Study On The Mechanism Of Selective Inhibition Of PI3K/AKT/RANKL Signal Axis In Prevention And Treatment Of Bone Loss In Ovariectomized Mice

Objective:The aim of this study was to explore the effect of PI3K inhibitor BYL-719 on the formation and function of osteoclasts and the corresponding mechanism,and to preliminarily study the therapeutic effect of BYL-719 on osteoporosis in ovariectomized mice.Method:1.Identify the phenomenon: study the effect of BYL-719 on the formation of osteoclasts.Under the induction of cytokine M-CSF(macrophage colony stimulating factor)and RANKL(nuclear factor-κB receptor activating ligand),osteoclast precursor cells BMMs(bone marrow monocytes)differentiate into osteoclasts.In the process,the effect of different concentrations of BYL-719 on the formation of osteoclasts at a concentration without cytotoxicity: a.Use the CCK-8 method to determine the safe concentration of BYL-719;b.Study the effect of different concentrations of BYL-719 on the formation of osteoclasts.2.Confirm Effects: study the effect of BYL-719 on the bone resorption function of osteoclasts.After clarifying the effect of BYL-719 on the formation of osteoclasts,this part of the study will use immunofluorescence staining and scanning electron microscopy to observe the effect of BYL-719 on the fold edge of osteoclasts(F-actin ring)and the formation of osteoclast bone resorption lacuna.3.Clarify the mechanism: study the molecular mechanism of BYL-719’s inhibition of osteoclast formation and bone resorption.This part of the research will use q PCR and Western Blot technology to explore the molecular mechanism of BYL-719 in inhibiting osteoclast formation and bone resorption from two aspects of gene and protein.4.Exploring the application: To study the therapeutic effect of BYL-719 on osteoporosis in ovariectomized mice in vivo.We randomly divided 28 8-week-old female mice into 4 groups,three of which were removed after anesthesia,and the other group only removed the adjacent fat tissue.Four weeks after the removal of the ovaries,the sham-operated group and the control group were injected with saline intraperitoneally,the low-dose group was intraperitoneally injected with 5 mg/kg BYL-719,and the high-dose group was intraperitoneally injected with 10 mg/kg BYL-719.Animals were sacrificed at 4 weeks after different treatments,specimens were obtained,and Micro-CT,H&E staining,TRAP staining and other methods were used to clarify the bone protective effect of BYL-719 on ovariectomized mice.Results:1.Phenomenon: a.BYL-719 is cytotoxic to BMMs at concentrations of 25μM and above;b.BYL-719 inhibits the formation of osteoclasts in a concentrationdependent manner.2.Effects: as the concentration of BYL-719 increases,the function of osteoclasts gradually weakens,manifested by the reduction of actin loops and bone resorption lacunae.3.Mechanism: a.BYL-719 inhibits the expression of osteoclast-specific genes in a concentration-dependent manner.b.BYL-719 inhibits the phosphorylation of P38,ERK and AKT stimulated by RANKL.4.Application: BYL-719 inhibits bone loss in vivo induced by ovariectomized mice.The bone loss of the mice in the simple ovariectomized control group was obvious,and the bone loss was significantly reduced after BYL-719 treatment,and it was more obvious in the high-dose group.Conclusion:BYL-719 by inhibiting the PI3K / AKT,NF-κB and MAPK pathway inhibition of RANKL-induced osteoclastogenesis and bone loss caused by ovariectomy in mice.