Scutellarin Prevents Nonalcoholic Fatty Liver Disease (NAFLD) and Hyperlipidemia via PI3K/AKT-Dependent Activation of Nuclear Factor (Erythroid-Derived 2)-Like 2 (Nrf2) in Rats

Abstract

BACKGROUND Nonalcoholic fatty liver disease (NAFLD) is a condition characterized by excessive fat accumulation in the form of triglycerides. The incidence of NAFLD and hyperlipidemia, with their associated risks of end-stage liver and cardiovascular diseases, is increasing rapidly. This study aimed to investigate the effects of scutellarin on the experimental NAFLD in high-fat diet fed and chronic stress rats, and its possible mechanism. MATERIAL AND METHODS Sprague-Dawley rats were fed with high-fat diet and subjected to chronic stress for 12 weeks, and administered orally with scutellarin for 4 weeks (n=8), and then blood and livers were harvested for analyzing. Enzyme activity assay, immunofluorescence, Western blot, and quantitative RT-PCR were performed to analyze the factors of the oxidant/antioxidant system and pathway. RESULTS After the high-fat diet and chronic stress administration for 12 weeks, serum and liver lipid metabolism of treatment groups with the different doses of SCU effectively improved and the degree of oxidative damage reduced. Using Western blot assay and immunofluorescence (IF) staining assay, Nrf2, HO-1, and PI3K, and AKT proteins significantly increased after SCU treatment for 4 weeks (P<0.01). The hepatic mRNA expression of HO-1, NQO1, and Nrf2 in SCU treatment groups was upregulated significantly through quantitative RT-PCR assay (P<0.05). However, compared to the positive control group, no difference was detected in the SCU (100 or 300 mg/kg) groups (P>0.05). These results indicate that SCU protects against NAFLD in rats via attenuation of oxidative stress. CONCLUSIONS The antioxidant effects of SCU on NAFLD are possibly dependent on PI3K/AKT activation with subsequent Nrf2 nuclear translocation, which increases expression of HO-1 and NQO1. We therefore suggest that breviscapine may be a potentially useful therapeutic strategy for NAFLD and hyperlipidemia.

Figure 1

The effect of serum TC, TG, HDL-C and LDL-C concentrations after SCU treatment.Under high-fat diet and chronic stress, rats were treated with different concentrations of SCU (50, 100, 300 mg/kg) for 4 weeks, and serum biochemistry was determined by clinical chemistry analyzer. All data were represented as means±standard deviation (n=8).Statistical analysis of TC, TG, HDL-C and LDL-C (* P<0.05, ** P<0.01, Veh-h-S group versus Veh group; ^# P<0.05, ^## P<0.01, treatment groups versus Veh-h-S group).

Figure 2

The effect of hepatic tissue blood flow (HTBF) after SCU treatment. (A) Laser speckle images of hepatic tissueon the dual intervention of high-fat diet and chronic stress 12 weeks and SCU treatment 4 weeks. The area of the dotted line is the location of collecting hepatic tissue blood flow. Relative perfusion in the arbitrary unit flux was portrayed in a color-coded laser speckle image (red=high flow, blue=low flow). (B) Bar graphs showing the HTBF in different groups.All data were represented as means ± standard deviation (n=8). Statistical analysis of HTBF (* P<0.05, ** P<0.01, Veh-h-S group versus Veh group; ^# P<0.05, ^## P<0.01, treatment groups versus Veh-h-S group; ^& P<0.05, ^&& P<0.01, SCU treatment groups versus LIP5 group).

Figure 3

The effects of oxidative damage through MDA, T-SOD and GSH in serum and liver after SCU treatment.Under high-fat diet and chronic stress, rats were treated with different concentrations of scutellarin (SCU, 50, 100, 300 mg/kg) for 4 weeks, and these contents were determined by acommercial assay kits. All data were represented as means ± standard deviation (n=8). Statistical analysis of MDA, T-SOD and GSH (* P<0.05, ** P<0.01, Veh-h-S group versus Veh group; ^# P<0.05, ^## P<0.01, treatment groups versus Veh-h-S group; ^& P<0.05, ^&& P<0.01, SCU treatment groups versus LIP5 group).

Figure 4

The effect of the liver function indicator through ALT, AST and hepatosmatic index in different groups. Under high-fat diet and chronic stress rats were treated with various concentrations of SCU (50, 100, 300 mg/kg) for 4 weeks, and serum ALT and AST were determined by clinical chemistry analyzer, and hepatosmatic indexwas the ratio of liver to body weight. All data were represented as means ± standard deviation (n=8).Statistical analysis of these date (* P<0.05, ** P<0.01, Veh-h-S group versus Veh group; ^# P<0.05, ^## P<0.01, treatment groups versus Veh-h-S group).

Figure 5

Histologic evaluation of livers from Sprague Dawley rats after SCU treatment. (A–F) Representative hematoxylin and eosin-stained liver sections (original magnification 200×) in different groups. (A) Veh group, (B) Veh-h-S group, (C) LIP5 group, (D) SCU50 group, (E) SCU100 group, (F) SCU300 group. Veh-h-S group rats had exacerbated hepatic steatosis (↑) and hepatocyte ballooning (▲) relative to Veh group. SCU reduced these parameters, but the effects had no dose-dependent relationship. Histologic scoring of liver steatosis (G), hepatocyte ballooning (H) and inflammatory infiltrates (I). All data were represented as means±standard deviation (n=8). Statistical analysis of these date (*P<0.05, **P<0.01, Veh-h-S group versus Veh group; ^# P<0.05, ^## P<0.01, treatment groups versus Veh-h-S group; ^& P<0.05, ^&& P<0.01, SCU treatment groups versus LIP5 group).

Figure 6

The evaluation on Nrf2-mediated antioxidant defense system of livers from Sprague Dawley rats after SCU treatment by western blotting. The levels of AKT/Nrf2 pathway proteins were evaluated by western blotting. β-actin was used as the internal control, respectively. (A) HO-1, (B) NQO1, (C) Nrf2, (D) p-AKT, and (E) PI3K proteins in different groups. All data were represented as means ± standard deviation (n=6).Statistical analysis of these date. (* P<0.05, ** P<0.01, Veh-h-S group versus Veh group; ^# P<0.05, ^## P<0.01, treatment groups versus Veh-h-S group; ^& P<0.05, ^&& P<0.01, SCU treatment groups versus LIP5 group). (F) The immunoblotting results of these proteins.

Figure 7

The effect of key protein on Nrf2-mediated antioxidant defense system of livers from Sprague Dawley rats after SCU treatment by immunofluorescence. Anti-NRF2 antibody (up) and anti-p-AKT antibody (under) were measured in livers, respectively. Green fluorescence was positive expression, and nuclei were stained with DAPI. Images of different groups were photographed using confocal fluorescence microscopy (original magnification 200×).

Figure 8

The gene expression of cytokines on Nrf2 pathway in livers from Sprague Dawley rats after SCU treatmentby quantitative RT-PCR.Quantitative values were obtained from the threshold cycle value (^ΔΔCt). (A–C) The quantitative values of HO-1, NQO1, Nrf2 gene expression in different groups.All data were represented as means±standard deviation (n=3).Statistical analysis of these date (* P<0.05, ** P<0.01, Veh-h-S group versus Veh group; ^# P<0.05, ^## P<0.01, treatment groups versus Veh-h-S group; ^& P<0.05, ^&& P<0.01, SCU treatment groups versus LIP5 group). (F) The immunoblotting results of these proteins.