The FoxO1-Mediated Inhibitory Effects Of Resveratrol And Puerarin On Osteoclastogenesis And Osteoclastic Activity

Oxidative stress is an independent risk factor for osteoporosis.The redox regulatory factor FoxO1 plays a fundamental regulatory role in the homeostasis of bone by up-regulating the expression levels of antioxidant enzymes such as catalase,superoxide dismutase（SOD）,glutathione peroxidase（GSH-PX）and so on.Resveratrol and puerarin,as natural antioxidants,can prevent bone loss by means of their antioxidant effects.Therefore,it is of important clinical significance to elucidate the role and molecular mechanisms of resveratrol and puerarin in anti-osteoporotic oxidative stress injury.Our research has showed that resveratrol regulates the expression of FoxO1 in white adipose tissue to ameliorate overweight status caused by high fat diet by inhibiting the PI3K/AKT pathway.Osteoclasts are the only kind of cells that mediate bone resorption in vivo,and their activity is the core of bone mass regulation.However,the effects and related molecular mechanisms of resveratrol and puerarin on the differentiation,activity,and bone resorption of osteoclasts by regulating the FoxO1 signaling pathway are still not fully clear.AimThis work explores the effects of resveratrol and puerarin on osteoclasts differentiation,activity and bone resorption by regulating PI3K/AKT/FoxO1 signaling pathway,and resveratrol regulating Sirt1/FoxO1 signaling pathway,and also provides a novel scientific and experimental basis for the resveratrol and puerarin clinical application in the prevention and/or treatment of osteoporosis.Methods1)A osteoporosis rat model was established by ovariectomy,and then drug intervention was performed in order to observe the effects of resveratrol and puerarin on osteoclastogenesis and its activity in vivo.Fifty-two healthy three-month-old female SD rats were divided into four groups randomly,including sham-operated group（Sham）,OVX,resveratrol（the dosage being 40mg/kg/d,RES）and puerarin（the dosage being 100mg/kg/d,PUE）.Except the Sham group,they were used to establish the OVX rat model of osteoporosis.Postoperative one week,all the rats were given medicine or vehicle in subcutaneous injection,lasting ten weeks.The following indexes were measured including boneμ-CT scanning,bone mineral density（BMD）by double energy X-ray absorptiometry（DEXA）,bone tissue tartrate-resistant acid phosphatase（TRAP）staining,bone histomorphometric analysis using Van Gieson staining,and serum osteocalcin（OC）,osteoprotegerin（OPG）,cross-linked C-telopeptides of type I collagen（CTX-I）,TRAP-5b,receptor activator of nuclear factor kappa B ligand（RANKL）,reactive oxygen species（ROS）and urinary hydroxyproline（HOP）detected employing enzyme-linked immunosorbent assay（ELISA）,and serum malondialdehyde（MDA）,SOD,and GSH-PX assayed by kits.2)RAW264.7 cells were induced differentiation to osteoclasts by 100ng/mL RANKL for 7d,in order to explore the molecular mechanisms of resveratrol and puerarin inhibiting bone resorption of osteoclasts.The cultured RAW264.7 cells were divided into ten groups:the Control group,100ng/mL RANKL（RANKL）group,10^-4M H₂O₂（H₂O₂）group,10^-5M RES（RES）group,10^-5M PUE（PUE）group,10^-4M H₂O₂+10^-5M RES（H₂O₂+RES）group,10^-4M H₂O₂+10^-5M PUE（H₂O₂+PUE）group,10^-5M LY294002（LY）group,10^-5M EX-527（EX-527）group,10^-5M RES+10^-5M EX-527（RES+EX-527）group.The following indexes were detected including cytotoxicity assay within 24,48,72h by CCK-8,osteoclasts marker enzymes（matrix metallopeptidase 9（MMP-9）,TRAP,and cathepsin K）mRNA expression levels by qRT-PCR,ROS by flow cytometry,the contents of MDA,SOD and GSH-PX detected by kits,and the protein expression levels of PI3K,AKT,p-AKT,FoxO1,p-FoxO1,Bim,caspase-3,catalase,Sirt1and acetyl-p53 detected by Western Blot,early apoptosis detected using Annexin-V/PI double staining by flow cytometry.Results1)The cancellous bone and cortical bone BMD measured byμ-CT and DEXA in OVX group was lower than that in Sham group（P<0.05,P<0.01）.Compared with the Sham group,the number of osteoclasts in OVX group is increased（P<0.01）,and the levels of OC and OPG in serum are decreased（P<0.01）,while the levels of CTX-I,TRAP-5b,RANKL and HOP are enhanced（P<0.01）,and OPG/RANKL is significantly decreased in response to OVX（P<0.01）.When rats were under the OVX operation,the MDA and ROS concentrations are increased,while the contents of SOD and GSH-PX are reduced,as compared with the Sham group（P<0.05,P<0.01）.Feeding the resveratrol or puerarin to the OVX rats,the changes induced by OVX are reversed.Compared with the OVX group respectively,the cancellous bone and cortical bone BMD of RES and PUE group is increased（P<0.05,P<0.01）,the number of osteoclasts in the bone tissue of RES and PUE group was decreased（P<0.05,P<0.01）,the contents of OC and OPG are elevated（P<0.05,P<0.01）,yet the CTX-I and TRAP-5b are reduced（P<0.05,P<0.01）.Moreover,the resveratrol and puerarin effectively decreased the level of RANKL and boost the ratio of OPG/RANKL（P<0.05,P<0.01）.Meanwhile,treating the OVX rats with resveratrol or puerarin,changes are also observed when compared with the OVX group;that is,MDA and ROS levels were highly decreased while the anti-oxidant enzymes such as SOD and GSH-PX are markedly elevated（P<0.05,P<0.01）.2)In vitro,RAW264.7 cells are induced by 100ng/mL RANKL differentiation to osteoclasts.（1）Compared with the RANKL group,10^-4M H₂O₂ induces oxidative stress,promotes osteoclasts formation（P<0.01）,and increases the mRNA expression levels of osteoclasts marker enzymes including MMP-9,TRAP and cathepsin K（P<0.01）.However,when the cells are treated with resveratrol or puerarin at the density of 10^-5M,the MDA and ROS production is inhibited to some degree,yet the SOD and GSH-PX levels are higher compared with the H₂O₂ group（P<0.05,P<0.01）.Furthermore,the number of TRAP-staining positive multinucleated osteoclasts and the mRNA expression levels of osteoclasts marker enzymes are lower（P<0.01）.（2）Compared with the Control group,in RANKL group,the ROS production of RAW264.7cells increases（P<0.01）,and the protein expression levels of p-AKT and of p-FoxO1 increases,while FoxO1 decreases.Yet,H₂O₂ enhances the above effects of RANKL.（3）Compared with RANKL group,the number of osteoclasts differentiated from RAW264.7cells is significantly decreased via the treatments of resveratrol,puerarin and LY294002respectively（P<0.01）.The same effects are performed on the mRNA expressions of MMP-9,TRAP and cathepsin K（P<0.01）.Moreover,the levels of ROS in RES,PUE,and LY groups are all novelly decreased（P<0.01）.Additionally,the protein expressions of p-AKT and p-FoxO1 are decreased.FoxO1,Catalase,Bim and caspase-3 however have inverse changes.Furthermore,they have been shown to induce apoptosis of RAW264.7 cells according to the analysis of Annexin-V/PI double staining by flow cytometry（P<0.01）.（4）The results of Western Blot shows that,resveratrol up-regulates the Sirt1 expression and down-regulates the acetylated-p53 expression,and the protein levels of FoxO1 and its downstream targets,Catalase and Bim,in RES+EX-527 group are lower than the RES group.After the treatments of resveratrol combined with EX-527,the ROS level,the number of TRAP-staining positive multinucleated osteoclasts and the mRNA expression levels of osteoclasts marker enzymes is much higher than the RES group（P<0.05）,yet the apoptosis level is significantly lower than the RES group（P<0.01）.Conclusion1)Resveratrol and puerarin due to their antioxidant activity,reduce the abnormal increase of RANKL caused by OVX,up-regulate the expression of OPG,and then suppress the formation,activity and bone resorption of osteoclasts,and prevent the destruction of bone microstructure.2)By activating AKT,RANKL inhibits the transcriptional activity of FoxO1,and hence promotes the osteoclasts formation and enhances the osteoclasts activity.10^-4M H₂O₂ enhances the effect of RANKL induced osteoclast formation.3)By suppressing the PI3K/AKT signaling pathway,resveratrol and puerarin up-regulate the protein expression level and transcriptional activity of FoxO1,then suppress the RAW264.7 cells differentiation into osteoclasts induced by RANKL,and finally induce the apoptosis of RAW264.7cells.The redox regulator FoxO1 is a pivotal target of the resveratrol and puerarin to play a role of inhibiting bone resorption.4)By activating Sirt1,resveratrol is able to up-regulate the protein expression level and transcriptional activity of FoxO1,and thus it suppresses the osteoclasts formation,decreases the osteoclasts activity,and increases the osteoclasts apoptosis.