MafG Modulates The Function Of Osteoblast Cells Under Simulated Weightlessness And Irradiation Environment

BackgroundWeightlessness and radiation are the two major factors of bone loss induced by the space environment.With the rapid and continuous development of the China Space Station and deep space exploration missions,the astronauts are facing a more complex health risk.Bone fracture caused by bone loss is one of the primary risks for astronauts in the space environment.However,the mechanisms of bone loss induced by the space environment are still not well been understood and need to be further studied and illustrated.ObjectiveIn this study,the effects of weightlessness,radiation and their combination（weightlessness plus radiation）on osteoblasts were investigated by establishing cellular and animal models to simulate weightlessness,radiation and their combination in the laboratory.The apoptosis induced by the environments and changes in MafG and other molecules in MC3T3-E1 cells under simulated weightlessness,radiation and their combination were observed.The important role of MafG in the regulation of osteoporosis induced by the space environment was illustrated via a study on the regulation of Nrf2/Maf/HO-1 signaling pathway components,and the mechanism of MafG in the process was preliminarily discussed.Methods1.Cellular models of weightlessness,radiation and their combination environment were established in MC3T3-E1 cells cultured in vitro by using clinostat and X-ray irradiation.RT-qPCR and Western blotting assays were conducted separately to evaluate the m RNA and protein expression of Runx2,an osteogenic differentiation transcription factor,and caspase-3,an apoptosis-related molecule.Apoptosis rate was assessed by flow cytometry.SD rat models of weightlessness,radiation and their combination were established by using tail suspension and X-ray irradiation on SD rats.Micro-CT was used to detect the ultrastructural changes,and immunohistochemistry staining and RTqPCR were used to assess the m RNA and protein expression of Runx2 and Caspase-3in rat femur tissues.2.In the cellular models of weightlessness,radiation and their combination,RT-qPCR and Western blotting were used to measure the m RNA and protein expression of important molecules in the Nrf2/MAF/HO-1 pathway,namely,MafG,Nrf2 and HO-1 and the apoptosis-related molecules Bax,Bcl-2 and Caspase-3.3.ROS level in MC3T3-E1 cells was changed by exposing cells to the oxidizing agent H₂O₂,the antioxidant Nacetylcysteine or both reagents with appropriate concentrations,respectively.Then,the protein levels of MafG,Nrf2,HO-1,Caspase-3,Bax and Bcl-2were examined by Western blotting,and the apoptosis rates of the treated MC3T3-E1 cells were evaluated by flow cytometry.4.MafG,Nrf2 and HO-1 were overexpressed or silenced by the CRISPR/d Cas9 ACT system or si RNA respectively,in cellular models.The protein expression levels of MafG,Nrf2,HO-1,Bax,Bcl-2 and Caspase-3 were tested by Western blotting,and the apoptosis rates of the treated MC3T3-E1 cells were evaluated by flow cytometry.Results1.Simulated weightlessness,radiation and their combination decreased the expression of MafG and increased the expression of HO-1 but had little effect on Nrf2 in the cellular model.It demonstrated that the expression levels of the apoptosis-related proteins Caspase-3 and Bax were increased in the simulated weightlessness,radiation and combination environments.The cellular model showed the highest increase in the expression of Caspase-3 and Bax under the combined environment.2.Simulated weightlessness,radiation and their combination could induce bone loss.The bone loss observed under the combined environment was more serious than observed with MG or RA alone.The levels of the bone metabolism markers BALP and CTX-I in serum and the levels of Runx2 and Caspase-3 indicated that bone formation was decreased and osteoblast apoptosis was increased.3.The oxidizing agent H₂O₂ significantly increased the expression levels of Cleaved caspase-3,Bax and HO-1 but significantly decreased the protein expression level of MafG.In contrast,the antioxidant NAC significantly decreased the expression levels of Cleaved caspase-3,Bax and HO-1 but significantly increased the protein expression level of MafG.However,H₂O₂ or NAC did not affect the protein expression of Nrf2.H₂O₂ significantly increased the apoptosis rate but NAC did not affect the apoptosis rate.4.Overexpression of MafG did not affect the protein expression of Nrf2 but significantly decreased the protein expression level of HO-1.Apoptosis was significantly decreased in the MafG-overexpressing cells under simulated weightlessness,radiation and their combination.The protein expression level of Nrf2 did not change significantly when MafG was silenced,and the protein expression level of HO-1 was significantly increased.The apoptosis rate in the MafG-silenced cells was significantly increased under the conditions of simulated weightlessness,radiation and their combination.5.In the Nrf2-overexpressing cells,MafG protein expression was significantly increased,while HO-1 protein was not altered.The levels of the apoptosis-related proteins Cleaved caspase-3 and Bax were decreased,Bcl-2 was increased,and the apoptosis rate was also decreased significantly.When Nrf2 was silenced,the protein level of MafG was not significantly altered,and the protein level of HO-1 was significantly increased.In the silenced Nrf2 cells,the Cleaved caspase-3 and Bax protein levels were significantly increased,that of the Bcl-2 was significantly decreased,and the apoptosis rate was accordingly increased.6.In the HO-1-overexpressing and silenced cells,Nrf2 and MafG protein expression were not significantly altered.When HO-1 was overexpressed,the levels of the apoptosisrelated proteins Cleaved caspase-3 and Bax were increased,Bcl-2 was decreased,and the apoptosis rate was also increased significantly.When HO-1 was silenced,the Cleaved caspase-3 and Bax protein levels were significantly decreased,that of the Bcl-2 was significantly increased,and there was no significantly difference in the apoptosis rate.Conclusion1.Simulated weightlessness,radiation and their combination induce bone loss and promote apoptosis.Simulated weightlessness and radiation combination have an effect of encouraging single factor.2.MafG participates in the pro-apoptotic regulation of simulated weightlessness,radiation and their combination on osteoblasts,and plays a role in the ROS/Nrf2-MafG/HO-1signaling pathway.3.ROS/Nrf2-MafG/HO-1 is the common signal pathway in the bone loss induced by simulated weightlessness or radiation.In conclusion,the pro-apoptotic effects of simulated weightlessness,radiation and their combination on osteoblasts and rat bone tissues were confirmed in this study,and the combined effect encourages that of the single factor.The simulated weightlessness,radiation and combination environments regulate the Nrf2/Maf/HO-1 signaling pathway and promote osteoblast apoptosis by inhibiting the expression of MafG.This study further elucidates the cellular apoptotic mechanism of bone loss induced by simulated weightlessness,radiation and their combination environment and provides a new molecular target for the development of an effective countermeasure of bone loss induced by the space environment.