Fig. 1Statin treatment attenuates methionine- and choline-deficient diet (MCDD)-induced hepatic steatosis and steatohepatitis. Representative histological images of each experimental group. H&E (×200; scale bar=100 µm), MT (×100; scale bar=50 µm). ND, normal chow diet; MT, Masson's trichrome.
Fig. 2Histologic scores and hepatic triglyceride (TG) content and plasma alanine aminotransferase (ALT) level. (A) Histologic scores for location and severity of steatosis, inflammation, and fibrosis, according to criteria of Kleiner et al. [18]. (B) Hepatic TG content and plasma ALT level in mice fed normal chow diet (ND) and methionine- and choline-deficient diet (MCDD) with or without various statins for 6 weeks. F, fluvastatin; P, pravastatin; S, simvastatin; A, atorvastatin; R, rosuvastatin. aP<0.05 compared with ND, bP<0.05 compared with MCDD.
Fig. 3Treatment with statins recovers methionine- and choline-deficient diet (MCDD)-induced suppression of peroxisomal proliferator-activated receptor α (PPARα) and target gene expression levels in the liver. (A) mRNA expression levels and (B) protein expression levels of PPARα. Expression levels of genes involved in mitochondrial and peroxisomal (C) fatty acid oxidation (FAO), and (D) peroxisomal biogenesis factor (Pex) 7. ND, normal chow diet; F, fluvastatin; P, pravastatin; S, simvastatin; A, atorvastatin; R, rosuvastatin; CPT, carnitine palmitoyltransferase; ACOX, acyl CoA oxidase; DBP, D-bifunctional protein. aP<0.05 compared with ND, bP<0.05 compared with MCDD.
Fig. 4Measurement of hepatic fatty acid oxidation (FAO) in mice fed methionine- and choline-deficient diet (MCDD) with or without statins. (A) Mitochondrial FAO. (B) Peroxisomal FAO. DPM, disintegrations per minute; ND, normal chow diet; F, fluvastatin; P, pravastatin; S, simvastatin; A, atorvastatin; R, rosuvastatin. aP<0.05 compared with ND, bP<0.05 compared with MCDD.
Fig. 5Changes in hepatic lipid peroxidation and gene expression levels of several anti-oxidative enzymes. (A) Hepatic malondialdehyde (MDA) levels in mice fed methionine- and choline-deficient diet (MCDD) and fluvastatin supplementation. (B) mRNA expression levels of catalase, (C) manganese superoxide dismutase (MnSOD), and (D) glutathione peroxidase 1 (GPx-1). ND, normal chow diet; F, fluvastatin; P, pravastatin; S, simvastatin; A, atorvastatin. aP<0.05 compared with ND, bP<0.05 compared with MCDD.
Table 1Primer sets for real-time polymerase chain reaction analysis
Gene |
Mouse primer sequences |
Pparα
|
5'-AGAGCCCCATCTGTCCTCTC-3' |
5'-ACTGGTAGTCTGCAAAACCAAA-3' |
Pex7
|
5'-TGGTGACAGGTGCGGTTGAC-3' |
5'-ATAGGAGCAGGAGGCCAGCA-3' |
Cpt-1α
|
5'-TGGCCGCATGTCAAGCCAGA-3' |
5'-AGGAGAGCAGCACCTTCAGCGA-3' |
Cpt-1β
|
5'-TTGGTCCCGTGGCGGATGA-3' |
5'-AAAGCGCTGGGCGTTCGTCT-3' |
Acox1
|
5'-CACGCACATCTTGGATGGTAGTCCG-3' |
5'-ACGCTGGCTTCGAGTGAGGAAGTTA-3' |
Dbp1
|
5'-ACGCCCTGGCGTTTGCAGAA-3' |
5'-TGGCCACTGCTTTTCCGCCT-3' |
Catalase
|
5'-GTGGCCAACTACCAGCGTGA-3' |
5'-ATCTACAGCGCACTGGACGC-3' |
MnSOD
|
5'-GGTGGAGAACCCAAAGGAGA-3' |
5'-CTTGGACTCCCACAGACACG-3' |
Gpx-1
|
5'-CGTGCAATCAGTTCGGACAC-3' |
5'-TAAAGAGCGGGTGAGCCTTC-3' |
Table 2Metabolic characteristics of mice fed methionine- and choline-deficient diet with or without statins
Characteristic |
Dietary group |
ND |
MCDD |
MCDD+F |
MCDD+P |
MCDD+S |
MCDD+A |
MCDD+R |
BW, g |
26.08±0.39 |
15.61±0.23a
|
14.82±0.31 |
14.89±0.36 |
15.13±0.42 |
14.34±0.24 |
14.96±0.27 |
LW, g |
1.22±0.06 |
0.84±0.05a
|
0.67±0.05b
|
0.69±0.04b
|
0.63±0.03b
|
0.67±0.05b
|
0.61±0.07b
|
LW/BW, % |
4.68±0.16 |
5.38±0.24a
|
4.52±0.24b
|
4.63±0.16 |
4.16±0.08b
|
4.67±0.27 |
4.08±0.39b
|
Plasma cholesterol, mg/dL |
143.96±23.25 |
53.25±12.88a
|
60.34±15.62 |
55.74±10.21 |
45.10±13.15 |
59.34±9.43 |
56.48±10.64 |
Plasma FFA, µEq/L |
526.46±23.12 |
344.88±19.26a
|
295.24±39.62 |
312.84±20.31 |
322.57±16.45 |
298.76±15.07 |
312.70±11.83 |
Plasma TG, mg/dL |
140.15±10.38 |
86.87±4.36a
|
88.68±3.67 |
86.54±2.93 |
83.67±6.39 |
90.81±7.12 |
86.80±5.24 |