Study On The Anti-Osteoporosis Effect Of Bortezomib And Its Mechanism

Part? the Protective Effect of Bortezomib on Bone Loss in Ovariectomized Mice and Its Related MechanismObjective:Proteasome inhibitors,such as bortezomib,are often used as antitumor drugs in clinic.Their changes in enzyme activities in mice may also have a considerable regulatory effect on the pathways related to bone metabolism regulation.There is little evidence to support bortezomib to be a anti-osteoporosis drug.In this study,we injected bortezomib into the ovariectomized mice to investigate the protective effect of bortezomib on bone loss in ovariectomized mice.Methods:the experimental group was divided into(1)SHAM group;(2)OVX group;(3)OVX+BZB group(n=7).In the SHAM operation group,only the tissues around the ovaries were excised,and in the other two groups,the ovaries were excised.From the 21st day after operation,OVX group was injected with 2%DMSO solution twice a week,OVX+BZB group was injected with bortezomib dissolved in 2%DMSO solution twice a week.The dose standard of bortezomib is 0.5mg/kg for 4 weeks.Four weeks later,the mice were killed and bilateral femurs were taken out.Five days later,the femurs were fixed with POM.The mice were examined with Micro-CT and analyzed with bone structural parameters according to the total number of groups.After the sections were made,he,trap and immunohistochemical staining were performed to detect the number of osteoclasts and the expression of Smurfl and Smurf2.Results:in vivo,we can observe that the bone loss of ovariectomized mice is more obvious than that of sham operated mice.Intraperitoneal injection of bortezomib can effectively reduce the bone loss of mice after ovariectomy.The results showed that the bone structure parameters,the number of trabeculae in epiphyseal end of femur in he stained mice and the number of trabeculae in trap stained epiphyseal end of femur were reversed by bortezomib.The expression of Smurfl and Smurf2 protein in OVX+BZB group was lower than that in OVX group.Conclusion:bortezomib can reduce bone loss after ovariectomy and inhibit osteoclast formation in mice.The part ? the Effect of Bortezomib on Osteoblasts Differentiation in Vitro and Its Related MechanismObjective:in order to study the effect of bortezomib on the differentiation of osteoblasts and its possible molecular mechanism,we need more research on osteogenesis in vitro.Methods:MC3T3-E1 cells were used in osteoblast related experiments,and the experimental groups were divided into(1)control group,(2)osteogenic group,(3)osteogenic+ bortezomib intervention group.After induction,osteoblast differentiation was determined by osteogenic related staining and quantitative methods such as alkaline phosphatase staining and activity measurement.RT-PCR and Western blot were used to detect the expression of differentiation related genes,Runx2,CoL-I,Smurfl,2 and Smad 1/5/8.Results:in vitro,bortezomib could promote the differentiation of MC3T3-E1 cells into osteoblasts.The results showed that ALP staining and ALP activity in the drug intervention group were more than that in the simple osteogenic induction group,and the number of calcium nodules stained by Alizarin Red S was significantly more than that in the osteogenic induction group.RT-PCR showed that the transcription levels of CoL-I,ALP and Runx2 in the drug intervention group were higher than those in the bone induction group,and the transcription levels of Smurf1 and Smurf2 were lower.Western blot showed that the expression of Smurf1 and Smurf2 in the drug intervention group was lower than that in the induction group,and the expression of Runx2.Smad1/5/8,OPG protein was increased.Conclusion:Bortezomib may activate BMP/Smads signal pathway by inhibiting Smurfl and Smurf2.It can also promote the expression of osteogenic related genes and proteins.The part ? the Effect Of Bortezomib on Osteoclast Differentiation in Vitro and Its Related MechanismObjective:more studies on osteoclast in vitro are needed to confirm the effect of bortezomib on osteoclast differentiation and its possible molecular mechanism.Methods:BMMCs were extracted from the bone marrow of C57BL/6 mice.The experimental groups were(1)control group,(2)RANKL group,(3)RANKL+Bzb.M-CSF was added to each group,and determined by tartrate resistant acid phosphatase(TRAP)staining To determine the number of osteoclasts,F-actin ring staining was used to observe whether bortezomib could inhibit the formation of osteoclast actin ring.The same number of BMMCs cells were inoculated into the bone plate of hydroxyapatite and treated in groups according to the pre experiment.The differences in the area and number of absorption pores were observed to determine whether the differentiation of osteoclasts was inhibited.Results:in the aspect of osteoclast differentiation,the experimental results showed that bortezomib could inhibit mononuclear macrophages in bone marrow,and the number of osteoclasts differentiated from BMMCs was decreased in the RANKL+BZB group compared with the RANKL group in trap staining,and the F-actin ring formation experiment confirmed that bortezomib could inhibit the formation of actin ring in osteoclasts.The absorption area of osteoclasts on hydroxyapatite bone plate in RANKL+BZB group was much lower than that in RANKL group.Western blot showed that the expression of trap,NFATc1,MMP-9,catheosink(ctsk)protein in the drug intervention group was lower than that in the induction group.Conclusion:Bortezomib can inhibit the differentiation of BMMCs into osteoclasts,down regulate the expression of osteoclast related genes and proteins,and inhibit the formation of osteoclasts.