摘要：

目的：探讨小鼠牙周炎骨组织中线粒体自噬水平变化和脂多糖（lipopolysaccharide，LPS）诱导下线粒体自噬水平对小鼠胚胎成骨细胞前体细胞（mouse embryo osteoblast precursor cells，MC3T3-E1 cells）成骨分化能力的影响。方法：采用丝线结扎法构建小鼠牙周炎模型，分为丝线结扎组和对照组，通过 Micro-CT 和苏木精-伊红（hematoxylineosin，HE）染色法观察牙槽骨吸收情况，通过实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测小鼠牙周炎骨组织中炎症相关基因和线粒体自噬相关基因的 mRNA 相对表达水平，免疫荧光（immunofluorescence，IF）染色法观察 PTEN 诱导假定激酶 1（PTEN-induced putative kinase 1，Pink1）在小鼠牙槽骨组织中的表达。体外实验采用 LPS 诱导 MC3T3-E1 细胞，分为对照组和 LPS 组；根据是否接受自噬干预将 MC3T3-E1细胞分为二甲基亚砜（dimethyl sulfoxide，DMSO） +LPS 组、雷帕霉素（rapamycin，Rapa） +LPS 组、3-甲基腺嘌呤（3-methyladenine，3-Ma） +LPS 组和 LPS 组；根据是否采用小干扰 RNA（small interfering RNA，siRNA）敲降 Pink1 将MC3T3-E1 细胞分为 siPink1 组和 NC 组。RT-qPCR 检测线粒体自噬和成骨相关基因的 mRNA 相对表达水平，透射电子显微镜（transmission electron microscope，TEM）观察线粒体和自噬体，IF 染色法观察线粒体自噬相关蛋白 Pink1 的表达变化，蛋白质印迹法检测自噬相关蛋白的表达水平，通过碱性磷酸酶（alkaline phosphatas，ALP）染色和茜素红染色观察各组细胞的成骨分化能力。结果：体内实验中，丝线结扎组牙槽骨颊侧吸收明显增多；丝线结扎组的骨组织中线粒体自噬相关蛋白 Pink1 表达水平升高，炎症和线粒体自噬相关基因的 mRNA 相对表达水平升高。体外实验结果示，成骨诱导 14 d 后，相较于对照组，LPS 组成骨相关基因的 mRNA 相对表达水平显著下降，且 LPS 组 ALP 染色着色更浅，茜素红染色着色面积较小。与对照组相比，LPS 组 Pink1、Parkin E3 泛素蛋白连接酶（E3 ubiquitin-protein ligase Parkin，Parkin）（与线粒体自噬相关）的 mRNA 和蛋白表达水平均显著升高，而微管相关蛋白 1 轻链 3（microtubuleassociated protein 1 light chain 3，LC3）、螯合体 1（sequestosome 1，P62）（与自噬相关）的 mRNA 和蛋白表达水平均显著下降；TEM 观察到 LPS 组的线粒体出现肿胀、损伤；IF 染色结果显示，LPS 组的 Pink1 阳性细胞的比例较对照组高。相较于 Dmso+LPS 组，Rapa+LPS 组 MC3T3-E1 细胞的 ALP 染色着色更深，茜素红染色着色面积较大；与 LPS 组比较，3-Ma+LPS 组 ALP 染色着色更浅，茜素红染色着色面积减小。与 NC 组比较，siPink1 组自噬和成骨相关基因的 mRNA相对表达水平下降，ALP 染色较浅，茜素红染色着色面积更小。结论：本研究条件下，LPS 刺激 MC3T3-E1 细胞可致线粒体自噬相关基因 Pink1、Parkin 的相对表达水平升高，激活自噬可部分恢复细胞的成骨分化能力。

关键词:

线粒体自噬, Pink1, Parkin, 牙周炎, 成骨细胞

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 andmitophagy-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 mitophagyrelated andosteogenesis-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.

Key words:

mitophagy, Pink1, Parkin, periodontitis, osteoblast

中图分类号: 

• R782