Vinpocetine Inhibits RANKL-induced Osteoclastogenesis And Attenuates Ovariectomy-induced Bone Loss

Objective1.To explore the influence of Vinp on osteoclast formation and function in vitro and further analyze relevant molecular mechanism.2.To investigate the variation of bone tissue and related serum indexes in mice with ovariectomy-induced osteoporosis after Vinp intervention.Methods1.CCK8 assay was used to test the effects of different concentrations of Vinp (40μM,20μM,10μM,5μM,2.5μM,0μM)on the proliferation of BMMCs.The effect of Vinp on RANKL(100 ng/ml)induced osteoclast formation was detected by TRAP staining.Bone resorption assay,actin-ring formation assay, real-time reverse transcription-PCR(RT-PCR)and Western blot were carried out to determine the effects of Vinp on osteoclast differentiation and function.2.The effects of Vinp on oxidative stress during RANKL-induced osteoclast differentiation were detected by measuring the production of reactive oxygen species(ROS)and the changes of related antioxidant enzymes including HO-1, NQO-1 and Nrf2 by Western blot.Further more,western blot was also used to detect the changes of MAPK signaling(ERK,p-ERK,JNK,p-JNK,P38,p-P38),NF-k(33)signaling(p-IKKa(14)b(11)IKKa(11)IKKb(11)IκBa(11)p-IκBa,P65,p-P65),and AKT signaling(AKT and p-AKT)after Vinp treatment.3.The osteoporosis model of C57BL/6J mice(10 weeks)was established by ovariectomy,which was divided into 4 groups(n=10): Sham group,OVX group,OVX+Vinp(2.5mg/kg)group and OVX+Vinp(10mg/kg)group.Intraperitoneal injection with normal saline or Vinp(2.5 or 10mg/kg)began after one week,at least five times a week.After 2 months,bilateral femurs were taken,followed by micro-CT scanning,H&E staining and TRAP staining of bone sections,and blood was taken from the eyes for ELISA detection.Results1.CCK8 assay confirmed that Vinp had no significant toxicity to BMMCs within the concentration range used.TRAP staining showed that Vinp significantly inhibited the formation of osteoclasts,while the formation of actin-ring and the absorptive area of bone plates decreased after Vinp treatment.2.RT-PCR and Western blot confirmed that the expression of osteoclast marker genes(TRAP,NFATc1,c-Fos,CTSK and MMP-9)was notably inhibited by Vinp.At the same time,Vinp reduced the activation of ERK and JNK in the MAPK pathway,inhibited the activation of the NF-κB pathway(IKK,IκBa,P65),the AKT pathway and the generation of reactive oxygen species,and promoted the expression of Nrf2,HO-1 and NQO-1.3.Micro-CT scan and bone tissue staining showed that Vinp could reduce bone loss in mice induced by OVX.ELISA showed that Vinp could reduce the RANKL/OPG ratio and the level of inflammatory cytokines in serum.ConclusionsVinp can inhibit RANKL-induced osteoclast differentiation and improve OVX-induced osteoporosis by down-regulating NF-κB,MAPK and AKT pathways and inhibiting oxidative stress and related gene expression.This study provides a potential new drug for the treatment of postmenopausal osteoporosis and other osteoclast related diseases,and may expand the clinical application of Vinp.