DHA Protects Hepatocytes from Oxidative Injury through GPR120/ERK-Mediated Mitophagy

Abstract

Oxidative stress occurs in a variety of clinical liver diseases and causes cellular damage and mitochondrial dysfunction. The clearance of damaged mitochondria by mitophagy may facilitate mitochondrial biogenesis and enhance cell survival. Although the supplementation of docosahexaenoic acid (DHA) has been recognized to relieve the symptoms of various liver diseases, the antioxidant effect of DHA in liver disease is still unclear. The purpose of our research was to investigate the antioxidant effect of DHA in the liver and the possible role of mitophagy in this. In vitro, H₂O₂-induced injury was caused in AML12 cells. The results showed that DHA repressed the level of reactive oxygen species (ROS) induced by H₂O₂ and stimulated the cellular antioxidation response. Most notably, DHA restored oxidative stress-impaired autophagic flux and promoted protective autophagy. In addition, PINK/Parkin-mediated mitophagy was activated by DHA in AML12 cells and alleviated mitochondrial dysfunction. The ERK1/2 signaling pathway was inhibited during oxidative stress but reactivated by DHA treatment. It was proven that the expression of ERK1/2 was involved in the regulation of mitophagy by the ERK1/2 inhibitor. We further proved these results in vivo. DHA effectively alleviated the liver oxidative damage caused by CCl₄ and enhanced antioxidation capacity; intriguingly, autophagy was also activated. In summary, our data demonstrated that DHA protected hepatocytes from oxidative damage through GPR120/ERK-mediated mitophagy.

Keywords: DHA; ERK1/2 signaling; liver injury; mitophagy; oxidative stress.

Figure 1

Docosahexaenoic acid (DHA) activated the cellular antioxidant response and alleviated the oxidative damage caused by H₂O₂ in hepatocytes. AML12 cells were pre-treated with DHA (50 μM) and TUG891 (10 μM) for 12 h, and then treated with H₂O₂ (400 μM) for another 2 h. (A,B) The cell viability of AML12 cells after treated with H₂O₂ and DHA was detected by CCK-8 assay kit. (C) Cellular reactive oxygen species (ROS) was stained with DCFH-DA fluorescent probe and detected by flow cytometry. (D) The apoptosis of AML12 cells was evaluated with Annexin V-FITC Apoptosis Staining kit. (E) The protein expression of Superoxide dismutase1 (SOD1) and Heme Oxygenase-1 (HO-1) was determined by Western-blotting. All data are expressed as mean ± SEM of three independent experiments. Significant differences between each group are shown with different letters (e.g., a, b, and c mean a statistically significant difference between each other, ** p < 0.05).

Figure 2

The effect of DHA on scavenging ROS is associated with restoration of protective autophagy. AML12 cells were pre-treated with DHA (50 μM) and TUG891 (10 μM) for 12 h, and then treated with H₂O₂ (400 μM) for another 2 h. (A) The protein expression of LC3II, Beclin1, and P62/SQSTM2 was determined by Western blotting. (B) The autophagic flux was detected by using confocal microscopy after transfection of GFP-mCherry-LC3II. (C) The lysosomes in AML12 cells were stained with LysoSensor™ and detected by using confocal microscopy after treatment. (D) Cells were pre-treated with 3-Methyladenine (3-MA) (5 mM) for 2 h, and then treated with DHA (50 μM) for 12 h followed by H₂O₂ (400 μM) 2 h challenge. Cellular ROS was stained with DCFH-DA fluorescent probe and detected by flow cytometry. All data are expressed as mean ± SEM of three independent experiments. Significant differences between each group are shown with different letters (e.g., a, b, and c mean a statistically significant differences between each other, p < 0.05).

Figure 3

DHA against H₂O₂-induced hepatocyte injury though PINK/Parkin-mediated mitophagy. AML12 cells were pre-treated with DHA (50 μM) and TUG891 (10 μM) for 12 h, and then treated with H₂O₂ (400 μM) for another 2 h. (A) The protein expression of PINK1, Parkin, and Mito-LC3II was determined by Western-blotting. (B) The immunofluorescence detected the expression of PINK in AML12 cells. All data are expressed as mean ± SEM of three independent experiments. Significant differences between each group are shown with different letters (e.g., a, b, and c mean a statistically significant differences between each other, p < 0.05).

Figure 4

DHA reversed H₂O₂-induced mitochondrial injury in hepatocytes. AML12 cells were pre-treated with DHA (50 μM) and TUG891 (10 μM) for 12 h, and then treated with H₂O₂ (400 μM) for another 2 h. (A) The mitochondrial membrane potential (ΔΨm) was detected with JC-1 staining assay. (B) The protein expression of Drp1 and PGC1α was determined by Western-blotting. All data are expressed as mean ± SEM of three independent experiments. Significant differences between each group are shown with different letters (e.g., a, b, and c mean a statistically significant differences between each other, p < 0.05).

Figure 5

Inhibiting ERK1/2 signaling decreased the effect of DHA against oxidative stress. AML12 cells were pre-treated with DHA (50 μM) and TUG891 (10 μM) for 12 h, and then treated with H₂O₂ (400 μM) for another 2 h. (A) The protein expression of ERK1/2, P-ERK1/2, AMPK, and P-AMPK was determined by Western-blotting. (B) The protein expression of ERK1/2 and P-ERK1/2 in AML12 cells after treated with U0126. (C) Cells were pre-treated with U0126 (10 μM) for 2 h, and then treated with DHA (50 μM) for 12h followed with H₂O₂ (400 μM) 2h challenge. Cellular ROS was stained with DCFH-DA fluorescent probe and detected by Flow cytometry. (D) The cell viability of AML12 cells. All data were expressed as mean ± SEM of three independent experiments. Significant differences between each group are shown with different letters (e.g., a, b, and c mean a statistically significant differences between each other, p < 0.05).

Figure 6

The inhibition of ERK1/2 signaling blocked DHA-mediated mitophagy. Cells were pre-treated with U0126 (10 μM) for 2 h, and then treated with DHA (50 μM) for 12 h followed with H₂O₂ (400 μM) 2h challenge. (A) The protein expression of Beclin1, P62/SOSTM1, LC3B, PINK, and Parkin was determined by Western-blotting. (B) The immunofluorescence detected the expression of PINK in AML12 cells. (C) The lysosomes in AML12 cells were stained with LysoSensor™ and detected by using confocal microscopy after treatment. All data are expressed as mean ± SEM of three independent experiments. Significant differences between each group are shown with different letters (e.g., a, b, and c mean a statistically significant differences between each other, p < 0.05).

Figure 7

Supplements of DHA protect against CCl₄-induced liver injury in mice by activating autophagy. (A) Experimental design All mice were randomly divided into three groups (n = 10). (B) The HE stains images of liver. (C) The level of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in serum. (D) The activities of SOD and glutathione (GSH) in liver. (E) The protein expression of Beclin1, P62/SOSTM1, and LC3B in liver were determined by Western-blotting. (F) The immunofluorescence detected the expression of LC3B in liver. The data are expressed as mean ± SEM and the significant differences between each group are shown with different letters (e.g., a, b, and c mean a statistically significant differences between each other, p < 0.05).

Figure 8

The schematic representation shows that DHA attenuates H₂O₂-induced hepatocyte injury through ERK1/2-dependent mitophagy. Oxidative stress would induce hepatocyte apoptosis by impairing mitochondrial function. DHA and TUG891 (a agonist of GPR120) supplementation activates GPR120/ERK1/2 signaling pathway which regulates PINK1/Parkin-mediated mitophagy. The up-regulation of protective mitophagy level promotes mitochondrial renewal and maintains mitochondrial homeostasis during oxidative injury. However, inhibiting the activation of ERK1/2 and autophagy by selective inhibitor U0126 or 3-MA, respectively, could block the protective effect of DHA.