The Mechanism Of Bone Loss Induced By Simulated Microgravity And Its Countermeasures

With the launch of the deep space exploration project of "Tianwen-1" and "Chang’e-5",China’s ability to explore space is becoming stronger and stronger,and space is attracting us to continue to explore and move forward with infinite charm.Meanwhile,the negative impact of the negative space environment on astronauts can not be ignored."Space bone loss" is the first major risk,the maintenance of astronauts’ bone health has become a vital task.The main macroscopic effect of microgravity exposure on bone tissue is osteopenia.This effect is due to the modification induced by microgravity at the cellular level,which may lead to the impairment of the activity and signal transduction of osteoclasts,osteoclasts,and osteo cyte in bone tissue.However,the specific mechanism of bone loss caused by space microgravity remains to be solved as to how the bone tissue cells sense the extracellular physical signal of microgravity and transmit it to the cell to produce a series of cascade reactions.As an antenna-like organelle on various cell surfaces,primary cilia are known to be sensitive to various physical and chemical factors such as light,taste,water flow,electromagnetic field,etc.What changes will happen in the process of bone loss caused by microgravity? Whether primary cilia can be used as a target to study drugs related to bone loss in the microgravity environment.Based on the above argument,the following studies were carried out to explore the mechanism and Countermeasures of bone loss induced by the simulated microgravity environment.The specific methods used in this experiment are as follows: 1.The osteoblasts extracted and isolated from the skulls of neonatal SD rats by multiple enzymatic digestion were used as the research object in this ex periment;2.The SM-31 3D random positioning machine by the Chinese Academy of Sciences was used to simulate the microgravity environment to treat osteoblasts.3.Immunofluorescence staining was used to detect the effects of simulated microgravity environm ent on primary cilia of osteoblasts.Western blot was used to detect the effects of simulated microgravity environment on osteoblasts’ osteogenic active proteins and BMP-2/Smads pathway related proteins;4.The relationship between primary cilia and BMP-2/Smads pathway was analyzed by immunofluorescence co-staining;5.The sh RNA were used to interfere with primary cilia,western blot and RT-PCR were used to analyze the influence of primary cilia on BMP-2/Smads pathway induced by BMP-2,and alkaline phosphatase and alizarin red staining were used to analyze the influence of primary cilia on the mineralization and maturation ability of BMP-2 induced osteoblasts.;6.Cytorelaxation D and overexpression of mi R-129-3p were used to promote ciliogenesis,and their effects on primary cilium,osteogenic activity,BMP-2/Smads pathway,and mineralization and maturation ability of osteoblasts in simulated microgravity environment were detected.7.U sed chloral hydrate to remove primary cilia,then detected the effects of overexpression of mi R-129-3p on osteoblastic activity and mineralization and maturation ability of osteoblasts in simulated microgravity environment after primary cilia disappeared.8.The expression of mi R-129-3p was detected under simulated microgravity and after treatment with cytochalasin D.The results are as follows:1.The results of immunofluorescence showed that the length and incidence of primary cilia of osteoblasts decreased significantly after 6 h treatment.Western blot results showed that the expression of osteoblast active protein and key protein of BMP-2 / Smads pathway were significantly down-regulated with the prolongation of treatment time.Alkaline phosphatase and alizarin red staining results also showed that simulated microgravity enviro nment inhibited the mineralization maturation ability of osteoblasts.2.Immunofluorescence staining analysis BMP-2/ Smads pathways related proteins and primary cilia were positioning situation,found that BMP-2,BMPR Ⅱ,p-Smad1/5/8 and Smad1/5/8 distribution exists in the primary cilia.3.The BMP-2/ Smads pathway induced by BMP-2 was inhibited and the mineralization and maturation ability of osteoblasts were decreased by sh RNA.4.When using cytochalasin D and overexpression of mi R-129-3 p promote cilia,the length and the incidence of primary cilia was significantly increased in the simulated microgravity environment,Western blot test results showed that osteoblast active protein with BMP-2/Smads pathway of protein expression quantity also rebounded,alkaline phosphatase and alizarin red staining results mature osteoblast mineralization ability are also protected.5.When the primary cilia of osteoblasts were removed with chloral hydrate,the protective effect of overexpression of mi R-129-3p on the activity and mineralization maturation of osteoblasts in the simulated microgravity environment disappeared.6.The expression of mi R-129-3p in osteoblasts treated with simulated microgravity was analyzed by RT-PCR.The results showed that simulated microgravity significantly decreased the content of mi R-129-3p in osteoblasts,while cytochalasin D could increase the content of mi R-129-3p in osteoblasts.These results suggest that the loss of primary cilia is cl osely related to the inhibition of osteogenic activity in simulated microgravity environment.Several key proteins of the BMP-2 / Smads pathway located in the primary cilia participate in this process.The complete structure of primary cilia is necessary f or BMP / Smads signal transduction.BMP-2/ Smads signal transduction is necessary for the normal development of osteoblasts.Cytochalasin D and overexpressed mi R-129-3p can maintain the normal transduction of the BMP-2/ Smads pathway by protecting primary cilia,to protect the osteogenic activity inhibited by the simulated microgravity environment.Primary cilia can be used as an important target to prevent bone loss under microgravity.