未经授权:糖皮质激素(GC)是常用的抗炎药之一,但不幸的是,它的管理伴随着骨质流失。尽管零星研究表明,在GC应激下,骨细胞会发生一系列病理变化,包括组织蛋白酶K的过度表达,骨细胞在GC诱导的骨丢失中的确切作用仍不清楚。
UNASSIGNED:在暴露于不同时间(0、12、24小时)和剂量的地塞米松(Dex)的MLO-Y4细胞系中,通过RT-qPCR和Western印迹评估了Ctsk的基因表达和组织蛋白酶K的蛋白质水平(0、10-8和10-6M)疗程,分别。然后进行共聚焦成像和免疫染色,以评估骨细胞来源的组织蛋白酶K对原代骨细胞离体培养系统中I型胶原蛋白的影响。使用MitoTrackerRed对线粒体进行染色以进行线粒体形态评估,并采用JC-1测定来评估Dex处理后MLO-Y4细胞中的线粒体膜电位。通过PINK1蛋白的免疫染色和CytoID测定评估PINK1介导的线粒体自噬的激活。Mdivi-1用于抑制线粒体自噬,siRNA用于抑制Pink1和Atg5。
UNASSIGNED:GC触发骨细胞产生过量的组织蛋白酶K,进而导致原代骨细胞离体培养系统中胞外基质中I型胶原蛋白的降解。同时,GC给药增加了骨细胞的线粒体分裂和膜去极化。Further,PINK1介导的线粒体自噬的激活被证明是骨细胞中功能失调的线粒体减少的原因.对线粒体自噬与组织蛋白酶K产生之间关系的检查表明,通过敲除Pink1基因来抑制线粒体自噬可以消除GC触发的组织蛋白酶K的产生。有趣的是,发现GC通过线粒体自噬对组织蛋白酶K的激活作用独立于经典自噬,因为当通过Atg5抑制抑制经典自噬时,这种作用不会受到阻碍。
UNASSIGNED:GC诱导的PINK1介导的线粒体自噬实质上调节了骨细胞中组织蛋白酶K的产生,这可能是骨细胞在骨丢失过程中促进细胞外基质降解的潜在机制。
UNASSIGNED:当前研究的结果表明,骨细胞有丝分裂在GC诱导的骨丢失中可能发挥作用,这提供了一种通过靶向PINK1介导的骨细胞有丝分裂来缓解GC诱导的骨质疏松症的潜在治疗方法。
UNASSIGNED: Glucocorticoid (GC) is one of frequently used anti-inflammatory agents, but its administration is unfortunately accompanied with bone loss. Although sporadic studies indicated that osteocytes are subject to a series of pathological changes under GC stress, including overexpression of cathepsin K, the definite role of osteocytes in GC-induced bone loss remains largely unclear.
UNASSIGNED: Gene expression of Ctsk and protein levels of cathepsin K were assessed in MLO-Y4 cell lines exposed to dexamethasone (Dex) of different time (0, 12, 24 hours) and dose (0, 10-8 and 10-6 M) courses by RT-qPCR and western blotting, respectively. Confocal imaging and immunostaining were then performed to evaluate the effects of osteocyte-derived cathepsin K on type I collagen in a primary osteocyte ex vivo culture system. MitoTracker Red was used to stain mitochondria for mitochondria morphology assessment and JC-1 assay was employed to evaluate the mitochondria membrane potential in MLO-Y4 cells following Dex treatment. Activation of PINK1-mediated mitophagy was evaluated by immunostaining of the PINK1 protein and CytoID assay. Mdivi-1 was used to inhibit mitophagy and siRNAs were used for the inhibition of Pink1 and Atg5.
UNASSIGNED: GC triggered osteocytes to produce excessive cathepsin K which in turn led to the degradation of type I collagen in the extracellular matrix in a primary osteocyte ex vivo culture system. Meanwhile, GC administration increased mitochondrial fission and membrane depolarization in osteocytes. Further, the activation of PINK1-mediated mitophagy was demonstrated to be responsible for the diminishment of dysfunctional mitochondria in osteocytes. Examination of relationship between mitophagy and cathepsin K production revealed that inhibition of mitophagy via knocking down Pink1 gene abolished the GC-triggered cathepsin K production. Interestingly, GC\'s activation effect towards cathepsin K via mitophagy was found to be independent on the canonical autophagy as this effect was not impeded when inhibiting the canonical autophagy via Atg5 suppression.
UNASSIGNED: GC-induced PINK1-mediated mitophagy substantially modulates the production of cathepsin K in osteocytes, which could be an underlying mechanism by which osteocytes contribute to the extracellular matrix degradation during bone loss.
UNASSIGNED: Findings of the current study indicate a possible role of osteocyte mitophagy in GC-induced bone loss, which provides a potential therapeutic approach to alleviate GC-induced osteoporosis by targeting PINK1-mediated osteocytic mitophagy.