Unveiling p65 as the target of diphyllin in ameliorating metabolic dysfunction-associated steatotic liver disease via targeted protein degradation technology

Abstract

Introduction: Metabolic dysfunction-associated steatotic liver disease (MASLD), characterized by hepatic steatosis, inflammation and fibrosis, is becoming a global epidemic. However, the currently available effective clinical strategies remain limited.

Methods: We conducted the choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) induced MASH mouse model to explore the effects of diphyllin on MASLD mice. We employ the targeted protein degradation technology applied for the discovery of compound/protein-protein interaction to identify p65 as a potential target protein.

Results: We determine that diphyllin, a natural arylnaphthalene lignan lactone, is effective on MASLD, evidenced by the inhibition of hepatic lipid accumulation through promoting fatty acid oxidation in vivo and in vitro. To uncover the underlying mechanisms, we design and synthesis diphyllin-based protac and identify p65 as a potential target protein. Under p65 deficiency, the effects of diphyllin on lipid metabolism are blocked in vitro. As p65 as an antagonist of NRF2, diphyllin interacts with p65, leading to the induction of the NRF2 transcriptional activity and the enhancement of antioxidant capacity. When NFR2 is inhibited, the lowering effects of diphyllin on lipid is abolished.

Discussion: Our study presents diphyllin as a potential lead compound for MASLD therapy but also offers a novel approach for elucidating the mechanisms of action of natural products.

Keywords: NRF2; diphyllin; metabolic dysfunction-associated steatotic liver disease; p65; targeted protein degradation technology.

FIGURE 1

Diphyllin treatment ameliorates liver injury and hepatic steatosis in CDAHFD-induced MASH mice. (A) Schematic of mice fed with CDAHFD for 4 weeks and treated with 100 mg/kg diphyllin for 5 weeks together with CDAHFD. (B) Body weight. (C) Food intake. (D–F) Levels of plasma LDH, AST, and ALT from mice treated with diphyllin. (G, H) Liver weight and liver index. (I) Oil-red staining of liver of mice treated with diphyllin (scale bars, 100 μm). (J, K) Content of liver triglycerides and cholesterol in mice treated with diphyllin. (L, M) Gene levels in the liver from mice treated with diphyllin. For all, n = 6–7 mice for all mice groups. Bar graphs are presented as mean ± SEM, and one-way ANOVA was used for all comparisons.

FIGURE 2

Diphyllin treatment improves inflammation and fibrosis in CDAHFD-induced MASH mice. (A, B) Inflammation- and fibrosis-related gene levels in the liver from mice treated with diphyllin. (C) Sirius red staining of liver of mice treated with diphyllin (scale bars, 100 μm). (D) Content of liver hydroxyproline in mice treated with diphyllin. For all, n = 6–7 mice for all mice groups. Bar graphs are presented as mean ± SEM, and one-way ANOVA was used for all comparisons.

FIGURE 3

Diphyllin inhibits lipid accumulation via the promotion of fatty acid oxidation. (A) Content of triglyceride of AML 12 cells treated with 1.5 mM FFA and diphyllin for 24 h. (B) Curve of the oxygen consumption rate of AML 12 cells treated with diphyllin for 24 h. (C) Basal oxygen consumption rate. (D) Oxygen consumption rate of AML 12 cells treated with FCCP. (E) Gene levels in AML 12 cells treated with diphyllin for 24 h. For all, n = 3–4 for all groups. Bar graphs are presented as mean ± SEM, and one-way ANOVA was used for all comparisons.

FIGURE 4

Identification of the target protein of diphyllin by the TPD strategy. (A) Structure of diphyllin–PROTAC. (B) Content of triglycerides of AML 12 cells treated with 1.5 mM FFA and diphyllin or diphyllin–PROTAC for 24 h. For all, n = 3–4 for all groups. (C) Analyses of protein–protein interaction (PPI) networks in L02 cells treated with diphyllin–PROTAC for 24 h. (D) Blotting and quantification of p65 in L02 cells treated with diphyllin–PROTAC for 24 h. (E) Blotting and quantification of p65 protein stability in L02 cells treated with 40 μM diphyllin under different temperature. For (C–E), all blotting assays were repeated three times. Bar graphs are presented as mean ± SEM, and one-way ANOVA was used for all comparisons. (F) Model of the interaction between diphyllin and p65.

FIGURE 5

P65 is the target protein of diphyllin regulating lipid metabolism. (A) p65 gene expression in AML 12 cells treated with si p65 for 24 h. (B) Content of triglyceride in AML 12 cells treated with diphyllin combined with si p65 or not for 24 h. (C) Curve of the oxygen consumption rate of AML 12 cells treated with diphyllin combined with si p65 or not for 24 h. (D) Basal oxygen consumption rate. (E) Oxygen consumption rate of AML 12 cells treated with FCCP. For all, n = 3–4 for all groups. Bar graphs are presented as mean ± SEM, and one-way ANOVA was used for all comparisons.

FIGURE 6

Effect of diphyllin on lipid metabolism depends on NRF2. (A) Structure of 4xARE luciferase plasmid. (B) Luciferase activity of AML 12 cells with p65 knockdown transfected with pGL-3 4xARE luciferase plasmid and treated with diphyllin for 24 h. (C) Sod gene expression in AML 12 cells treated with diphyllin combined with NRF2 inhibitor (10 μM Nrf2-IN-30) or not for 24 h. (D) Acox1 and Cpt1α gene expression in AML 12 cells treated with diphyllin together with NRF2 inhibitor (10 μM Nrf2-IN-30) or not for 24 h. (E) Content of triglycerides in AML 12 cells treated with diphyllin combined with the NRF2 inhibitor (10 μM Nrf2-IN-30) or not for 24 h. (F) Curve of the oxygen consumption rate of AML 12 cells treated with diphyllin combined with the NRF2 inhibitor (10 μM Nrf2-IN-30) or not for 24 h. (G) Basal oxygen consumption rate. (H) Oxygen consumption rate of AML 12 cells treated with FCCP. For all, n = 3–4 for all groups. Bar graphs are presented as mean ± SEM, and one-way ANOVA was used for all comparisons.