| BackgroundMechanical stimuli are crucial for maintaining the equilibrium between bone formation and resorption.The weightless environment of spaceflight disrupts bone homeostasis,resulting in persistent bone loss phenomena that seriously endanger astronauts’ health.With the completion of China’s Tiangong-1 space station’s in-orbit assembly and construction,long-term in-orbit flight has become the norm for our astronauts.Therefore,further revealing the molecular mechanism of bone loss induced by weightlessness and providing effective countermeasures for astronaut bone loss in weightless environments are the main problems that need to be solved in the field of aerospace medicine at present.Bone is a highly vascularized tissue.Studies have shown that skeletal capillaries are critical for maintaining bone homeostasis,where blood circulation and vascular regeneration are essential for bone growth and development,shaping and remodeling,and injury healing.In terms of time,angiogenesis precedes bone formation,which is a prerequisite for ossification.Spatially,skeletal capillaries can also serve as a communication network between bone and adjacent tissues,providing nutrients and removing metabolic waste.This suggests a tight spatiotemporal link between angiogenesis and bone formation,known as “angiogenesis-osteogenesis coupling”.Reduced microvessels and angiogenesis in bone can lead to impaired bone formation,reduced bone mass,and reduced fracture healing ability.It has been reported that conditioned media obtained from microvascular endothelial cells under simulated microgravity inhibited osteoblast function.However,the exact mechanism has not been elucidated.Exosomes,an important pathway for intercellular communication,play regulatory roles by delivering nucleic acids,proteins,or other regulatory factors into receptor cells and participate in various physiological activities,such as the immune response,bone reconstruction,and angiogenesis.In the bone remodeling microenvironment,exosomes can deliver mi RNAs to osteoblasts and thus affect bone formation.However,it has not been reported whether microvascular endothelial cells under simulated microgravity can regulate osteoblast function through exosomes and the possible mechanisms involved.ObjectiveThe aim of this study is to explore the effect of exosomes derived from microvascular endothelial cells under simulated microgravity on osteoblast function and its molecular mechanism from the perspective of “angiogenesis-osteogenesis coupling”,to enrich the theoretical system of the mechanisms of bone loss induced by weightlessness,and to provide potential intervention targets for the prevention and treatment of bone loss induced by weightlessness.Methods1.To explore the effect of exosomes derived from microvascular endothelial cells under simulated microgravity on osteoblasts,the following experimental methods were adopted.2D clinorotation was used to perform simulated microgravity treatment on microvascular endothelial cells for 48 h.Exosomes were extracted from cell culture supernatants by ultracentrifugation.Transmission electron microscopy was used to observe exosome morphology,a nanoanalyzer was used to analyze the diameter distribution of exosomes,and western blotting was used to detect exosomal markers and cellular markers in exosomes and cells,respectively.An immunofluorescence assay was conducted to observe whether osteoblasts can take up microvascular endothelial cell-derived exosomes.Con Exos and Clino Exos were incubated with osteoblasts for 48 h to detect the effects of exosomes derived from microvascular endothelial cells under simulated microgravity on osteogenic differentiation.q RT-PCR was used to detect the m RNA expression of the osteogenic differentiation markers Alp,Osx,Runx2 and Ocn,and western blotting was used to detect the protein levels of OSX,RUNX2 and OCN.ALP staining and ALP activity were conducted to detect the enzymatic function of ALP.2.To screen significantly differentially expressed mi RNAs in exosomes derived from microvascular endothelial cells under simulated microgravity,the following experimental methods were adopted.After microvascular endothelial cells were treated with simulated microgravity for 48 h by 2D clinorotation,cell culture supernatants were collected,and exosomes were extracted using ultracentrifugation.mi RNA sequencing was performed on two sets of microvascular endothelial cell-derived exosomes to screen differentially expressed mi RNAs.q RT-PCR was performed to further validate differentially expressed mi RNAs in microvascular endothelial cells and their secreted exosomes,thus clarifying the candidate mi RNA.Osteoblasts were cocultured with Con Exos and Clino Exos for 48 h.mi R-92b-3p expression levels in osteoblasts were measured by q RT-PCR.3.To explore the role of mi R-92b-3p in the regulation of osteoblast function by microvascular endothelial cells under simulated microgravity,the following experimental methods were adopted.The effect of mi R-92b-3p on osteogenic differentiation was examined after transfection of mi R-92b-3p mimics or inhibitors and corresponding NCs into MC3T3-E1 cells.The mi R-92b-3p inhibitor and NC were transfected into Clino Exos through electroporation.Then,the modified exosomes were added to MC3T3-E1 cells.q RT-PCR,western blotting,ALP staining,and ALP activity were used to detect whether microvascular endothelial cell-derived exosomes could regulate osteogenic differentiation by transporting mi R-92b-3p.4.To explore the mechanism by which mi R-92b-3p regulates osteoblast function,the following experimental methods were adopted.Bioinformatics analysis was used to predict the target genes of mi R-92b-3p.q RT-PCR and western blotting were used to detect whether mi R-92b-3p and Clino Exos regulate the m RNA and protein expression of ELK4.A dual luciferase reporter gene assay further verified whether mi R-92b-3p could bind to the Elk4 m RNA 3’UTR.si RNA-Elk4 and NC were transfected into MC3T3-E1 cells to detect the effect of ELK4 on osteogenic differentiation.mi R-92b-3p inhibitor and si RNA-Elk4 were cotransfected into MC3T3-E1 cells to verify whether mi R-92b-3p could regulate osteogenic differentiation by targeting Elk4.Results1.Exosomes derived from microvascular endothelial cells under simulated microgravity were extracted by ultracentrifugation and identified.Exosomes derived from microvascular endothelial cells under simulated microgravity were taken up by osteoblasts and inhibited osteogenic differentiation.2.The mi RNA expression profile of microvascular endothelial cell-derived exosomes under simulated microgravity changed significantly.mi R-92b-3p was significantly elevated in both microvascular endothelial cells and their secreted exosomes under simulated microgravity.The level of mi R-92b-3p in osteoblasts was increased by microvascular endothelial cell-derived exosomes under simulated microgravity.3.mi R-92b-3p significantly inhibited osteogenic differentiation,mainly by suppressing the m RNA and protein expression of OSX,RUNX2,and OCN,markers of osteogenic differentiation,and suppressing the expression and activity of ALP.Microvascular endothelial cell-derived exosomes under simulated microgravity could inhibit osteogenic differentiation by transporting mi R-92b-3p into MC3T3-E1 cells.4.In osteoblasts,mi R-92b-3p inhibited ELK4 expression.Microvascular endothelial cell-derived exosomes under simulated microgravity inhibited ELK4 expression in osteoblasts.A dual luciferase reporter gene assay confirmed that mi R-92b-3p targeted Elk4.ELK4 promoted osteogenic differentiation.mi R-92b-3p inhibited osteogenic differentiation by targeting Elk4.Conclusions1.Exosomes derived from microvascular endothelial cells under simulated microgravity inhibit osteogenic differentiation,and the mi RNA expression profile of exosomes is significantly altered by simulated microgravity.2.Exosomes derived from microvascular endothelial cells under simulated microgravity inhibit osteogenic differentiation by transporting mi R-92b-3p into osteoblasts.3.mi R-92b-3p inhibits osteogenic differentiation by suppressing ELK4 expression.In summary,this study reveals that exosomes derived from microvascular endothelial cells under simulated microgravity are taken up by osteoblasts and inhibit osteogenic differentiation.mi R-92b-3p is significantly upregulated in microvascular endothelial cell-derived exosomes under simulated microgravity.mi R-92b-3p is transferred into osteoblasts by microvascular endothelial cell-derived exosomes,which inhibits osteogenic differentiation by suppressing ELK4 expression.This study takes the intercellular communication between microvascular endothelial cells and osteoblasts as an entry point to deepen the understanding of the mechanisms of bone loss induced by weightlessness and to provide new ideas and a theoretical basis for the prevention of bone loss in astronauts in weightless environments. |
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