Lipopolysaccharide induces parkin expression and mitophagy in murine peritoneal macrophages / 中国免疫学杂志

ABSTRACT

Objective: To investigate whether lipopolysaccharide induced parkin expression and mitophagy in macrophages.Methods:The murine peritoneal primary macrophages were aseptically isolated from Kunming mice and cultured in complete medium.The mitochondrial membrane potential of macrophages was detected by flow cytometry,after the cells were stimulated with 200 ng/ml LPS and labeled mitochondria with JC-1.The parkin mRNA level of macrophages was detected by RT-PCR, protein levels of parkin and autophagic related protein LC3 Ⅱ and LC3 Ⅰ were determined by Western blot.The distribution and co-localization of parkin with LC3 and mitochondria in macrophages were respectively observed by laser scanning confocal microscope, before and after the cells were treated with LPS.Results: Flow cytometry results after JC-1 staining showed that mitochondrial membrane potential in macrophages was declined after stimulation with 200 ng/ml LPS, and continuously decreased with prolonged treatment time.The mRNA levels of parkin were increased slightly within 6 h after LPS stimulation,but parkin proteins were increased significantly within 6 h after LPS stimulation.The results of parkin distribution showed that parkin was evenly distributed in the cytoplasm at normal status, but became the obvious punctate distribution after LPS stimulation in macrophages.Western blot results showed LC3 Ⅱ/LC3 Ⅰ levels were increased after LPS stimulation, indicating the appearance of macrophage autophagy.Confocal microscopy showed that there were co-localization of parkin,LC3 and mitochondrial in macrophages after LPS stimulation.Conclusion:Parkin expression is increased significantly and mediated mitochondrial autophagy in macrophages after LPS stimulation, which is involved in the clearance of damaged mitochondria,thereby playing a role in regulating macrophage inflammatory response.