Role of HO-1 against Saturated Fatty Acid-Induced Oxidative Stress in Hepatocytes

Abstract

Increased circulating levels of free fatty acids, especially saturated ones, are involved in disease progression in the non-alcoholic fatty liver. Although the mechanism of saturated fatty acid-induced toxicity in the liver is not fully understood, oxidative stress may be deeply involved. We examined the effect of increased palmitic acid, the most common saturated fatty acid in the blood, on the liver of BALB/c mice via tail vein injection with palmitate. After 24 h, among several anti-oxidative stress response genes, only heme oxygenase-1 (HO-1) was significantly upregulated in palmitate-injected mice compared with that in vehicle-injected mice. Elevation of HO-1 mRNA was also observed in the fatty liver of high-fat-diet-fed mice. To further investigate the role of HO-1 on palmitic acid-induced oxidative stress, in vitro experiments were performed to expose palmitate to HepG2 cells. SiRNA-mediated knockdown of HO-1 significantly increased the oxidative stress induced by palmitate, whereas pre-treatment with SnCl₂, a well-known HO-1 inducer, significantly decreased it. Moreover, SB203580, a selective p38 inhibitor, reduced HO-1 mRNA expression and increased palmitate-induced oxidative stress in HepG2 cells. These results suggest that the HO-1-mediated anti-oxidative stress compensatory reaction plays an essential role against saturated fatty acid-induced lipotoxicity in the liver.

Keywords: fatty liver; hem oxygenase-1; lipotoxicity; oxidative stress; palmitic acid.

Figure 1

Effect of palmitate injection through the tail vein on BALB/c mice liver. Control, n = 10; palmitate, n = 11. Values are expressed as means ± SD. N.S.: not significant, *** p < 0.001, **** p < 0.0001. (A), Representative hepatic histological findings (HE staining: original magnification, ×20, bar = 50 μm. (B) The level of malondialdehyde (MDA) in mouse liver. (C) Plasma alanine transaminase (ALT) levels in mice. (D) mRNA levels of anti-oxidative stress genes in the livers of mice treated with palmitate relative to those in control mice. (E) HO-1 protein and 4-hydroxy-2-nonenal (4-HNE) levels in mouse livers were compared by immunoblotting. β-actin was used as a loading control. Densitometric analysis was performed.

Figure 2

Effect of high-fat diet (HFD) on the livers of C57/BL6J mice fed for 12 weeks compared with normal diet (ND) mice (n = 10/group). Values are represented as means ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001. (A) Representative hepatic histological findings (HE staining: original magnification, ×20, Bar = 100 μm. (B) The effect of HFD on the level of malondialdehyde (MDA) in mouse livers. (C) Plasma alanine transaminase (ALT) levels in mice. (D) mRNA levels of HO-1 in the livers of mice fed a high-fat diet relative to those in ND mice. (E) HO-1 protein and 4-hydroxy-2-nonenal (4-HNE) levels in mice liver were measured by immunoblotting. β-actin was used as a loading control. Densitometry was performed.

Figure 3

Effect of siRNA-mediated HO-1 knockdown on palmitate-induced oxidative stress in HepG2 cells. Cells were transfected with an HO-1 siRNA or control siRNA for 12 h, followed by treatment with 400 μM palmitate (PA) or vehicle (Control) for an additional 12 h. Values are expressed as means ± SD, n = 3. ** p < 0.01, *** p < 0.001, **** p < 0.0001. (A) After treatment with the vehicle or palmitate, cells were incubated with 5 μM of 2′,7′-dicholordihydrofluorescein diacetate (H₂DCFDA) to detect reactive oxygen species (ROS) and were examined by fluorescence microscopy. Representative images of cells from three independent experiments are shown. Bar = 100 μm. (B) Relative levels of malondialdehyde (MDA) in cells. (C) 4-hydroxy-2-nonenal (4-HNE) levels in HepG2 cells were analyzed by immunoblotting. β-actin was used as a loading control. The positive control is presented on the right (immunoblotting of HepG2 cells treated with 100 μM of tert-butyl hydroperoxide (t-BHP) for 6 h). Densitometric analysis was performed. (D) Relative lactate dehydrogenase (LDH) release was calculated in the supernatant of cells.

Figure 4

Effect of HO-1 inducer, SnCl₂, on palmitate-induced oxidative stress in HepG2 cells. Cells were treated with a vehicle (Control) or 400 μM palmitate (PA) with or without pre-treatment with SnCl₂ 100 μM for 8 h. Values are represented as means ± SD, n = 3. N.S.: not significant, **** p < 0.0001. (A) After Control or PA treatment, cells were incubated with 5 μM of 2′,7′-dicholordihydrofluorescein diacetate (H₂DCFDA) to detect reactive oxygen species (ROS) and were observed by fluorescence microscope. Representative images from three independent experiments are shown. Bar = 50 μm (B) Relative levels of malondialdehyde (MDA) in cells are shown. (C) 4-hydroxy-2-nonenal (4-HNE) levels were compared by immunoblotting. β-actin was used as a loading control. Densitometric analysis was performed. (D) Relative lactate dehydrogenase (LDH) release was measured in the supernatant of HepG2 cells.

Figure 5

Involvement of the p38MAPK pathway in palmitate-induced oxidative stress in HepG2 cells. Values are expressed as means ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. (A) Immunoblotting data of phospho-p38MAPK (Thr180/Tyr182) and total p38 in HepG2 cells treated with the vehicle (Control) or 400 μM palmitate (PA) for indicated time. (B) Relative HO-1 mRNA expression in HepG2 cells treated with Control or PA with or without pre-treatment of SB203580 (SB) at 10 μM for 1 h. (C) Relative lactate dehydrogenase (LDH) release was calculated in the supernatant of cells exposed to the Control or PA with or without pre-treatment with SB. (D) 4-hydroxy-2-nonenal (4-HNE) levels in HepG2 cells treated with Control or PA with or without pre-treatment with SB were analyzed by immunoblotting. β-actin was used as a loading control. Densitometric analysis was performed.