| Background With the launch of the series of Tiangong space laboratories,China’s manned aerospace industry has gradually developed into a new era of long-term space flight.During the space flight,the medical problems induced by weightlessness and radiation are very prominent and complicated,which brings new tasks and chall enges to aerospace medicine research.Weightlessness has an important effect on bone formation.With the extension of the flight time,the body appears reduced bone density,bone demineralization,change of bone structure and reduction of bone mechanical strength.All of these changes may seriously affect the health of astronauts.The most significant effect of weightlessness is on the weight-bearing bones.The calcium contents of the weight-bearing bones will be lost at a rate of 1% to 2% per month and this phenomenon is not self-limited.Osteoblasts are the key factor for the formation of bone,and the changes of their function will directly affect the dynamic balance of bone metabolism.In recent years,the function and molecular mechanism of FoxO fami ly have gradually become a focus of immunology,genetics,and oncology.Studies have shown that FoxO family plays an important role in the regulation of bone remodeling,which is not only involved in the regulation of osteoblasts,but also in the regulation of osteoclasts.FoxO family can activate the expression of genes related to cell cycle,apoptosis,and oxidative stress.As an important member of the FoxO family,FoxO3 a has been shown to have a close connection with the functions of osteoblasts.However,the role of FoxO3 a in the regulation of bone loss induced by weightlessnesss has not been clarified.Objective This research aimed to investigate the regulation mechanism of FoxO3 a on osteoblast apoptosis under simulated weightlessness.For this purpose,we studied the effect of simulated weightlessness on osteoblast apoptosis,observed the changes of FoxO3 a in osteoblasts under simulated weightlessness,clarified the effect of the alteration of FoxO3 a on osteoblast apoptosis under simulated weightlessness and its mechanism,and the n explored the upstream regulation mechanism of FoxO3 a.Methods 1.The apoptosis rate of osteoblasts was detected by flow cytometry.Western Blot and q RT-PCR techniques were used to detect the expression of Bax,Bcl-2 and Caspase-3 protein and their m RNA in osteoblasts under simulated weightlessness.q RT-PCR was used to detect the time-dependent changes of FoxO3 a m RNA under simulated weightlessness.Then Western Blot was used to detect the time-dependent changes of total FoxO3 a,p-FoxO3 a and nuclear FoxO3 a protein under simulated weightlessness.Additionally,the expression changes of FoxO3 a in the nucleus and cytoplasm of osteoblasts under simulated weightlessness were detected by Immunofluorescence technique.2.After knocking down FoxO3 a in osteoblasts under normal gravity or over-expressing FoxO3 a under simulated weightlessness,Flow cytometry was used to detect the effects of FoxO3 a on the apoptosis rate of osteoblasts and Western Blot technique was used to detect the expression of Bax,Bcl-2 and Cleaved Caspase-3 proteins.3.Flow cytometry was used to detect the time-denpendent changes of ROS experession in osteoblasts under simulated weightlessness.After knocking down FoxO3 a in osteoblasts under nor mal gravity or over-expressing FoxO3 a under simulated weightlessness,Flow cytometry was used to detect the effects of FoxO3 a on the ROS expression and Western Blot was used to detect protein expression of Mn SOD in osteoblasts.Then,after over-expressing the expression of ROS by H2O2 under normal gravity environment or inhibiting the expression of ROS by NAC under simulated weightlessness,the expression changes of Bax,Bcl-2 and Cleaved Caspase-3 proteins were detected.4.Luciferase assay was performed to prove the direct combination of miR-132-3p and its predicted target gene FoxO3 a.Mimic-132 and inhibitor-132 were used to over-express or knock down the miR-132-3p expression.q RT-PCR and Western blot were used to detect the regulatory effect of miR-132-3p on FoxO3 a m RNA and protein level.Flow cytometry was used to detect the effects of miR-132-3p on the ROS expression and apoptosis rate of osteoblasts.Western Blot was used to detect the changes of Mn SOD,Bax,Bcl-2 and Cleaved Caspase-3 proteins in osteoblasts.Results 1.Under simulated weightlessness,the apoptosis rate of osteoblasts was increased,the m RNA and protein expression of Bax and Cleaved Caspase-3 were increased,but the m RNA and protein expression of Bcl-2 was decreased.FoxO3 a m RNA expression decreased in a time-dependent manner under simulated weightlessness.The total amount of FoxO3 a protein and the amount of FoxO3 a protein in nucleus were decreased,but the expression of p-FoxO3 a was increased.Immunostaining analysis showed FoxO3 a protein expression decreased in a time-dependent manner under simulated weightlessness.2.Under normal gravity,the apoptosis rate of osteoblasts was increased by knocking down the expression of FoxO3 a.The protein expression of Bax and Cleaved Caspase-3 were increased,but the protein expression of Bcl-2 was decreased.Over-expression FoxO3 a could partly reverse the increase of apoptosis rate induced by simulated weightlessness.And it could also partly reverse the increase of Bax and Cleaved Caspase-3 and the decrease of Bcl-2 induced by simulated weightlessness.3.The expression of ROS was increased in a time-dependent manner under simulated weightlessness.Under normal gravity,the expression of ROS and protein expression of Mn SOD were decreased by knocking down the expression of FoxO3 a.Over-expression of FoxO3 a could partly reverse the increase of ROS and the decrease of Mn SOD under simulated weightlessness.Under normal gravity,the apoptosis rate of osteoblasts was increased by over-expression of ROS.The protein expression of Bax and Cleaved Caspase-3 were increased,the protein expression of Bcl-2 was decreased.Inhibiting the expression of ROS could partly reverse the increase of apoptosis rate induced by simulated weightlessness.And it could also partly reverse the increase of Bax and Cleaved Caspase-3 and the decrease of Bcl-2 induced by simulated weightlessness.4.miR-132-3p bonded with the 3’UTR of FoxO3 a directly,and it inhibited the protein expression of FoxO3 a without influencing the m RNA level.miR-132-3p significantly increased under simulated weightlessness for 48 h.Under normal gravity,supplement of miR-132-3p in osteoblasts could increase the expression of ROS,the apoptosis rate and the protein expression of Bax and Cleaved Caspase-3.It also decreased the protein expression of Bcl-2 and Mn SOD.Under simulated weightlessness,knocking down the miR-132-3p expression could partly reverse the increase of ROS,the increase of apoptosis rate and the increases of Bax and Cleaved Caspase-3 protein expression.It also partly reverse the decreases of Bcl-2 and Mn SOD protein expression.Conclusion 1.Simulated weightlessness can increase apoptosis and decrease the expression of FoxO3 a in osteoblasts.2.The decreased expression of FoxO3 a can induce apoptosis in osteoblasts.Over expression of FoxO3 a can partly reverse the increase of apoptosis induced by simulated weightlessness.3.The regulatory effect of FoxO3 a on apoptosis in osteoblasts is accomplished b y inhibiting ROS expression.4.FoxO3 a is the target gene of miR-132-3p.Simulated weightlessness can induce apoptosis in osteoblasts via miR-132-3p/FoxO3a/ROS pathway.In summary,our study shows that simulated weightlessness can lead to the increase of apoptosis in osteoblasts via miR-132-3p/FoxO3a/ROS pathway.This study not only further clarifies the mechanism of apoptosis induced by weightlessness in osteoblasts,but also provides a new direction for research on the protective measures of osteopenia induced by weightlessness. |
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