Role of mitophagy in hydrogen-induced reduction of LPS-caused mitochondrial injury to macrophages of mice

Abstract

Objective To evaluate the role of mitophagy in hydrogen-induced reduction of lipopolysaccharide(LPS)-caused mitochondrial injury to macrophages of mice. Methods Macrophage line RAW264.7 cells of mice were routinely cultured and divided into 4 groups(n=6 each)using a random number table method: control group(Con group), LPS group, LPS plus hydrogen group(LPS+ H2 group) and LPS plus hydrogen plus mitophagy inhibitor 3-methyladenine(3-MA) group(LPS+ H2+ 3-MA group). Cells were incubated for 6 h with LPS at the concentration of 1 μg/ml in LPS group.Cells were incubated for 6 h with LPS 1 μg/ml and hydrogen-rich medium 0.6 mmol/L.Cells were incubated for 1 h with 2 mmol/L 3-MA and then incubated for 6 h with LPS 1 μg/ml and hydrogen-rich medium 0.6 mmol/L in LPS+ H2+ 3-MA group.Mitochondrial respiratory control ratio(RCR)was measured using a Clark-type electrode. Mitochondrial membrane potential(MMP)was determined by JC-1 staining.Autophagosomes were counted with a transmission electron microscope.The expression of PTEN-induced putative kinase 1(PINK1), E3 ubiquitin ligase(Parkin), microtubule-associated protein 1 light chain 3 Ⅱ(LC3 Ⅱ)and Beclin-1 was determined by Western blot. Results Compared with Con group, RCR and MMP were significantly decreased, the expression of PINK1, Parkin, LC3Ⅱ and Beclin-1 was up-regulated, and the number of autophagosomes was increased in LPS group(P<0.05). Compared with LPS group, RCR and MMP were significantly increased, the expression of PINK1, Parkin, LC3Ⅱ and Beclin-1 was up-regulated, and the number of autophagosomes was increased in LPS+ H2 group(P<0.05). Compared with LPS+ H2 group, RCR and MMP were significantly decreased, the expression of PINK1, Parkin, LC3Ⅱ and Beclin-1 was down-regulated, and the number of autophagosomes was decreased in LPS+ H2+ 3-MA group(P<0.05). Conclusion Enhanced mitophagy is involved in hydrogen-induced reduction of LPS-caused mitochondirial injury to macrophages of mice. Key words: Hydrogen; Endotoxins; Macrophages; Mitochondria; Autophagy