Protective Effect Of Pine Polyphenols Polyelectrolyte Nanoparticles On Bone Loss Induced By Simulated Weightlessness

Astronauts suffer bone loss during long-term space missions.And such bone loss is difficult to recover after returning to the ground.Therefore,to find weightlessness bone loss protectant is imminent,and became hot spot of manned space technology.In this study,polyphenol of Pinus koraiensis was screened out to be research object.The activity section(S3)with alleviating decreased osteoblast activity effect in pine total polyphenols was found by activity tracking,and the molecular composition and structure of S3 was analysised by UPLC-MS.Comparation study of materials for encapsulation S3 was carried out based on the self-assembly principle of polyelectrolyte.Then the acidic fragments of Auricularia auricula(AAP I?)and poly-?-L-lysine(PLL)were screened out.The encapsulation structure of PP was determined using FT-IR and DLS followed by optimization of PP preparation through Box-Behnken response surface method which the PP with the highest S3 encapsulation efficiency was prepared.The protective effect of PP on simulated weightlessness-induced bone loss in rats was tested by electronic universal testing machine and ?-CT.The OPG/RANK/RANKL,Wnt/?-catenin and Keap1/Nrf2/ARE signaling pathways and osteogeneis genes in rat femur were quantitatively analyzed by ELISA,western blot and q RT-PCR,respectively.Consequently,the protective effect of PP on bone loss induced by simulated weightlessness in rats was systematically studied.The model for simulating the osteoblasts dysfunction induced by weightlessness was established using 2D-RWVS.And then the model was used to verify that TP has a protective effect on the osteoblasts from dysfunction induced by simulated weightlessness.After that TP was divided into 8 active sections(S1-S8)by macroporous resin.S3 exhibited the highest proliferation rate of osteoblasts(97.02±4.05%),and better portective effect than TP.Analysis of the molecular composition and structure of the main active components in S3 by mass spectrometry showed that all the compounds in S3 had a flavonoid core.PLL was chosen as cationic polymer and AAP I? was separated and purified as anionic polymer by DEAE-52 and Sephadex G-100 for S3 encapsulation.The PP preparation was optimized by response surface methodology.The structure analysis of PP indicated that AAP I?,PLL,and S3 are assembled into PP by electrostatic interaction.In vitro studies have found that PP releases slowly in simulated gastric environment but rapidly in simulated intestinal environment,which demonstrated that biopolymers formed by polyelectrolyte networks around PP protect S3 from simulated gastrointestinal tract damage.The elastic modulus,shear modulus,rigidity,toughness,maximum strain and maximum stress of rat femur were significantly enhanced by both S3 and PP after simulated weightlessness.Damage induced by simulated weightlessness to the structural and integrity of cancellous which contained BMD,BV/TV,Con.D,SMI,BS/BV,Tb.N,Tb.Sp and Tb.Th at rat femur were significantly reduced by both S3 and PP.The decreased BALP and PINP in the serum of rats were significantly increased by both S3 and PP under simulated weightlessness.However,S3 and PP had no effect on TRAP-5b and NTX.This result indicated that S3 and PP exert a protective effect on bone loss in rat induceed by simulated weightlessness through promoting bone formation rather than inhibiting bone resorption.And these result further indicated that PP can protect S3 from gastrointestinal damage in vivo.PP decreased MDA level,activated SOD,CAT,GSH-Px in femur under simulated weightlessness.In addition,PP enhanced the expression of Nrf2,indicating that the antioxidant enzyme defense system was enhanced and Keap1/Nrf2/ARE signaling pathway was activated by PP in rat femur.Phosphorylation degree of GSK3? and ?-catenin expression in rat femoral tissue were increased by PP after simulated weightlessness,indicating that the antagonism of Wnt/?-catenin signaling pathway can be relieved by PP.The expression of osteogeneis genes in rat femur was promoted by PP after simulated weightlessness,indicating that PP can promote the differentiation and maturation of osteoblasts,reduce the loss of minerals and organic matter of bone,enhance the mineralization ability of bone.From these three aspects,PP plays a protective role in simulated weightlessness-induced bone loss in rats.In conclusion,S3 has anti-bone loss activity under weightlessness which can be prepared as a novel weightlessness bone loss protectant(PP)by polyelectrolyte self-assembly with reduceed damage of gastrointestinal tract on S3 activity,thereby improving the protective effect of S3 on weightlessness-induced bone loss.PP can alleviate bone loss induced by simulated weightlessness through reduce the inhibition of the oxidative stress on Wnt/?-catenin signaling pathway and then promote bone homeostasis to bone formation.These investigations reveal the molecular mechanism of PP that alleviating bone loss induced by simulated weightlessness,which provide important theoretical and practical insights for the further development of weightlessness bone loss protectant.