The Ameliorating Effect Of Osthole On Bone Loss Induced By Simulated Microgravity

Space environment is of great harm to astronauts’health.Specifically,the skeletal metabolic disorder is one of the most serious damages,during long-term space flight,which results in bone mineral density reduction,or even disused bone loss.Dietary supplementation and resistive exercise remain the primary countermeasures in the International Space Station to date although its effectiveness is controversial.On the other hand,adverse reactions of pharmacological interventions have greatly limited their application in spaceflight.Therefore,the development of effectiveness countermeasures against space flight-related bone loss is still one of the most urgent issues for human deep space exploration.Traditional Chinese medicine Cnidium monnieri is widely applied in clinic with significant curative effect and few toxic side effects for senile osteoporosis therapy.Osthole,an active ingredient from Cnidium monnieri fruit,has been proven to effectively relieve osteoporosis in ovariectomized or aged mice.However,whether osthole can prevent bone loss elicited by space microgravity and the related molecular mechanisms remain unknown.The aims of this study are to investigate the effect of osthole on preventing and treating bone loss in hindlimb-suspended rat model,and illustrate the related mechanisms by using osteoblast treated with a random positioning machine.This research is able to provide experimental and theoretical basis for prevention and treatment of bone loss in space environment.Forty three-month-old Wistar rats were randomly divided into 5 groups（n=8）:the control group（Ctrl）,the simulated microgravity model group（SM）,the simulated microgravity with low（SM+L,10 mg/kg/d）,medium（SM+M,30 mg/kg/d）and high（SM+H,90 mg/kg/d）concentrations of osthole administration group.After 4 weeks treatment,the whole blood was collected from the abdominal aorta to collect serum,and the changes of organ indexes were evaluated,as well as the femoral,tibia,and lumbar vertebrae which were shaved adherent muscles were also collected.Bone mineral density and biomechanics of femurs and lumbar spines were measured using a dual-energy x-ray absorptiometry and three-point bending test,respectively.Serum bone formation maker bone-specific alkaline phosphatase（BALP）and bone resorption markers tartrate-resistant acid phosphatase 5b（TRACP-5b）and type I collagen C-terminal peptide（CTX-1）were detected using ELISA.And the expression levels of alkaline phosphatase（ALP）,bone glaprotein（BGP）,cathepsin K（Ctsk）and chloride channel 7（Clcn7）were analyzed using qRT-PCR.The micro-architectures in bone were observed by micro-CT and Van Gieson（VG）staining.On the other hand,the concentration of osthole was selected by MTT assay,and an appropriate concentration would carry out in next experiment.Namely,MC3T3-E1pre-osteoblasts treated with oshtole in random positioning machine to evaluate calcium nodule formation and alkaline phosphatase activity.Furthermore,gene and protein expression levels were analyzed by qRT-PCR and western blot,respectively.In vivo studies,all group had no significant effects on organ indexes and bone mineral density in lumbar spine,but femoral bone mineral density was significantly increased for osthole treatment,whereas it was decreased in the SM group.A three-point bending test suggested that bone mechanical strength was also enhanced by osthole treatment with increased elastic modulus and maximum load compared to those of the SM group.Deterioration of bone trabecular microstructure was improved by osthole with increased morphological parameters such as bone volume fraction,trabecular thickness,and trabecular number,as well as decreased trabecular separation compared to the SM group.Besides,serum BALP levels were significantly incaresed by osthole treatments compared to SM group.Instead,serum TRACP-5b and CTX-1 levels were obviously suppressed by osthole treatments.Moreover,low and medium concentrations of osthole significantly up-regulated the expression levels of ALP and BGP,but only middle concentration down-regulated the expression levels of Ctsk and Clcn7.Furthermore,in vitro experiment demonstrated that osthole(1×10^-6mol/L)remarkably increased the formation of mineralized nodules and alkaline phosphatase activity in MC3T3-E1 pre-osteoblasts treated with random position machine.The gene and protein expression levels of well-known markers and regulators for osteoblasts differentiation,including runt-related transcription factor 2（Runx2）and bone morphogenetic protein-2（BMP-2）were up-regulated when treated with osthole in simulated microgravity condition.In summary,osthole can effectively prevent bone loss induced by simulated microgravity through promoting bone formation and inhibiting bone resorptionat a dosage of 30 mg/kg in handlimb suspension rats.In addition,osthole(1×10^-6 mol/L)relieves the inhibition of osteogenic differentiation of MC3T3-E1 cells treated with random position machine through upregulating the expression of RUNX2 and BMP-2.