections. (B) Serum ALT and AST and hepatic TG, TC, LDL-C, and HDL-C levels. The results are expressed as the mean ± SD (n = 6). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group.
Figure 4
SAL modulates lipid metabolism via the AMPK signaling pathway in HFD-induced NAFLD mice. (A) Representative Western blotting results and densitometric analysis of p-AMPK, AMPK, p-ACC, SREBP-1c, and PPAR_α_ (n = 3). (B) qRT-PCR analysis of Acc, Scd1, Fas, Dgat1, Dgat2, and Cpt-1 gene expression (n = 5). The results are expressed as the mean ± SD. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group.
Figure 5
SAL decreases hepatic miR-802 expression in HFD-induced NAFLD mice. (A) Volcanic plots showing the analysis of different genes. (B) Venn diagram verifying different genes among groups, including known mature miRNAs with the opposite expression trend. (C) KEGG signaling pathway analysis of miR-802 target genes. (D) qRT-PCR analysis of miR-802 expression in the livers of mice from the control, HFD, and SAL-H groups (n = 3 per group). The results are expressed as the mean ± SD. ###P < 0.001 vs. control group; ∗∗P < 0.01 vs. model group.
Figure 6
SAL improves hepatic steatosis in HFD-treated mice through downregulating miR-802. (A) Representative images of H&E, Oil red O, and Nile red staining of liver sections. (B) Hepatic TG, TC, LDL-C, and HDL-C levels. The results are expressed as the mean ± SD (n = 6). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group; NS, no significance.
Figure 7
SAL activates the AMPK pathway through miR-802 downregulation. (A) qRT-PCR analysis of Prkab1, Acc, and Fas genes expression (n = 5). (B) Representative Western blotting results and densitometric analysis of PRKAB1, PRKAA1, PRKAA2, p-AMPK, p-ACC, SREBP-1c, and PPAR_α_ (n = 3). The results are expressed as the mean ± SD. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group; NS, no significance.
Figure 8
SAL activates AMPK via inhibiting miR-802 and its binding to PRKAA1 or Prkab1. (A) Targeted miR-802 and Prkab1/PRKAA1 WT or Prkab1/PRKAA1 Mut sequences. (B) Dual-luciferase reporter assay. (C) Representative Western blotting results and densitometric analysis of PRKAA1, p-AMPK, p-ACC, SREBP-1c, and PPAR_α_. The results are expressed as the mean ± SD (n = 3–5). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. model group; NS, no significance.
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