Diabetes Metab J.  2023 Jul;47(4):500-513. 10.4093/dmj.2022.0110.

Beneficial Effects of a Curcumin Derivative and Transforming Growth Factor-β Receptor I Inhibitor Combination on Nonalcoholic Steatohepatitis

• Ha KB
• Lee ES
• Park NW
• Jo SH
• Shim S
• Kim DK
• Ahn CM
• Chung CH

• KMID: 2544729
• DOI: http://doi.org/10.4093/dmj.2022.0110

Abstract

Background
Curcumin 2005-8 (Cur5-8), a derivative of curcumin, improves fatty liver disease via AMP-activated protein kinase activation and autophagy regulation. EW-7197 (vactosertib) is a small molecule inhibitor of transforming growth factor β (TGF-β) receptor I and may scavenge reactive oxygen species and ameliorate fibrosis through the SMAD2/3 canonical pathway. This study aimed to determine whether co-administering these two drugs having different mechanisms is beneficial.
Methods
Hepatocellular fibrosis was induced in mouse hepatocytes (alpha mouse liver 12 [AML12]) and human hepatic stellate cells (LX-2) using TGF-β (2 ng/mL). The cells were then treated with Cur5-8 (1 μM), EW-7197 (0.5 μM), or both. In animal experiments were also conducted during which, methionine-choline deficient diet, Cur5-8 (100 mg/kg), and EW-7197 (20 mg/kg) were administered orally to 8-week-old C57BL/6J mice for 6 weeks.
Results
TGF-β-induced cell morphological changes were improved by EW-7197, and lipid accumulation was restored on the administration of EW-7197 in combination with Cur5-8. In a nonalcoholic steatohepatitis (NASH)-induced mouse model, 6 weeks of EW-7197 and Cur5-8 co-administration alleviated liver fibrosis and improved the nonalcoholic fatty liver disease (NAFLD) activity score.
Conclusion
Co-administering Cur5-8 and EW-7197 to NASH-induced mice and fibrotic hepatocytes reduced liver fibrosis and steatohepatitis while maintaining the advantages of both drugs. This is the first study to show the effect of the drug combination against NASH and NAFLD. Similar effects in other animal models will confirm its potential as a new therapeutic agent.

Keyword

ALK5 inhibitor; Curcumin; Fibrosis; Non-alcoholic fatty liver disease; Transforming growth factor beta

Figure

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  Fig. 1. Curcumin 2005-8 (Cur5-8) reduces lipid accumulation in hepatocytes. (A, B, C) Cell morphology and BODIPY staining were observed under a microscope following 24 hours of oleic acid (OA) treatment. In alpha mouse liver 12 (AML12) cells, OA was combined with Cur, Cur5-8, and EW-7197. (D, E) Levels of adipose differentiation-related protein (ADRP), sterol regulatory element-binding protein 1c (Srebp1c), and fatty acid synthase (Fas) were measured using a Western blotting assay. aP<0.001 vs. control (Con), bP<0.001 vs. oleic acid, cP<0.001 vs. oleic acid+Cur5-8, dP<0.05 vs. Con, eP<0.05 vs. oleic acid.

• 

  Fig. 2. EW-7197 relieves hepatocellular fibrosis but has side effects. Hepatocellular fibrosis was induced by treating human hepatic stellate cell (LX-2) cells with transforming growth factor β (TGF-β; 2 ng/mL). (A, B) The ability of EW-7197 to restore the original state was tested by expressing fibrosis markers such as p-SMAD2/3, α-smooth muscle actin (α-SMA), and ColI. (C, D) Alpha mouse liver 12 (AML12) cells were treated with EW-7197 combined with TGF-β. (E, F) AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (Erk), and Akt activities were measured to determine the effect of EW-7197 treatment on the TGF-β non-canonical pathway. aP<0.05 vs. control (Con), bP<0.05 vs. TGF-β, cP<0.001 vs. Con, dP<0.001 vs. TGF-β.

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  Fig. 3. Curcumin 2005-8 (Cur5-8) and EW-7197 improves transforming growth factor β (TGF-β)-induced hepatocellular fibrosis and lipid accumulation. Hepatocellular fibrosis was induced in alpha mouse liver 12 (AML12) cells using TGF-β. The cells were then treated with Cur, Cur5-8, and EW-7197, individually or in combination (EW+Cur or EW+Cur5-8). (A) Changes in cell morphology were then visualized. (B, D) Lipid accumulation in hepatocytes was visualized using BODIPY staining. (C, E) The degree of fibrosis and lipid droplet accumulation was determined using α-smooth muscle actin (α-SMA) staining and by measuring the BODIPY-positive stained area. Veh, vehicle. aP<0.05 vs. control (Con), bP<0.05 vs. TGF-β, cP<0.001 vs. Con, dP<0.001 vs. TGF-β.

• 

  Fig. 4. Combinning curcumin 2005-8 (Cur5-8) and EW-7197 reduces hepatic fibrosis. To determine the effect of co-administering different combinations, we added Cur, Cur5-8, and EW-7197 separately to human hepatic stellate cell (LX-2) cells treated with transforming growth factor β (TGF-β). We also added EW-7197+Cur and EW-7197+Cur5-8 combinations. Levels of p-SMAD 2/3 proteins, members of the canonical pathway of TGF-β, α-smooth muscle actin (α-SMA), and ColI proteins, which are markers of fibrosis, were measured in (A, B) LX-2 cells and (C, D) AML12 cells. aP<0.05 vs. control (Con), bP<0.05 vs. TGF-β.

• 

  Fig. 5. Curcumin 2005-8 (Cur5-8) and EW-7197 co-administration improves hepatic lipid accumulation. (A, B) Accumulation of lipid droplets and variations in liver sizes were measured using H&E staining and liver imaging, respectively. (C) Nonalcoholic fatty liver disease (NAFLD) activity was scored by quantifying the degree of steatosis, hepatocyte ballooning, and lobular inflammation based on the results of H&E staining. (D, E, F) The NAFLD activity score is assigned as described in the Methods section. Metabolism-related proteins were determined by measuring rho-associated coiled-coil kinase 1 (Rock1) and sterol regulatory elementbinding protein 1c (Srebp1c) levels, AMP-activated protein kinase (AMPK) activity, and reactive oxygen species (ROS) scavenging effect. Endoplasmic reticulum stress regulation was determined by measuring Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and activating transcription factor 6α (ATF6α) levels. aP<0.05 vs. control (Con), bP<0.05 vs. methionine-choline-deficient diet (MCD), cP<0.001 vs. Con, dP<0.001 vs. MCD.

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  Fig. 6. Curcumin 2005-8 (Cur5-8) and EW ameliorate liver fibrosis. (A, B, C) Liver fibrosis was measured using Sirius red and trichrome staining. The degree of liver fibrosis in the experimental groups was compared by quantifying the positively-stained area. (D, E) Levels of extracellular matrix-related α-smooth muscle actin (α-SMA), collagen I (ColI), fibronectin, and transforming growth factor β (TGF-β) canonical pathway-related p-SMAD2/3 were measured. aP<0.05 vs. control (Con), bP<0.05 vs. methionine-choline-deficient diet (MCD), cP<0.001 vs. Con, dP<0.001 vs. MCD.

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