Department of endocrinology, Shengli Clinical Medical College of Fujian Medical University, Fuzhou, China.
Department of Endocrinology, Fujian Provincial Hospital, Fuzhou, China.
Biosci Biotechnol Biochem. 2020 Jun;84(6):1191-1200. doi: 10.1080/09168451.2020.1737505. Epub 2020 Mar 6.
The molecular mechanism of autophagy in Lactoferrin (LF) induced osteoblast differentiation is not fully demonstrated. In this study, alkaline phosphatase (ALP) activity, alizarin red S staining and ELISA were used to study N-terminal propeptide of type I procollagen (PINP) expression. mRFP-GFP-LC3 adenoviruses, mono-dansylcadaverine (MDC) staining, scanning electron microscopy, and western blot analysis was employed to probe the LF induced autophagy. The interaction between autophagy receptor Neighbor of Brca1 gene (Nbr1) and pp38 was studied. 3-methyladenine (3-MA) and chloroquine (CQ) could inhibit the activity of ALP, PINP and the autophagy in LF group. LF treatment could up-regulate and down-regulate the expressions of pp38 and Nbr1with a dose-dependent manner, respectively. LF could inhibit the recognition of pp38 and Nbr1. In addition, LF can prompt Nbr1-medicated autophagy and prevent pp38 degradation by autophagy. LF can induce Nbr1-mediated autophagy and inhibit pp38 entering into autophagy flux in the physiological process of osteoblast differentiation. CQ:chloroquine；LF: Lactoferrin; 3-MA: 3-methyladenine; ALP: Alkaline phosphatase; ANOVA: Analysis of variance; CCK-8: Cell Counting Kit-8; LC3: Microtubule-associated protein light chain3; MDC: Monodansylcadaverine; Nbr1: neighbor of Brca1 gene; PINP: N-terminal propeptide of type I procollagen; PVDF: Polychlorotrifluoroethylene; pp38: phosphorylation p38; RAPA: Rapamycin; SDS: sodium dodecyl sulfate.
乳铁蛋白（LF）诱导成骨细胞分化中的自噬分子机制尚未完全阐明。本研究采用碱性磷酸酶（ALP）活性、茜素红 S 染色和 ELISA 检测方法研究 I 型前胶原 N 端前肽（PINP）的表达。采用 mRFP-GFP-LC3 腺病毒、单丹磺酰尸胺（MDC）染色、扫描电子显微镜和 Western blot 分析方法研究 LF 诱导的自噬。研究自噬受体 Neighbor of Brca1 gene（Nbr1）与 pp38 的相互作用。3-甲基腺嘌呤（3-MA）和氯喹（CQ）可抑制 LF 组的 ALP、PINP 和自噬活性。LF 处理可呈剂量依赖性地上调和下调 pp38 和 Nbr1 的表达。LF 可抑制 pp38 和 Nbr1 的识别。此外，LF 可抑制 pp38 降解，促进 Nbr1 介导的自噬。LF 可在成骨细胞分化的生理过程中诱导 Nbr1 介导的自噬，抑制 pp38 进入自噬流。CQ：氯喹；LF：乳铁蛋白；3-MA：3-甲基腺嘌呤；ALP：碱性磷酸酶；ANOVA：方差分析；CCK-8：细胞计数试剂盒-8；LC3：微管相关蛋白轻链 3；MDC：单丹磺酰尸胺；Nbr1：Brca1 基因邻接物；PINP：I 型前胶原 N 端前肽；PVDF：聚偏二氟乙烯；pp38：磷酸化 p38；RAPA：雷帕霉素；SDS：十二烷基硫酸钠。
