A Study On The Mechanism Of GPF Alleviating Osteoporosis In Ovariectomized Mice By Regulating ROS Activity

Background: Physiologically,the maintenance of bone homeostasis remains in a dynamic balance,in which osteoblasts and osteoclasts play a vital role.This homeostasis balance gets disrupted as a result of diseases such as hyperparhyroidism or Cushing’s disease;as part of natural processes such as menopause or aging;or as a side effect of medications such as corticosteroids.The osteoclast-induced bone resorption outperforms the osteoblast-mediated bone generation,leading to skeletal disorders,such as osteoporosis.Osteoporosis,a common disease affecting a huge population all over the world,is characterized by reduced bone mass,destruction of bone microstructure,and increased susceptibility to fragility fracture;it has caused severe morbidity or even mortality to patients,as well as numerous medical and economic burden to our aging population in society.Emerging evidence suggests bright prospects of some natural antioxidants in the treatment of osteoporosis.6′-O-Galloylpaeoniflorin(GPF),an antioxidant isolated from peony roots(one of very widely used Oriental medicines,with various anti-inflammatory,antitumor,and antioxidant activities),shows a series of potential clinical applications.However,its effects on osteoporosis remain poorly investigated.The current study aimed to explore whether GPF can attenuate osteoclastogenesis and relieve ovariectomy-induced osteoporosis via attenuating reactive oxygen species(ROS),and investigate the underlying mechanisms.Methods: After the culture of primary murine bone marrow-derived macrophages/monocytes were induced by the use of macrophage colony-stimulating factor(M-CSF)and the receptor activator of NF-κB ligand(RANKL)and then treated with GPF.Cell Counting Kit-8(CCK-8)assay was performed to evaluate cell proliferation and viability.Thereafter,tartrate-resistant acidic phosphatase(TRAP)staining,podosome belt formation,and resorption pit assay were carried out to assess the role of GPF in the production of osteoclasts and the osteogenic resorption of mature osteoclasts.Western blotting and q RT-PCR examination were performed to evaluate proteins’ generation and osteoclast-specific gene levels,respectively.The ROS generation in cells was measured in vitro by 2’,7’-Dichlorodi-hydrofluorescein diacetate(DCFH-DA).Ovariectomyinduced osteoporosis mouse administered with GPF or vehicle was performed to explore the in vivo potential of GPF,then a micro-CT scan was performed in combination with histological examination for further analysis.Results:(ⅰ)GPF suppresses the RANKL-mediated formation of osteoclasts depending on its dose,especially at the early stage,with no significant cytotoxicity;(ⅱ)GPF attenuates bone-resorptive function of mature osteoclasts in vitro;(ⅲ)GPF suppresses osteoclast-specific gene expression;(ⅳ)GPF markedly inhibits the activation of ERK,JNK,c-Fos,and NFATc1;(ⅴ)GPF scavenges RANKL-mediated intracellular ROS generation;and(ⅵ)GPF relieves ovariectomy-induced osteoporosis.Conclusion: GPF suppressed the formation of osteoclasts and podosome belts,as well as bone resorption when induced by RANKL through affecting intracellular ROS activity,MAPKs signaling pathway,and subsequent NFATc1 translocation and expression,as well as osteoclast-specific gene expression in vitro.In vivo study suggested that exposure to GPF prevented osteoporosis-related bone loss in the ovariectomized mice.These findings indicate that GPF attenuates osteoclastogenesis and relieves ovariectomy-induced osteoporosis by inhibiting ROS and MAPKs/c-Fos/NFATc1 signaling pathway.This suggested that GPF may be potentially used to treat bone diseases like periodontitis,rheumatoid arthritis,and osteoporosis associated with osteoclasts.