口腔颌面外科杂志2025，Vol.35Issue(3)：171-180,10.DOI:10.12439/kqhm.1005-4979.2025.03.002
线粒体自噬在牙周炎骨组织中的作用研究
The role of mitophagy in periodontitis bone tissue
摘要
Abstract
Objective:To investigate changes in mitophagy levels in bone tissue during periodontitis in mice and to explore the effects of lipopolysaccharide(LPS)-induced mitophagy on the osteogenic differentiation capacity of mouse embryonic osteoblast precursor(MC3T3-E1)cells.Methods:A mouse periodontitis model was established using silk ligatures,and mice were divided into ligation and control groups.Micro-CT and hematoxylin-eosin(HE)staining were used to assess alveolar bone resorption.Real-time quantitative polymerase chain reaction(RT-qPCR)was employed to detect the relative mRNA expression levels of inflammation-related genes and mitophagy-related genes in the periodontal bone tissue.Immunofluorescence(IF)staining was used to observe the PTEN-induced putative kinase 1(Pink1)expression in the alveolar bone.For in vitro experiments,MC3T3-E1 cells were induced with LPS and divided into the control group and the LPS group.According to whether autophagy intervention was applied,MC3T3-E1 cells were further divided into the dimethyl sulfoxide(DMSO)+LPS group,rapamycin(Rapa)+LPS group,3-methyladenine(3-MA)+LPS group,and LPS group.Additionally,MC3T3-E1 cells were divided into the siPink1 group and the NC group based on whether Pink1 was knocked down using small interfering RNA(siRNA).RT-qPCR was used to detect the relative mRNA expression levels of mitophagy-related and osteogenesis-related genes.Transmission electron microscopy(TEM)was employed to observe mitochondria and autophagosomes.IF staining was performed to examine changes in the expression of the mitophagy-related protein Pink1.Western blotting was conducted to detect the expression levels of autophagy-related proteins.Alkaline phosphatase(ALP)staining and alizarin red staining were used to evaluate the osteogenic differentiation capacity of cells in each group,respectively.Results:In vivo experiments showed that the silk ligation group exhibited significantly increased buccal alveolar bone resorption.The relative expression level of the mitophagy-related protein Pink1 was elevated in the bone tissue of the ligation group,along with increased relative mRNA expression levels of inflammation-related and mitophagy-related genes.In vitro experiments demonstrated that,after 14 days of osteogenic induction,compared with the control group,the LPS group showed significantly decreased relative mRNA expression levels of osteogenesis-related genes,weaker ALP staining,and smaller alizarin red staining areas.Compared with the control group,the LPS group exhibited significantly increased mRNA and protein expression levels of Pink1 and E3 ubiquitin-protein ligase Parkin(Parkin)(related to mitophagy),while showing significantly decreased mRNA and protein expression levels of microtubule-associated protein 1 light chain 3(LC3)and sequestosome 1(P62)(related to autophagy);TEM observation revealed swollen and damaged mitochondria in the LPS group;IF staining results indicated a higher proportion of Pink1-positive cells in the LPS group compared with the control group.Compared with the DMSO+LPS group,the Rapa+LPS group displayed darker ALP staining and larger alizarin red staining areas in MC3T3-E1 cells.In contrast,the 3-MA+LPS group showed lighter ALP staining and smaller alizarin red staining areas compared with the LPS group.Compared with the NC group,the siPink1 group exhibited decreased relative mRNA expression levels of autophagy-and osteogenesis-related genes,lighter ALP staining,and smaller alizarin red staining areas.Conclusion:Under the conditions of this study,LPS stimulation of MC3T3-E1 cells led to increased relative expression levels of mitophagy-related genes Pink1 and Parkin.Activation of autophagy can partially restore the osteogenic differentiation capacity of cells.关键词
线粒体自噬/Pink1/Parkin/牙周炎/成骨细胞Key words
mitophagy/Pink1/Parkin/periodontitis/osteoblast分类
口腔医学引用本文复制引用
蔡宇艺,王海丞,孙斌,徐亦凡,王佐林..线粒体自噬在牙周炎骨组织中的作用研究[J].口腔颌面外科杂志,2025,35(3):171-180,10.基金项目
科技部重点研发专项(2018YFE0202200) （2018YFE0202200）
国家自然科学基金(81600836) （81600836）
