Pristimerin Inhibits Osteoclast Differentiation And Function In Vitro And In Vivo

Objective 1.To investigate the effect of Pristimerin on osteoclast differentiation and function in vitro and to elucidate its mechanism.2.To study the effect of Pristimerin on bone loss in ovariectomized osteoporosis model mice and the changes of bone tissue and serum inflammatory factors in mice after intraperitoneal injection of pristimerin.Methods 1.In vitro experiments: BMMCs of mice were maintained in ?-MEM medium and treated with different doses of Pristimerin(0n M,5n M,10 n M,25 n M,50 n M,75 n M)at 1,3,5days,CKK8 kit was used to detect the effect of Pristimerin on the proliferation of BMMCs.In the process of osteoclast differentiation induced by RANKL and M-CSF,BMMCs were given different concentrations of Pristimerin for intervention,and TRAP staining and Actin ring cytoskeleton staining were performed 5 days later.The effect of Pristimerin on osteoclast function was detected by bone plate absorption assay.Real-time quantitative PCR and Western blot were used to detect the effect of Pristimerin on the expression of genes related to osteoclast activity such as NFATc1 and c-Fos.Finally,Western blot was used to detect the expression of NF-?B and MAPKs pathway proteins after Pristimerin treatment.2.In vivo experiment: osteoporosis model was established by removing both ovaries of C57 mice(7 weeks),and the mice were divided into three groups: sham operation group,ovariectomized group,and ovariectomized +Pristimerin group.Drug administration was started on the 3th day after ovariectomy,and Pristimerin(1mg/kg)or DMSO solution was intraperitoneally injected 6 times a week.After 8 weeks of administration,mice were sacrificed and bilateral femurs were taken.Micro-CT scanning was performed on the left femur to analyze the bone structure,and TRAP staining and H&E staining were performed on the right femur to analyze the bone structure,and the serum levels of inflammatory cytokines were detected by ELISA..Results CKK8 experiment showed that the use of Pristimerin at a concentration of 75 n M had no effect on the proliferation of BMMCs,and the difference was not statistically significant(P > 0.05).TRAP staining results confirmed that Pristimerin can significantly inhibit the formation of osteoclasts,and bone plate absorption experiment and Actin ring staining confirmed that Pristimerin can significantly inhibit the function of osteoclast bone resorption.Q-PCR and Western blot confirmed that Pristimerin significantly inhibited the expression of osteoclast marker genes such as NFATc1,CK,TRAP and MMP-9.Western blot analysis of pathway proteins showed that Pristimerin inhibited the phosphorylation levels of IKK,P65,IKB,P38 and ERK,and blocked the activation of NF-?B and MAPKs pathways.In vivo experiments,Micro-CT scan results and tissue section staining suggested that Pristimerin had a protective effect on bone loss caused by ovariectomy.After the intervention with Pristimerin,the levels of TNF-?,RANKL,IL-1? and IL-6 in serum of mice decreased,while the levels of OPG increased.Conclusions Pristimerin can inhibit osteoclast differentiation and function in vitro and in vivo,and improve bone loss in mice induced by ovariectomy.The mechanism of this action is that Pristimerin blocks the activation of NF-?B and MAPKs pathways.This study provides a new potential drug for the prevention and treatment of osteoporosis in the future.