The Study Of Peimine On Improving Osteoporosis Via MAPK Pathway Downregulation In Osteoclast

Purpose : Osteoporosis is a systemic disease characterized by the decrease of bone mineral density and the degradation of bone microstructure,which will lead to the increase of bone fragility and the incidence of fracture.With the trend of aging population,the incidence rate of osteoporosis is increasing year by year.At present,drugs widely used in clinic,such as bisphosphonates,have curative effects,but they are also accompanied by certain side effects.Studies have shown that peimine is a compound with anti-inflammatory,analgesic,antioxidant and stress effects,which can play a wide range of biological roles in human body.It is reported that peimine can inhibit MAPK signaling pathway in osteoarthritis model.More and more studies have shown that MAPK signaling pathway plays an important role in the pathogenesis of osteoporosis.We explored the anti osteoporosis effect of peimine by treating RANKL induced osteoclast differentiation with peimine in vitro,and further clarified the effect and related mechanism of peimine on osteoclasts.Methods:（1）Effect of peimine on osteoclasts: CCK-8 method was used to detect the cytotoxicity of peimine with different concentration gradients.（2）experimental design of peimine on MAPK signal pathway during osteoclast differentiation: it can be divided into two groups: 1)Western blot experimental group,pretreated with peimine with different concentration gradients and added with RANKL with different time gradients,The expression of downstream proteins was detected.2)trap staining group was treated with peimine with different concentration gradients under the induction of the same concentration of RANKL,and the difference of differentiation was observed.（3）Animal experiment: ovariectomized（OVX）female mice were used to simulate postmenopausal osteoporosis,and peimine or blank control intervention was given to compare the changes of bone mass between pimine treated group and non treated group,so as to determine whether peimine has an effect on osteoporosis.Results:（1）Peimine can inhibit RANKL induced osteoclast differentiation in vitro:BMM cells were treated with 0.5% when the culture medium contained MCSF and RANKL 0μmol/L、5μmol/L、10μmol/L、20μmol/L The results of trap staining showed that with the increase of peimine concentration,the differentiation of BMM into multinucleated osteoclasts decreased significantly.（2）Peimine can inhibit the expression of MAPK signaling pathway during osteoclast differentiation: BMM cells were pretreated with peimine（20μmol/L）for 24 hours.After that,the cells were divided into blank group and peimine group,with five wells in each group.And they were treated with RANKL for 0,5,15,30 and 60 minutes respectively.Compared with the blank control group,the phosphorylation levels of JNK,Akt,p38 and p65 proteins decreased significantly after adding peimine.according to the results of Western-blot experiment.Conclusion: Peimine can inhibit osteoclast differentiation and bone resorption by inhibiting MAPK signal pathway,so as to achieve the effect of anti osteoporosis.And peimine has the potential as a new therapeutic agent against osteoporosis.