Aspirin Improves Nonalcoholic Fatty Liver Disease and Atherosclerosis through Regulation of the PPAR δ-AMPK-PGC-1 α Pathway in Dyslipidemic Conditions

Abstract

This study is aimed at elucidating how aspirin could systemically and simultaneously normalize nonalcoholic fatty liver disease (NAFLD) and atherosclerosis through both in vitro and in vivo studies in hyperlipidemic conditions. We evaluated the effects and mechanism of aspirin on the levels of various biomarkers related to NAFLD, atherosclerosis, and oxidative phosphorylation in cells and animals of hyperlipidemic conditions. The protein levels of biomarkers (PPARδ, AMPK, and PGC-1α) involved in oxidative phosphorylation in both the vascular endothelial and liver cells were elevated by the aspirin in hyperlipidemic condition. Also in the stimulation pathway of oxidative phosphorylation by aspirin, PPARδ was a superior regulator than AMPK and PGC-1α in HepG2 cells. In the vascular endothelial cells, the phosphorylated endothelial nitric oxide synthase level was increased by the treatment. The protein levels of biomarkers related to lipid synthesis were decreased by the treatment in the liver cells. In rabbits administered with cholesterol diet, the levels of triglyceride, HDL-cholesterol, and alanine amino transferase in serums were ameliorated by the aspirin treatment, the levels of ATP and TNFα were increased or decreased, respectively, by the aspirin in liver and aorta tissues, and mannose receptor and C-C chemokine receptor type 2 levels were increased or decreased by the aspirin in spleen, respectively. The elevated levels of macrophage antigen, angiotensin II type1 receptor, and lipid accumulation were decreased in both the liver and aorta tissues in the aspirin-treated group. In conclusion, aspirin can systemically and simultaneously ameliorate NAFLD and atherosclerosis by inhibiting lipid biosynthesis and inflammation and by elevating catabolic metabolism through the activation of the PPARδ-AMPK-PGC-1α pathway. Furthermore, aspirin may normalize atherosclerosis and NAFLD by modulating the mannose receptor and CCR2 in macrophages.

Figure 1

Protein levels of PPARδ, p-AMPK, PGC-1α, AT1R, and p-eNOS in HUVECs that were treated with high concentrations of palmitate, cholesterol, and aspirin. The oxygen consumption rate in HUVECs treated with aspirin. (a) (A-1, A-2, A-3, A-4) The protein levels of PPARδ (A-1), p-AMPK (A-2), PGC-1α (A-3), and p-eNOS (A-4) were higher in the CPA group, but they were reversed by a PPARδ antagonist. (A-5) The protein level of AT1R was higher in the CP group, but it was decreased in the CPA group. (b) Aspirin treatment increased the oxygen consumption rate in HUVECs. The results are expressed as means ± SEM (N = 3). Values were statistically analyzed by unpaired t-test and one-way ANOVA. An upper line on the three bars means one-way ANOVA analysis. All experiments were repeated three and over times. Meaning of indications: Ctrl is an untreated control group, CP is a cholesterol and palmitate-treated group, CPA is a cholesterol, palmitate, and aspirin-treated group, and CPAG is a cholesterol, palmitate, aspirin, and GSK0660-treated group. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 2

Protein levels of PPARδ, p-AMPK, PGC-1α, FAS, HMGCR, NF-κB, and AT1R in HepG2 cells that were treated with high concentrations of palmitate, cholesterol, and aspirin. The oxygen consumption rate in HepG2 cells treated with aspirin. The effects of AMPK antagonist on p-AMPK and PPARδ protein levels in HepG2 cells. (a) (A-1, A-2, A-3) The protein levels of PPARδ (A-1), p-AMPK (A-2), and PGC-1α (A-3) were higher in the CPA group than those of the CP group; however, the effects of aspirin were reversed by treatment with a PPARδ antagonist. (A-4, A-5, A-6, A-7) The protein levels of FAS (A-4), HMGCR (A-5), NF-κB (A-6), and AT1R (A-7) were lower in the CPA group than those of the CP group; however, the effects of aspirin were reversed by treatment with a PPARδ antagonist. (b) The oxygen consumption rate was increased by aspirin treatment. (c) AMPK antagonist, Compound C, decreased only the protein expression of p-AMPK. The results are expressed as means ± SEM (N = 3). Values were statistically analyzed by unpaired t-test and one-way ANOVA. An upper line on the three bars means one-way ANOVA analysis. All experiments were repeated three and over times. Meaning of indications: Ctrl is an untreated control group, CP is a cholesterol and palmitate-treated group, CPA is a cholesterol, palmitate, and aspirin-treated group, and CPAG is a cholesterol, palmitate, aspirin, and GSK0660-treated group. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 3

Immunocytochemistry for TNFα and Oil Red O staining in HepG2 cells treated with high concentrations of palmitate, cholesterol, and aspirin. (a) The elevated TNFα protein expression in the CP group was decreased by aspirin; however, the effect of aspirin was reversed by treatment with a PPARδ antagonist. (b) Lipid accumulation increased in the CP group was decreased by aspirin treatment; however, the effect of aspirin was offset by treatment with a PPARδ antagonist. Images were taken at ×200 magnification. The results are expressed as means ± SEM (N = 3 or 6). Values were statistically analyzed by unpaired t-test and one-way ANOVA. An upper line on the three bars means one-way ANOVA analysis. All experiments were repeated three and over times. Meaning of indications: Ctrl is an untreated control group, CP is a cholesterol and palmitate-treated group, CPA is a cholesterol, palmitate, and aspirin-treated group, and CPAG is a cholesterol, palmitate, aspirin, and GSK0660-treated group. ^∗∗∗p < 0.001.

Figure 4

CCR2 western blot analysis and immunocytochemistry of mannose receptor in RAW 264.7 cells treated with high concentrations of palmitate, cholesterol, and aspirin. (a) The protein level of CCR2 was lower in the CPA group than that of the CP group; however, the effect of aspirin was reversed by treatment with a PPARδ antagonist. (b) The protein expression of mannose receptor was increased by aspirin treatment. But the effect of aspirin was offset by a PPARδ antagonist. Images were taken at ×200 magnification. The results are expressed as means ± SEM (N = 3). Values were statistically analyzed by unpaired t-test and one-way ANOVA. An upper line on the three bars means one-way ANOVA analysis. All experiments were repeated three and over times. Meaning of indications: Ctrl is an untreated control group, CP is a cholesterol and palmitate-treated group, CPA is a cholesterol, palmitate, and aspirin-treated group, and CPAG is a cholesterol, palmitate, aspirin, and GSK0660-treated group. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 5

Concentrations of triglyceride (TG), high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) in sera from rabbits administered a cholesterol diet and aspirin. (a) TG level was higher in the cholesterol-diet rabbits than that of the control rabbits, and it was lower in the aspirin-administered rabbits. (b) HDL-cholesterol level was more significantly elevated in the aspirin-administered rabbits than that of the cholesterol-diet rabbits without aspirin. (c) LDL-cholesterol level was higher in the cholesterol-diet rabbits than that of the control rabbits, and it was not decreased in the aspirin-administered rabbits. (D, E) ALT and AST levels were higher in the cholesterol-diet rabbits than those of the control rabbits; however, they were decreased in the aspirin-administered rabbits. The results are expressed as means ± SEM (N = 3 or 4). Values were statistically analyzed by unpaired t-test or one-way ANOVA. An upper line on the three bars means one-way ANOVA analysis. All experiments were repeated three and over times. Meaning of indications: Ctrl means normal control rabbits administered with normal, C means rabbits fed with 1% cholesterol diet, and CA means rabbits administered with 1% cholesterol diet plus aspirin of 100 mg/kg/day. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 6

Concentrations of ATP and TNFα in the aorta and liver, and concentrations of mannose receptor and CCR2 in spleen from rabbits administered a cholesterol diet and aspirin. (a) ATP concentration in the aorta showed a tendency to increase in the aspirin-administered group than that of the cholesterol-diet group without aspirin. (b) ATP concentration in the liver was higher in the aspirin-administered group than that of the cholesterol-diet group without aspirin. (c) TNFα concentration in the aorta was lower in the aspirin-administered group than that of the cholesterol-diet group without aspirin. (d) TNFα concentration in the liver showed an increasing trend in the cholesterol-diet group than that of the control group; however, it was lower in the aspirin-administered group than that of the cholesterol-diet group without aspirin. (e) CCR2 concentration in the spleen was higher in the cholesterol-diet group than that of the control group; however, it was lower in the aspirin-administered group. (f) Mannose receptor concentration in the spleen was higher in the aspirin-administered group than that of the cholesterol-diet group without aspirin. The results are expressed as means ± SEM (N = 3 or 4). Values were statistically analyzed by unpaired t-test or one-way ANOVA. An upper line on the three bars means one-way ANOVA analysis. All experiments were repeated three and over times. Meaning of indications: Ctrl means normal control rabbits administered with normal, C means rabbits fed with 1% cholesterol diet, and CA means rabbits administered with 1% cholesterol diet plus aspirin of 100 mg/kg/day. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 7

Immunohistochemistry for macrophage antigen and AT1R and Oil Red O staining in frozen-sectioned aorta tissue slides from rabbits administered a cholesterol diet and aspirin. (a, b) The protein expressions for macrophage antigen and AT1R in aorta tissues were extremely increased in cholesterol diet group compared to control; however, they were decreased in aspirin-administered group. (c) The lipid accumulation in aorta tissues of cholesterol diet group was elevated compared to control; however, the accumulation was lowered in aspirin-administered group. Magnification is 200 times for images of immunohistochemistry and 40 times for images of Oil Red O staining. Densities for images were analyzed with the ImageJ program. The results of image density are expressed as means ± SEM (N = 10). Values were statistically analyzed by unpaired t-test or one-way ANOVA. An upper line on the three bars means one-way ANOVA analysis. All experiments were repeated three and over times. Meaning of indications: C means rabbits fed with 1% cholesterol diet and CA means rabbits administered with 1% cholesterol diet plus aspirin of 100 mg/kg/day. ^∗∗∗p < 0.001.

Figure 8

Immunohistochemistry for macrophage antigen and AT1R and Oil Red O staining in frozen-sectioned liver tissue slides from rabbits administered a cholesterol diet and aspirin. (a, b) The protein expressions for macrophage antigen and AT1R in liver tissues were increased in cholesterol-diet group compared to control; however, they were decreased in aspirin-administered group. (c) The lipid accumulation in liver tissues of cholesterol-diet group was elevated compared to control; however, the accumulation was lowered in aspirin-administered group. Magnification is 200 times. Densities for images were analyzed with the ImageJ program. The results of image density are expressed as means ± SEM (N = 10). Values were statistically analyzed by unpaired t-test or one-way ANOVA. An upper line on the three bars means one-way ANOVA analysis. All experiments were repeated three and over times. Meaning of indications: Ctrl means normal control rabbits administered with normal, cholesterol means rabbits fed with 1% cholesterol diet, and cholesterol+aspirin means rabbits administered with 1% cholesterol diet plus aspirin of 100 mg/kg/day. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 9

Hypothetical action mechanism of aspirin on nonalcoholic fatty liver and atherosclerosis. Simultaneous amelioration of both nonalcoholic fatty liver and atherosclerosis by aspirin is mainly achieved by catabolic activation, anti-inflammation, or vasodilation, which are the result of sequential regulation of the PPARδ→p-AMPK→PGC-1α→oxidative phosphorylation pathway. In addition, the effects of aspirin are achieved by macrophage modulations, which include the increase of mannose receptor and decrease of CCR2.