The Basic Research About Rapamycin Improving Osteogenesis And Angiogenesis In High-turnover Osteoporosis With Iron Accumulation

Part I: Effects of iron accumulation on mTOR level in mice and osteoblasts and on bone mass in miceObjective: To study the relationship between iron accumulation and mTOR expression,and to explore the possible role of mTOR on osteoporosis with iron accumulation.Methods: 1.Animal experiment: Mice models with exogenous iron accumulating need to be established.Twenty eight-week-old wild(Wt)male mice(C57/BL6)were randomly divided into two groups,10 mice were treated intraperitoneally with ferric ammonium citrate(Wt+FAC group)while the other 10 were treated with saline(Wt+saline group)as control.Both ferric ammonium citrate and saline with the same dose of 40mg/kg were used for three times a week for 2 months.Mice models with endogenous iron accumulating need to be established too.Ten 8-week-old(?Hep)transgenic female mice and ten 8-week-old wild type(Wt)female mice were ovariectomized(OVX).They were divided into ?Hep+OVX group and Wt+OVX group accordingly.After two-months feeding,the weight of the mice was measured and the serum and bone tissue samples were collected.The ferritin(Fer)and mTOR concentration in Serum were detected by enzyme linked immunosorbent assay(ELISA).The slices of liver and femur were stained with Prussian blue(Perls),the liver iron content was measured by inductively coupled plasma-optical emission spectrometry(ICP-OES).The rest femur specimens were scanned with micro computed tomography(micro-CT)to test the bone mass,bone mineral density(BMD)and then measured by inductively coupled plasma-mass spectrometry(ICP-MS)to test the bone iron content.The expression of mTOR gene and protein in tibia were detected by reverse transcription-polymerase chain reaction(RT-PCR)and western blot respectively.2.Cell experiment: h FOB1.19 cells and RAW264.7 cells in the experimental group were treated with 200?M and 50?M FAC-contained medium for 72 hours(iron group)respectively,while those in the control group were treated with PBS(cont group).The iron ion concentration in the cell culture medium of each group was detected by ELISA.The expression of mTOR gene and protein were detected by RT-PCR and western blot respectively.Results: 1.In vivo study: There was no significant difference in body weight of mice between two groups(p>0.05).After intervention with exogenous FAC,serum ferritin concentration,liver iron content,bone iron content,prussian blue iron deposition in liver and distal femur increased significantly in Wt+FAC group by comparing with Wt+Saline group(p<0.05),and serum mTOR concentration,mTOR gene expression and protein expression in liver and tibia also increased significantly(p<0.05).After the knocking-out of endogenous hepcidin,serum ferritin concentration,liver iron content,bone iron content,prussian blue iron deposition in liver and distal femur increased significantly in ?Hep+OVX group by comparing with Wt+OVX group(p<0.05),and serum mTOR concentration,mTOR gene expression and protein expression in liver and tibia also increased significantly(p<0.05).Bone mass and BMD decreased significantly in ?Hep+OVX group by comparing with Wt+OVX group(p<0.05).2.In vitro study: After the intervention of exogenous FAC,the concentration of iron ion in h FOB1.19 cell culture medium increased significantly(p<0.05),and the expression of mTOR gene and protein increased significantly too(p<0.05).The concentration of iron ion in RAW264.7 cell culture medium increased significantly(p<0.05),but there was no significant difference in mTOR gene expression and protein expression between the two groups(p>0.05).Conclusion: The mTOR level increase as iron concentration increase.Iron accumulation may promote bone loss in OVX mice through increased expression of mTOR,which may lead to osteoporosis with iron accumulation.Part ?: The osteogenic role of rapamycin on high-turnover osteoporosis with iron accumulationObjective: To study the osteogenic effect of rapamycin,an inhibitor of mTOR,on high-turnover osteoporosis with iron accumulationMethods: 1.Animal experiment: 16 8-week-old wild(Wt)female mice and 48 8-week-old(?Hep)transgenic female mice were ovariectomized(OVX).They were divided into four groups(Wt+OVX group,?Hep+OVX group,?Hep+OVX+CMC group and ?Hep+OVX+Rapa group).Rapa with a dose of 3 mg/kg/day was injected intraperitoneally for 2 months.CMC was the solvent of Rapa,and the dose and duration of injection were the same as ?Hep+OVX+Rapa group.?Hep+OVX+CMC group was used as sham group.After the last injection,four groups of mice were executed to collect serum and bone tissue samples.Bone mass,bone mineral density(BMD)and other indexes of femur were measured by micro-CT.Bone mass and trabecular structure were measured by scanning electron microscopy(SEM)and H-E staining.Osteoblastic ability of bone formation was detected by alkaline phosphatase(ALP)staining.The femoral bending modulus of elasticity,bending energy,maximum bending stress and bending rigidity coefficient were measured by biomechanical experiments.The expression of serum osteogenic-indexes(ALP,PINP,Ocn)were detected by ELISA,and the expression of osteogenic genes(Runx2,SP7,ALP)in bone were detected by RT-PCR.2.Cell experiment: h FOB1.19 cells were divided into four groups(cont group,iron group,iron + si-NC group and iron + si-MT group).The cont group was defined as control group and iron group were treated with 200?M FAC-contained medium for 72 hours.The iron + si-MT group and iron + si-NC group were treated with mTOR-specific si RNA transfection and blank transfection for osteoblasts respectively,and then treated with 200?M FAC-contained medium for 72 hours.Alkaline phosphatase(ALP)staining was used to assess the osteoblastic activity.Alizarin red staining(ARS)was used to evaluate the osteoblastic ability of bone mineralization.The expression of osteogenic genes(Runx2,SP7,ALP)were detected by RT-PCR.The expression of osteogenic proteins(Runx2,Ocn)were detected by western blot.Results: 1.In vivo study: Micro-CT of femer showed that the BMD,BV/TV,TB.N,TB.Th and Conn D significantly decreased but TB.Sp,SMI significantly increased in ?Hep+OVX group and ?Hep+OVX+CMC group by comparing with Wt+OVX group.The BMD,BV/TV,TB.N,TB.Th and Conn D significantly increased but TB.Sp,SMI significantly decreased in ?Hep+OVX+Rapa group by comparing with ?Hep+OVX group and ?Hep+OVX+CMC group(p<0.05).Compared with Wt+OVX group,femoral scanning electron microscopy and tibial HE staining showed that the bone mass and trabecular bone in ?Hep+OVX group and ?Hep+OVX+CMC group decreased significantly,but they were improved significantly after rapamycin treatment(p<0.05).Tibial ALP staining also showed that osteoblastic ability of bone formation increased significantly when the ?Hep+OVX group received rapamycin treatment(p<0.05).Compared with Wt+OVX group,the femoral bending modulus of elasticity,bending energy,maximum bending stress and bending rigidity coefficient in ?Hep+OVX group and ?Hep+OVX+CMC group decreased significantly,and recovered significantly after rapamycin injection(p<0.05).Compared with ?Wt+OVX group,the expression of serum osteogenic-indexes(ALP,PINP,Ocn)and bony osteogenic-genes(Runx2,SP7,ALP)in ?Hep+OVX group and ?Hep+OVX+CMC group decreased significantly,but increased significantly after rapamycin intervention(p<0.05).2.In vitro study: ALP staining and alizarin red staining showed that osteoblastic activity and osteoblastic ability of bone mineralization decreased significantly in iron group and iron + si-NC group by comparing with cont group.But these indexes of iron + si-MT group improved significantly compared with iron group and iron + si-NC group(p<0.05).The expression of osteogenic genes(Runx2,SP7,ALP)and osteogenic proteins(Runx2,Ocn)decreased significantly in iron group and iron + si-NC group by comparing with cont group.But these indexes of iron + si-MT group recovered significantly compared with iron group and iron + si-NC group(p<0.05).Conclusion: Rapamycin,which has positive effect on osteogenesis,can improve bone mass and osteoblastic activity in high-turnover osteoporosis with iron accumulation.Part ?: The angiogenic role of rapamycin on high-turnover osteoporosis with iron accumulationObjective: To study the angiogenic effect of rapamycin,an inhibitor of mTOR,on high-turnover osteoporosis with iron accumulationMethods: 1.Animal experiment: The samples from the second part of the experiment(Wt+OVX group,?Hep+OVX group,?Hep+OVX+CMC group and ?Hep+OVX+Rapa group)continue to be used.RT-PCR and Western Blot were used to test the expression of Slit3 gene and protein in bone.The spare tibias were were prepared for frozen sections and stained with platelet-endothelial cell adhesion molecule(CD31)antibody and endothelial mucin(EMCN)antibody.The number and distribution of H subtype blood vessels were observed by confocal microscopy.2.Cell experiment: The osteoblasts from the second part of the experiment(cont group,iron group,iron + si-NC group and iron + si-MT group)continue to be used.Four groups of the same human umbilical vein endothelial cells(HUVECs)were cultured with corresponding medium(CM)extracted from treated osteoblasts.The migration ability and tubular formation ability of HUVECs were detected by scratch test and tube-formation test,and the expression of EMCN in HUVEC were detected by immunofluorescence.Results: 1.In vivo study: Compared with Wt+OVX group,Slit3 gene expression,protein expression,H subtype vessel number and distribution decreased significantly in ?Hep+OVX group and ?Hep+OVX+CMC group,and increased significantly after rapamycin treatment(p<0.05),but there was no significant difference between ?Hep+OVX group and ?Hep+OVX+CMC group(p>0.05).2.In vitro study: Compared with HUVECs cultured with CM extracted from cont group,HUVECs had significantly decreased ability of migration and tube-forming,decreased EMCN expression which were cultured with CM extracted from iron group and iron+si-NC group,and above indexes recovered significantly when they cultured with CM extracted from iron+si-MT group(p<0.05).Conclusion: Rapamycin,which has positive effect on angiogenesis,can promote the expression of Slit3 gene and protein,increase the number and distribution of H subtype blood vessels,and increase the HUVEC activity in high-turnover osteoporosis with iron accumulation.Part ?: The mechanism of rapamycin in improving high-turnover osteoporosis with iron accumulationObjective: To study the underlying mechanism of rapamycin in improving high-turnover osteoporosis with iron accumulation.Methods: 1.Cell experiment: The osteoblasts from the second part of the experiment(cont group,iron group,iron + si-NC group and iron + si-MT group)continue to be used.Four groups of the same human umbilical vein endothelial cells(HUVECs)were cultured with corresponding medium(CM)extracted from treated osteoblasts.RT-PCR,Western Blot(or ELISA)were used to detect the expression of mTOR,STAT1,Cxcl9,VEGF,KDR(VEGF receptor),p KDR and other related genes and pathway proteins.Immunofluorescence was used to examine the phosphorylation of KDR in osteoblasts and HUVECs.2.Animal experiment: The samples from the second part of the experiment(Wt+OVX group,?Hep+OVX group,?Hep+OVX+CMC group and ?Hep+OVX+Rapa group)continue to be used.RT-PCR and Western Blot were used to examine the expression of mTOR,STAT1,Cxcl9,VEGF,KDR(VEGF receptor),p KDR and other pathway-related genes and proteins in bone.Results: 1.In vitro study: After FAC intervention(iron group),the expression of mTOR and its downstream STAT1,Cxcl9,and VEGF genes increased consistently,while the expression of p KDR protein decreased.After mTOR-specific si-RNA transfection(iron + si-MT group),the above indexes recovered(p<0.05).There was no significant difference between iron group and iron + si-NC group(p>0.05).2.In vivo study: Compared with Wt+OVX group,the expression of mTOR and its downstream STAT1,Cxcl9,and VEGF in bone increased,while the expression of p KDR protein decreased.After rapamycin treatment(?Hep+OVX+Rapa group),the above indexes recovered(p<0.05).There was no significant difference between ?Hep + OVX group and ?Hep + OVX + CMC group(p>0.05).Conclusion: Iron accumulation inhibits p KDR through mTOR/STAT1/Cxcl9 pathway,it reduces the effective binding of VEGF with KDR and affects the bone mass recovery of high-turnover osteoporosis.Rapamycin targeting mTOR can improve the bone mass through its positive effects on osteogenesis and angiogenesis.