The Role Of ClC-5 In Bone Loss Under Simulated Weightlessness And It's Preliminary Study Of The Mechanism

BackgroundWith the acceleration development of space in humans,the bone loss induced by long term space flight increasing seriously,and it is also a major medical problems limit the ability of human to explore the space.Some prevention and treatment measures have been found to solve the weightlessness-induced bone loss,such as protective equipment,nutritional supplement,physical exercise,Chinese herbal medicine therapeutic,etc.However,these therapeutic effects are not ideal and it is crucial to study the mechanism of weightlessness bone loss.Osteocytes,as the main cells in bone tissue,is not only the main factors to maintain the normal bone metabolism but also the menchanical sensitive cells.Research showed that some positive ion channels act roles under weightlessness including Ca2+,K+ and so on.However,there is little known about negative aion channels on bone loss under simulated weightlessness.The chloride ion,as the most important negative ion,participants in a variety of physiological processes of mammals.And the chloride ion gene mutations are related with some genetic diseases.ClCs are voltage-gated chloride channels.ClC-5 is belonged to the second branch of ClCs and controls the endocytotic acidification and chloride ion absorption.Recent studies found that ClC-5 gene knockout mice showed the retarded growth and abnormal bone formation,and the mutations of ClC-5 could lead to Dent's disease with the symptoms of hyperphosphaturia,hypercalciuria and kidney stones.This remind us that ClC-5 may play a role in osteogenic differentiation and associated with abnormal phosphorus metabolism.In our previous studies,we found that dramatic bone loss and significant increased serum phosphorus level in tail suspension rats,and this phenomenon also occured in rats after feeding with high phosphorous diet for 1 month.This suggests that abnormal phosphorus metabolism under simulated microgravity may be one of the reasons for bone loss.Then,whether ClC-5 plays a role in bone loss? If that,whether ClC-5 is related to the increased phosphorus level in suspended rats? What are the mechanisms? These are the main points for our research.ObjectiveThe purpose of our project is to observe the role of ClC-5 in bone loss and investigate the possible mechanisms during this process under simulated weightlessness.Methods 1.We used suspended rats and Two-dimensional gyrator to simulated weightlessness.Real-time PCR to detect the expression of ClC-5 in osteocytes under simulated weightlessness.2.By overexpressing of ClC-5 and RNAi of ClC-5,we analyzed the expression of osteogenesis related genes.We used Alizarin red staining to explore the change of extracellular mineralization after overexpression of ClC-5.Then,Real-time PCR was used to detect the effect of ClC-5 expression changes on osteogenesis related genes under simulated weightlessness.3.Real-time PCR was performed to detect the effect of ClC-5 expression changes on osteoclastic genes under nomal situation and simulated weightlessness.Meanwhile,Transmission electron microscope was used to observe the ultrastructure in osteocytes under simulated weightlessness.4.Energy spectrum analysis,Real-time PCR and intracellular p H staining were performed to discuss the correlation of ClC-5 and phosphorous metabolism.5.Real-time PCR was used to tested the levels of osteogenesis related genes and osteoclastic genes in osteocytes after culturing in high serum inorganic phosphorus.Results 1.The expression of ClC-5 in IDG-SW3 osteocytes under simulated weightlessness.The expression level of ClC-5 in IDG-SW3 osteocytes increased in response to simulated weightlessness.In addition,the immunohistochemical staining showed ClC-5 expression was also increased in unloaded tibia.2.The effects of ClC-5 on osteocyte osteogenic capacity under simulated weightlessness.(1)The expression of ALP,BSP,OC and Osx were increased in ClC-5 overexpressed osteocytes.Overexpressed ClC-5 also promoted the mineralization of osteocytes in vitro.(2)ClC-5 Si RNA down-regulated the ALP,BSP,OC and Osx expression.(3)Simulated weightlessness down-regulated the ALP,BSP,OC and Osx expression.(4)After overexpressed ClC-5 in osteocytes,the expression of ALP,BSP,OC and Osx were decreased under simulated weightlessness.(5)The expression of ALP,BSP,OC and Osx were increased in Cl C5 silence osteocytes under simulated weightlessness.3.The effects of ClC-5 on osteocyte osteoclastic capacity under simulated weightlessness.(1)The levels of TRAP?RANKL and RANKL/OPG were decreased in ClC-5 overexpressed osteocytes in vitro.(2)ClC-5 Si RNA up-regulated the expression of TRAP?RANKL and RANKL/OPG ratio.(3)Simulated weightlessness increased the levels of TRAP?RANKL and RANKL/OPG ratio.(4)After overexpressed ClC-5 in osteocytes,the expression of TRAP?RANKL and RANKL/OPG ratio were increased under simulated weightlessness.(5)The levels of TRAP?RANKL and RANKL/OPG ratio were decreased in Cl C5 silence osteocytes under simulated weightlessness.4.The relationship of ClC-5 and inorganic phosphorus on bone loss under simulated weightlessness(1)The expression of Pit-1 increased in osteocytes under simulated weightlessness.(2)The lever of Pit-1 and extracellular phosphorous increased in ClC-5 overexpressed osteocytes and decreased in ClC-5 silence osteocytes.(3)The expression of Pit-1 decreased in ClC-5 silence osteocytes under simulated weightlessness.(4)The p H of intracellular were both reduced in osteocytes and ClC-5 silence osteocytes under simulated weightlessness.Moreover,the intracellular p H of osteocytes under simulated weightlessness is relatively higher compared with that in ClC-5 silence osteocytes under simulated weightlessness.(5)After culturing IDG-SW3 osteocytes in 5 m M high phosphorous medium,the expression of ALP and BSP were decreased,TRAP?RANKL expression and RANKL/OPG ratio were increased.Conclusions 1.Simulated weightlessness up-regulates ClC-5 in IDG-SW3 osteocytes and the increased ClC-5 may participants in bone remodelling.2.ClC-5 could decrease the expression of osteogenesis related genes and increase the expression of osteoclastogenesis genes under simulated weightlessness,it plays an important role in bone loss.3.ClC-5 may through intracellular p H to effects the lever of extracellular phosphorous in osteocytes under simulated weightlessness4.ClC-5 may partially through intracellular p H to increase the lever of extracellular phosphorous,reduce the osteogenesis,increase osteoclastogenesis and eventually lead to bone loss under simulated weightlessness.