Exercise Intervention Mitigates Pathological Liver Changes in NAFLD Zebrafish by Activating SIRT1/AMPK/NRF2 Signaling

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a common disease that causes serious liver damage. Exercise is recognized as a non-pharmacological tool to improve the pathology of NAFLD. However, the antioxidative effects and mechanisms by which exercise ameliorates NAFLD remain unclear. The present study conducted exercise training on zebrafish during a 12-week high-fat feeding period to study the antioxidant effect of exercise on the liver. We found that swimming exercise decreased lipid accumulation and improved pathological changes in the liver of high-fat diet-fed zebrafish. Moreover, swimming alleviated NOX4-derived reactive oxygen species (ROS) overproduction and reduced methanedicarboxylic aldehyde (MDA) levels. We also examined the anti-apoptotic effects of swimming and found that it increased the expression of antiapoptotic factor bcl2 and decreased the expression of genes associated with apoptosis (caspase3, bax). Mechanistically, swimming intervention activated SIRT1/AMPK signaling-mediated lipid metabolism and inflammation as well as enhanced AKT and NRF2 activation and upregulated downstream antioxidant genes. In summary, exercise attenuates pathological changes in the liver induced by high-fat diets. The underlying mechanisms might be related to NRF2 and mediated by SIRT1/AMPK signaling.

Keywords: NAFLD; ROS; SIRT1/AMPK/NRF2; exercise; zebrafish.

Figure 1

Zebrafish receiving a high exercise regimen (HEX) had reduced body weight gain and lipid accumulation compared to high-fat diet (HFD) zebrafish. Normal diet (ND) zebrafish represent the control group. (A,B) Morphology and body weight of zebrafish. (C) Oil Red O staining of zebrafish livers (n = 3). (D) Quantitation of Oil Red O staining. (E) Fasting blood glucose (ND, n = 13; HFD, n = 10; HEX, n = 11). (F) Liver triglyceride (n = 5). (G) Liver cholesterol (n = 5). The above experiments were carried out using 9-month-old zebrafish. *, p < 0.05, **, p < 0.01, ***, p < 0.001. Data represent the mean, and error bars represent SEM. Scale bar, 20 μm. NAFLD, non-alcoholic fatty liver disease; ND, normal diet; HFD, high fat diet; HEX, high-fat diet plus exercise.

Figure 2

SIRT1/AMPK signaling and lipid metabolism in zebrafish livers. (A) Western blot representing SIRT1/AMPK signaling in 9-month-old zebrafish livers (n = 8). (B) Expression of lipogenesis-related genes (n = 6). (C) Expression of β-oxidation-related genes in 9-month-old zebrafish livers (n = 6). *, p < 0.05, **, p < 0.01, ***, p < 0.001. Data represents the mean, and error bars represent SEM. NAFLD, non-alcoholic fatty liver disease; ND, normal diet; HFD, high fat diet; HEX, high-fat diet plus exercise.

Figure 3

Quantitation of pathological changes in zebrafish livers. (A) H&E staining of 9-month-old zebrafish livers (n = 3). (B) NAFLD activity scores. *, p < 0.05, **, p < 0.01, ***, p < 0.001. Data represent the mean and error bars represent SEM. Scale bar, 20 μm. NAFLD, non-alcoholic fatty liver disease; ND, normal diet; HFD, high fat diet; HEX, high-fat diet plus exercise.

Figure 4

Quantitation of pathological changes in 9-month-old zebrafish livers. (A) Gene expression of il1β and tnfα (n = 6). (B) Masson’s staining and fibrosis score of zebrafish livers (n = 3). (C,D) The protein levels of α-smooth muscle actin (α-SMA) (n = 8). *, p < 0.05, **, p < 0.01, ***, p < 0.001. Data represent the mean and error bars represent SEM. Scale bar, 20 μm. NAFLD, non-alcoholic fatty liver disease; ND, normal diet; HFD, high fat diet; HEX, high-fat diet plus exercise.

Figure 5

Hepatocyte apoptosis in 9-month-old zebrafish livers. (A) Tunel staining of zebrafish livers, and (B) Quantitation of Tunel-positive cells (n = 3). (C) Gene expression of caspase3, bax, and bcl2 (n = 6). *, p < 0.05 **, p < 0.01, ***, p < 0.001. Data represent the mean, and error bars represent SEM. Scale bar, 20 μm. NAFLD, non-alcoholic fatty liver disease; ND, normal diet; HFD, high fat diet; HEX, high-fat diet plus exercise.

Figure 6

Oxidative stress in 9-month-old zebrafish livers. (A) NOX4 protein expression (n = 8). (B) Dihydroethidium (DHE) staining of zebrafish livers (n = 3), and (C) quantitation of DHE staining. (D) MDA levels in zebrafish livers (n = 5). *, p < 0.05, **, p < 0.01, ***, p < 0.001. Data represent the mean, and error bars represent SEM. Scale bar, 20 μm. NAFLD, non-alcoholic fatty liver disease; ND, normal diet; HFD, high fat diet; HEX, high-fat diet plus exercise.

Figure 7

NRF2 signaling in 9-month-old zebrafish livers. (A) Western blot of p-AKT and NRF2 expression in zebrafish livers (n = 8). (B) Gene expression of ho-1, nqo1, and cat (n = 6). *, p < 0.05, **, p < 0.01, ***, p < 0.001. Data represent the mean, and error bars represent SEM. NAFLD, non-alcoholic fatty liver disease; ND, normal diet; HFD, high fat diet; HEX, high-fat diet plus exercise.