背景:绝经后骨质疏松症(PMOP)是一种以低骨量和微结构损伤为特征的全身性骨病。巴林达(MO)含有各种具有抗PMOP活性的成分。巴林达衍生的细胞外囊泡样颗粒(MOEVLPs)是从MO中分离出的新活性成分,目前尚无相关研究探讨其抗骨质疏松作用及机制。
目的:探讨MOEVLPs对PMOP的缓解作用及其机制。
方法:使用差速离心和超速离心从MO中分离MOEVLP。透射电子显微镜(TEM),流量纳米分析仪,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),琼脂糖凝胶电泳,和薄层色谱法用于表征MOEVLP。利用PMOP小鼠模型来检查MOEVLP的抗PMOP作用。H&E和免疫组织化学染色用于药物安全性和成骨效果评估。在体外实验中使用小鼠胚胎成骨细胞前体细胞(MC3T3-E1)。CCK-8套件,茜素红染色,蛋白质组学,生物信息学分析,免疫印迹法探讨MOEVLPs的作用机制。
结果:在这项研究中,来自MO的MOEVLP被成功地分离和表征。动物实验表明,MOEVLP表现出特定的股骨靶向,对心脏无毒,肝脏,脾,脾肺,肾,和主动脉,并具有抗PMOP特性。MOEVLP增强骨形成的能力优于阿仑膦酸钠。体外实验,结果表明,MOEVLPs对MC3T3-E1细胞的成骨分化没有显着增强作用。相反,MOEVLPs促进MC3T3-E1细胞增殖。蛋白质组学和生物信息学分析表明,MOEVLPs的增殖效应与丝裂原活化蛋白激酶(MAPK)信号通路密切相关,特别是cAMP反应元件结合蛋白(CREB)和核糖体S6激酶1(RSK1)的表达改变。Western印迹结果进一步证实了这些发现。
结论:我们的研究成功分离出高质量的MOEVLP,并证明MOEVLP可以通过MAPK通路促进成骨细胞增殖来缓解PMOP。MOEVLPs有潜力成为一种新型的天然抗PMOP药物。
BACKGROUND: Postmenopausal osteoporosis (PMOP) is a systemic bone disease characterized by low bone mass and microstructural damage. Morinda Officinalis (MO) contains various components with anti-PMOP activities. Morinda Officinalis-derived extracellular vesicle-like particles (MOEVLPs) are new active components isolated from MO, and no relevant studies have investigated their anti-osteoporosis effect and mechanism.
OBJECTIVE: To investigate the alleviating effect of MOEVLPs on PMOP and the underlying mechanism.
METHODS: Differential centrifugation and ultracentrifugation were used to isolate MOEVLPs from MO. Transmission electron microscopy (TEM), flow nano analyzer, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), agarose gel electrophoresis, and thin-layer chromatography were employed to characterize MOEVLPs. PMOP mouse models were utilized to examine the anti-PMOP effect of MOEVLPs. H&E and immunohistochemical staining were used for drug safety and osteogenic effect assessment. Mouse embryo osteoblast precursor cells (MC3T3-E1) were used in vitro experiments. CCK-8 kit, alizarin red staining, proteomic, bioinformatic analyses, and western blot were used to explore the mechanism of MOEVLPs.
RESULTS: In this study, MOEVLPs from MO were successfully isolated and characterized. Animal experiments demonstrated that MOEVLPs exhibited specific femur targeting, were non-toxic to the heart, liver, spleen, lung, kidney, and aorta, and possessed anti-PMOP properties. The ability of MOEVLPs to strengthen bone formation was better than that of alendronate. In vitro experiments, results revealed that MOEVLPs did not significantly enhance osteogenic differentiation in MC3T3-E1 cells. Instead, MOEVLPs promoted the proliferation of MC3T3-E1 cells. Proteomic and bioinformatic analyses suggested that the proliferative effect of MOEVLPs was closely associated with the mitogen-activated protein kinase (MAPK) signaling pathway, particularly the altered expression of cAMP response element-binding protein (CREB) and ribosomal S6 kinase 1 (RSK1). Western blot results further confirmed these findings.
CONCLUSIONS: Our studies successfully isolated high-quality MOEVLPs and demonstrated that MOEVLPs can alleviate PMOP by promoting osteoblast proliferation through the MAPK pathway. MOEVLPs have the potential to become a novel and natural anti-PMOP drug.