The Role And Mechanism Of TGF-β1 Regulating Osteoclast/osteogenic Coupling In Disuse Osteoporosis

Background:Disuse osteoporosis is an important type of secondary osteoporosis,and its high incidence is closely related to the increase in long-term bedridden patients caused by aging.Disuse osteoporosis is characterized by uncoupling between bone resorption by osteoclast and bone formation by osteoblast.However,the key regulatory factors and regulatory mechanisms of the uncoupling remain unclear.TGF-β1 can affect the migration of BMSC to the bone resorption area and plays an important role in bone remodeling.Our previous study found that the level of active TGF-β1 in the bone marrow of mice with disuse osteoporosis was significantly increased,so the applicant proposed a hypothesis: TGF-β1 regulates coupling between bone resorption and formation by regulating BMSC migration,and TGF-β1 might be a potential target for disuse osteoporosis treatment.In this project,tail suspension method is to be used for mice disuse osteoporosis model construction,with which the role of TGF-β1 in regulating bone mass as well as bone resorption and formation coupling in mice is verified.GFP labelled BMSC is to be used for observing BMSC migration in mice with disuse osteoporosis.The activation of TGF-β1 and downstream signaling pathway is to be detected.The above experiments are all designed to clarify the mechanism of TGF-β1 regulated bone resorption and formation coupling in disuse osteoporosis.Objective:1.The disuse osteoporosis model was established in mice and osteoclast-osteogenesis decoupling phenomenon of disuse osteoporosis was determined.2.To prove the role of TGF-β1 in regulating BMSC migration in the occurrence and development of disuse osteoporosis.3.To explore the application value of TGF-β 1 as a target to regulate bone remodeling and treat disuse osteoporosis.Methods:Part I: Establishment of disuse osteoporosis model in mice and study of bone mass change and osteoclast-osteoblast decoupling in vivo.The tail suspension method was used to establish a mouse model of hind limb disuse osteoporosis.The specific method was to use 6-week-old MALE C57BL/6mice,and the tail suspension made the head of the mouse body at a 30 degree Angle downward to avoid the weight bearing of the hind limbs,and ensure that the forelimbs of the mice could obtain enough food and water.Two weeks later,the mice were sacrificed and the distal femur was taken for HE staining and Micro CT scanning to analyze the trabecular morphology,trabecular volume,bone mineral density,trabecular separation,and trabecular thickness.Part II: Study on the migration of BMSC in vivo and the mechanism of TGF-β1 affecting THE migration of BMSC in disuse osteoporosis mice.BMSCs from tail-suspended disuse osteoporosis and healthy mice were isolated and induced by osteoblasts in vitro.Osteoblast colonies were observed by ALP staining and mineralization deposition was observed by alizarin red staining,so as to evaluate the osteogenic differentiation potential of BMSCs in disuse osteoporosis model mice.BMSCS differentiated into osteoblasts from healthy mice were isolated,labeled with GFP(green fluorescent protein)and injected into the femoral bone marrow cavity of mice.Models of tail suspension disuse osteoporosis were made and divided into two groups.One group was intraperitoneally injected with TβR1 inhibitor as the control group.The number and distribution of GFP positive cells were observed by immunohistochemistry.Part III: Application of TβR1 inhibitor in the treatment of disuse osteoporosis.The mice were divided the mice into three groups:hind limb suspension group,and hind limb suspension + inhibitor treatment group and the control group,.The tail suspension mice model of disuse osteoporosis was established.The inhibitor group was injected with TβR1 inhibitor and normal saline every day,and bone loss and osteoclast-osteogenesis coupling were evaluated 2 weeks later.After 2 weeks of tail suspension:The distal femur was taken for HE staining and Micro CT scanning,and the bone mineral density,trabecular morphology,trabecular volume,trabecular separation,and other indicators were analyzed.The distal femur section was taken for TRAP staining to observe the distribution of osteoclasts.The levels of activated TGF-β1 in bone marrow were determined byELISA:Section of distal femur was taken and p-smad 2/3 staining was performed to observe the activation of TGF-β1 signaling pathway in the bone resorption region.Section of distal femur was taken for P-Rho A staining to observe Rho A phosphorylation level.The levels of active TGF-β1 in bone marrow of mice were detected by Elisa.Results:Part I: H&E staining trabecular bone area Tb.Area(mm2)of HLU mouse bone tissue was significantly reduced compared with blank control group(CON),and bone loss increased.TRAP staining showed increased osteoclasts in trabecular bone of femoral epimetaphysis.Bone volume fraction(BV/TV)significantly decreased,bone mineral density(BMD)significantly decreased,however,the trabecular separation(TB.SP)significantly increased,,El ISA showed that serum osteoclast related markers(RANKL,TRACP-5B,CTX-1)increased.The decrease of ALP and Osteocalcin and the increase of TGF-β1 activity in bone marrow proved that the disuse osteoporosis model of hind limb suspension mice was successfully constructed and osteoclast-osteoblast decoupling existed.Part II: The osteogenic differentiation of BMSC in the femur of the disuse osteoporosis model mice was normal.Compared with the disuse osteoporosis+TGF-β1 inhibitor treatment group,the number of GFP-positive labeled cells was significantly reduced,and the migration of BMSC was blocked.Part III: Compared with blank control group(CON)and TGF-β1 inhibitor intervention group(HLU+SB-505124),the number of trabecular bone in HLU mice was decreased and bone loss was increased,and TGF-β1 inhibitor could reduce bone loss of trabecular cancellous bone in disuse osteoporosis mice.In TRAP staining,TGF-β1 inhibitor can significantly reduce the number of osteoclasts around the epiphyseal trabecular bone in posterior limb suspension mice.In hindlimb suspension group,ELISA results showed that,the levels of CTX-1,TRAcp5 B and Osteoclast differentiation factor(RANKL)were significantly increased,while the levels of bone formation and osteoblast markers(ALP and Osteocalcin)were significantly decreased.After TGF-β1 intervention,the above indexes were significantly improved.P-SMAD2/3 and P-Rho A immunofluorescence staining showed that TGF-β1signaling pathway was overactivated in the disuse osteoporosis model,while TGF-β1inhibitor reduced the activation of SMAD2/3 conduction system.Compared with the blank control group and the TGF-β1 inhibitor intervention group,The level of Rho A around the trabecular bone of the distal femur decreased significantly,and no significant decrease was observed in the disused osteoporosis mice treated with TGF-β1 inhibitor compared with the blank control group.Conclusion:1.Osteogenic-osteoclast decoupling was observed in the mouse model of disuse osteoporosis and was associated with TGF-β1 overactivation.2.TGF-β1 plays a certain role in the occurrence and development of disuse osteoporosis by promoting osteoclast generation and inhibiting THE migration of BMSC in mice model of disuse osteoporosis.3.TGF-β1 inhibitor can inhibit the differentiation of osteoclasts in vivo,promote the migration of BMSC and the formation of osteoblasts,and improve the osteoclast-osteogenic decoupling in mice with disuse osteoporosis.