Effect Of Bx-912 On Osteoclast-related Osteoporosis

Experimental background & objective: Osteoporosis is a systemic metabolic disease associated with aging,especially in the elderly and postmenopausal women.The fragility fracture caused by osteoporosis not only brings huge economic and psychological burden to patients and families,but also brings many unstable factors to society.There are still many shortcomings in the current clinical treatment of osteoporosis.Therefore,it is very important to further study the pathogenesis of osteoporosis,find new targets,and improve the understanding of the pathogenesis of osteoporosis have very important economic and social significance.At present,the research on PDK1(3-phosphoinositoldependent protein kinase 1)at home and abroad mainly focuses on related medical fields such as tumor,energy metabolism,cell growth and metastasis,and has not been systematically studied in the field of orthopedics.In this study,we will explore BX-912(PDK1 inhibitor)against M-CSF(macrophage colonystimulating factor)and RANKL(nuclear factor-κB receptor activating factor)from both cellular and animal experiments.Ligand)co-induced osteoclastogenesis and effects on LPS(lipopolysaccharide)-induced mouse osteolysis model and OVX(ovarian-induced osteoporosis mice)-induced osteoporosis model,To explore the role of PDK1 in inhibiting osteoclast differentiation in vitro and the role of osteoporosis in vivo,to find new signaling pathways and potential therapeutic targets for osteoclast differentiation,and to provide new treatment for osteoclast-related osteoporosis Treatment ideas.Experimental methods:(1)C57BL/6 mouse bone marrow cells were extracted and induced to differentiate into osteoclasts by M-CSF+RANKL.After 5 days,osteoclast resistance to tartrate acid phosphatase staining(TRAP staining)was performed to observe the number of osteoclasts.(2)BMMs were treated with different concentrations of BX-912 for 5 days,and the effect of BX-912 on the growth activity of BMMs was detected by Cell Counting Kit-8(CCK-8).BX-912 at a concentration of 0.312 μM was used to differentiate osteoclasts.After 5 days,TRAP staining was performed to observe the number and morphology of osteoclasts,thus verifying the effect of BX-912 on osteoclastogenesis in vitro.(3)Construction of LPS(lipopolysaccharide)induced mouse skull osteolysis model.Male mice were randomly divided into 3 groups: control group,LPS group and LPS+BX-912 group.Under the condition of shallow anesthesia in each group,the mice were injected with the drug under the sagittal suture of the head of the mouse.After 28 days,the mice were sacrificed by cervical vertebrae.The skull was taken and stored with 4% PFA(paraformaldehyde)at 4 ° C for 24 hours.The bone parameters of each group of mouse skull specimens were analyzed by micro-CT three-dimensional reconstruction scan.The skulls of each group were decalcified,embedded,and sectioned,and subjected to H&E staining(hematoxylin-eosin staining)and TRAP staining to observe the changes of bone morphology and the number of osteoclasts in bone tissue,and perform statistical analysis.(4)Construction of OVX(Ovariectomy)induced mouse osteoporosis model.Female mice were randomly divided into three groups: control group,OVX group,OVX+BX-912 group.The mice were removed under bilateral anesthesia.The control group only had skin incision,and the ovaries were removed and placed back into the abdominal cavity.After 7 days,OVX+BX-912 group was intraperitoneally injected with BX-912 every other day.The control group and OVX group were injected with PBS every other day.All mice were sacrificed 28 days later.After lumbar vertebrae,4% paraformaldehyde was obtained at 4 °C,fixed for 24 hours.The lumbar spine specimens were subjected to micro-CT threedimensional reconstruction scanning to quantitatively analyze bone parameters.The lumbar spine specimens were decalcified,embedded,and sectioned,and subjected to H&E staining and TRAP staining respectively to observe the morphology of bone tissue and the number of osteoclasts in bone tissue,and statistical analysis was performed.Experimental results:(1)Mouse bone marrow whole cells were extracted,M-CSF culture medium was induced to differentiate into BMMs,BMMs were irregular,long spindle-shaped,adherent growth,induced by M-CSF+RANKL culture medium for 5 days.The cells gradually fuse to form a plurality of larger osteoclasts,which contain 3-20 closely packed nuclei.(2)The results of CCK-8 showed that when the concentration of BX-912 was 0.312μM or less,there was no significant effect on the growth activity of BMMs,and the OD value was not statistically different(P>0.05).The results of TRAP staining showed that BX-912 significantly inhibited osteoclastogenesis,and the difference was statistically significant(P<0.05).(3)Micro-CT three-dimensional reconstruction scan showed that the BX-912 treatment group can significantly increase the bone volume fraction,increase the number of trabecular bone,and reduce the trabecular bone separation.The difference was statistically significant(P<0.05).H&E staining showed BX-912 treatment can improve the bone microstructure of the skull,relieve the damage of the trabecular bone,and increase the bone density.TRAP staining shows that BX-912 can significantly inhibit the formation of osteoclasts in the skull and reduce the damage of bone structure.The significance of the study(P<0.05)indicates that BX-912 can improve LPS-induced osteolysis symptoms in mice.(4)Micro-CT three-dimensional reconstruction scan analysis showed that BX-912 treatment group can significantly increase bone volume fraction,increase trabecular bone number,reduce trabecular bone separation,and increase trabecular bone thickness difference(P <0.05),H&E staining showed that BX-912 treatment can improve lumbar vertebrae micro-structure,relieve trabecular bone destruction and increase bone density.TRAP staining showed that BX-912 can significantly inhibit lumbar intravertebral osteoclastogenesis and reduce the difference in bone structure destruction was statistically significant(P < 0.05),indicating that BX-912 can improve the symptoms of OVX-induced osteoporosis in mice.Experimental conclusion:(1)The two cytokines,M-CSF and RANKL,are involved in the process of osteoclast differentiation and maturation.(2)BX-912 at a concentration of 0.312 μM inhibited M-CSF and RANKL-induced osteoclast differentiation and maturation in vitro,suggesting that PDK1 plays an important role in the process of osteoclast differentiation and maturation.(3)BX-912 can inhibit LPS-induced osteolysis of mouse skull,indicating that BX-912 has potential for the treatment of osteoporosis.BX-912 can improve lumbar vertebrae bone loss in OVX mice,with potential anti-bone the role of osteoporosis.