Pseurotin A Inhibits Osteoclastogenesis and Prevents Ovariectomized-Induced Bone Loss by Suppressing Reactive Oxygen Species

Rationale: Growing evidence indicates that intracellular reactive oxygen species (ROS) accumulation is a critical factor in the development of osteoporosis by triggering osteoclast formation and function.Pseurotin A (Pse) is a secondary metabolite isolated from Aspergillus fumigatus with antioxidant properties, recently shown to exhibit with a wide range of potential therapeutic applications.However, its effects on osteoporosis remain unknown.This study aimed to explore whether Pse, by suppressing ROS levels, is able to inhibit osteoclastogenesis and prevent the bone loss induced by estrogen-deficiency in ovariectomized (OVX) mice. Methods:The effects of Pse on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis and bone resorptive function were examined by tartrate resistant acid phosphatase (TRAcP) staining and hydroxyapatite resorption assay.2',7'dichlorodihydrofluorescein diacetate (H2DCFDA) was used to detect intracellular ROS production in vitro.Western blot assay was used to identify proteins associated with ROS generation and scavenging as well as ROS-mediated signaling cascades including mitogenactivated protein kinases (MAPKs), NF-κB pathways, and nuclear factor of activated T cells 1 (NFATc1) activation.The expression of osteoclast-specific genes was assessed by qPCR.The in vivo potential of Pse was determined using an OVX mouse model administered with Pse or vehicle for 6 weeks.In vivo ROS production was assessed by intravenous injection of dihydroethidium (DHE) into OVX mice 24h prior to killing.After sacrifice, the bone samples were analyzed using μCT and histomorphometry to determine bone volume, osteoclast activity, and ROS level in vivo.Results: Pse was demonstrated to inhibit osteoclastogenesis and bone resorptive function in vitro, as well as the downregulation of osteoclast-specific genes including Acp5 (encoding TRAcP), Ctsk (encoding cathepsin K), and Mmp9 (encoding matrix metalloproteinase 9).Mechanistically, Pse suppressed intracellular ROS level by inhibiting RANKL-induced ROS production and enhancing ROS scavenging enzymes, subsequently suppressing MAPK pathway activation (ERK, P38, and JNK) and NF-κB pathways, as well as inhibiting NFATc1 signaling.μCT and histological results indicated that OVX resulted in significant bone loss, with dramatically increased numbers of osteoclasts on the bone surface as well as increased ROS levels in the bone marrow microenvironment; whereas Pse supplementation was capable of effectively preventing these OVX-induced changes. Conclusion:Pse was demonstrated for the first time as a novel alternative therapy for osteoclastrelated bone disease such as osteoporosis through suppressing ROS levels.