The mitochondria-targeted antioxidant MitoQ attenuates liver fibrosis in mice

Abstract

Oxidative stress plays an essential role in liver fibrosis. This study investigated whether MitoQ, an orally active mitochondrial antioxidant, decreases liver fibrosis. Mice were injected with corn oil or carbon tetrachloride (CCl4, 1:3 dilution in corn oil; 1 µl/g, ip) once every 3 days for up to 6 weeks. 4-Hydroxynonenal adducts increased markedly after CCl4 treatment, indicating oxidative stress. MitoQ attenuated oxidative stress after CCl4. Collagen 1α1 mRNA and hydroxyproline increased markedly after CCl4 treatment, indicating increased collagen formation and deposition. CCl4 caused overt pericentral fibrosis as revealed by both the sirius red staining and second harmonic generation microscopy. MitoQ blunted fibrosis after CCl4. Profibrotic transforming growth factor-β1 (TGF-β1) mRNA and expression of smooth muscle α-actin, an indicator of hepatic stellate cell (HSC) activation, increased markedly after CCl4 treatment. Smad 2/3, the major mediator of TGF-β fibrogenic effects, was also activated after CCl4 treatment. MitoQ blunted HSC activation, TGF-β expression, and Smad2/3 activation after CCl4 treatment. MitoQ also decreased necrosis, apoptosis and inflammation after CCl4 treatment. In cultured HSCs, MitoQ decreased oxidative stress, inhibited HSC activation, TGF-β1 expression, Smad2/3 activation, and extracellular signal-regulated protein kinase activation. Taken together, these data indicate that mitochondrial reactive oxygen species play an important role in liver fibrosis and that mitochondria-targeted antioxidants are promising potential therapies for prevention and treatment of liver fibrosis.

Keywords: Antioxidant; MitoQ; hepatic stellate cell; liver fibrosis; mitochondria; oxidative stress.

Figure 1

MitoQ attenuates liver injury after CCl₄ treatment in vivo. CCl₄ was administered to mice as in Methods, and MitoQ or decylTPP was added to the drinking water of some animals. The control group received vehicle (corn oil and decylTPP) for 6 weeks. A: Blood was collected at 5 and 6 weeks of CCl₄ treatment and alanine aminotransferase (ALT) was measured. Values are means ± SEM. a, p<0.05 vs control; b, p<0.05 vs the corresponding CCl₄ group. B-D: Representative images of H&E slides after 6 weeks of CCl₄ treatment (n = 4/group).

Figure 2

MitoQ inhibits apoptosis and inflammation in the liver after CCl₄ treatment in vivo. Mice were treated as in Figure 1, and livers were collected at 5 and 6 weeks. A: Representative immunoblot images of cleaved caspase-3 (CC3) and myeloperoxidase (MPO); B: Quantification of CC3 immunoblot images by densitometry; C: TUNEL-positive hepatocytes were counted in 10 random fields per slide as percentage of total; D: Quantification of MPO immunoblot images by densitometry. Values are means ± SEM. a, p<0.05 vs control; b, p<0.05 vs the corresponding CCl₄ group (n = 4/group).

Figure 3

MitoQ inhibits liver fibrosis after CCl₄ treatment in vivo. Mice were treated as in Figure 1. Livers were collected at 6 weeks of CCl₄ or vehicle treatment for histology. Left: representative images of sirius red-stained liver sections. Right: representative second harmonic generation (SHG) images (n = 4/group).

Figure 4

MitoQ decreases hydroxyproline and collagen 1α1 mRNA in the liver after CCl₄ treatment in vivo. Mice were treated as in Figure 1, and livers were collected at 5 and 6 weeks. A: Hydroxyproline. B: Collagen 1α1 mRNA detected by qPCR. Values are means ± SEM. a, p<0.05 vs control; b, p<0.05 vs the corresponding CCl₄ group (n = 4/group).

Figure 5

MitoQ inhibits stellate cell activation and TGF-β/Smad signaling in the liver after CCl₄ treatment in vivo. Mice were treated as in Figure 1, and livers were collected at 5 and 6 weeks. A: Representative immunoblot images of smooth muscle α-actin (α-SMA), Smad2/3, phospho-smad2/3 (pSmad2/3), and β-actin. B: Quantification of α-SMA immunoblot images by densitometry. C: Quantification of transforming growth factor-β1 (TGFβ1) mRNA by qPCR. D: Quantification of pSmad2/3 immunoblot images by densitometry. Values are means ± SEM. a, p<0.05 vs control; b, p<0.05 vs the corresponding CCl₄ group (n = 4/group).

Figure 6

MitoQ inhibits oxidative stress in the liver after CCl₄ treatment in vivo. Mice were treated as in Figure 1, and livers were collected at 5 and 6 weeks. Shown are representative immunoblot images of 4-hydroxynonenal adducts (4-HNE) and β-actin (n = 4/group).

Figure 7

MitoQ inhibits stellate cell activation in vitro. HSCs were isolated from normal rats and cultured in 20% serum-containing medium. After 2 days, serum was decreased to 0.5%. MitoQ (0.5-2 µM) or an equal volume of DMSO (Control) was added at days 2 and 4, and cells were harvested at 6 days. A: Representative immunoblot images of smooth muscle α-actin (α-SMA), collagen-I, and β-actin. B: Quantification of α-SMA immunoblot images by densitometry. C. Quantification of collagen-I immunoblot images by densitometry. D: Representative images of cultured HSCs at 6 days. Values are means ± SEM. a, p<0.05 vs corresponding controls; b, p<0.05 vs 0.5 µM MitoQ. c, p<0.05 vs 1 µM MitoQ (n = 3/group).

Figure 8

MitoQ decreases 4-hydroxynonenal adducts in cultured stellate cells. HSCs were isolated from normal rats and treated as described in Figure 7. After 6 days, cell lysates were subjected to immunoblotting to detect 4-hydroxynonenal adducts (4-HNE) and β-actin. Shown are representative immunoblot images (n = 3/group).

Figure 9

MitoQ inhibits TGF-β, Smad and ERK signaling in cultured stellate cells. HSCs were isolated from normal rats and cultured as described in Figure 7. After 6 days, cell lysates were collected to detect transforming growth factor-β1 (TGF-β1), Smad2/3, phospho-Smad2/3 (p-Smad2/3), extracellular signal-regulated protein kinase 1/2 (ERK1/2), phospho-ERK1/2 (p-ERK1/2) and β-actin. A: Representative immunoblot images. B: Quantification of TGF-β1 immunoblot images by densitometry. C: Quantification of p-Smad2/3 immunoblot images by densitometry. D: Quantification of p-ERK1/2 immunoblot images by densitometry. Values are means ± SEM. a, p<0.05 vs corresponding controls; b, p<0.05 vs 0.5 µM MitoQ. c, p<0.05 vs 1 µM MitoQ (n = 3/group).