The Effect Of Exercise On Hepatocyte Mitophagy In Hffc Diet Induced Nafld Mice

PURPOSE: Mitophagy can degrade damaged mitochondria, which playing an important role in NAFLD progression. This study aimed to investigate the effects of 8-weeks voluntary running wheel on hepatic mitophagy in High fat, High fructose, High cholesterol (HFFC) diet mice. METHODS: Thirty C57BL/6 J mice were divided into three groups: an 18-weeks normal diet group (NC), an 18-week HFFC diet group (HC), and an HFFC diet group with 8-weeks of exercise intervention (HE). The fluorescent probe was used to detect mitochondrial reactive oxygen species (ROS) and membrane potential (MMP) levels.Western blot was used to detect the expression levels of mitophagy proteins (P62, LC3, LAMP-2A, Parkin). The remaining liver samples were sectioned for immunofluorescence staining of P62, LC3, Parkin, and VDAC. RESULTS:1) Compared with the NC group, the LC3-II/I ratio (1.0 ± 0.61 vs 0.58 ± 0.17) and LAMP-2A (1.0 ± 0.39 vs 0.51 ± 0.23, P < 0.01) expression level were decreased in HC group mice, while P62 (1.0 ± 0.65 vs 2.27 ± 0.72, P < 0.01) was increased. The colocalization of LC3 and P62 (1.0 ± 0.44 vs 0.7 ± 0.24) were decreased. Exercise intervention significantly increased the LC3-II/I ratio (1.2 ± 0.25, P < 0.05), LAMP-2A expression level (1.14 ± 0.44, P < 0.01), and the co localization of LC3 and P62 (1.6 ± 0.55, P < 0.01), but decreased the P62 (0.65 ± 0.43, P < 0.01) content. 2) Mice in HC group showed increased ROS (1.0 ± 0.07 vs 1.2 ± 0.27) and decreased MMP levels (1.0 ± 0.08 vs 0.57 ± 0.19, P < 0.05). After exercise intervention, mitochondrial ROS levels significantly dropped (0.83 ± 0.3, P < 0.01) and MMP normalized (0.74 ± 0.27). 3) In the HC group, mitochondrial Parkin expression decreased (1.0 ± 0.28 vs. 0.74 ± 0.6), while P62(1.0 ± 1.02 vs 5.3 ± 3.36, P < 0.01), LC3-II(1.0 ± 0.6 vs 1.92 ± 0.99) levels increased markedly. Parkin and VDAC colocalization (1.0 ± 0.57 vs 0.46 ± 0.23, P < 0.01) also reduced significantly compared to NC. Conversely, in the HE group, mitochondrial Parkin (1.1 ± 0.35), LC3-II (3.46 ± 1.35, P < 0.05) expression and the colocalization of Parkin and VDAC (0.97 ± 0.6, P < 0.01) were significantly increased, but the P62 was reduced (1.57 ± 1.58, P < 0.01). CONCLUSIONS:1) 18-weeks HFFC diet reduced mitochondrial function and mitophagy levels. 2) 8-weeks voluntary wheel intervention improved mitochondrial function and mitophagy in HFFC diet mice. Supported by: This work was supported by Higher Education Institutions in Jiangsu Province (24KJA310002), the Youth Project of National Natural Science Foundation of China (32000839), Graduate Research and Innovation Projects of Jiangsu Province ( KYCX24_2420)