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Study On The Anti-Osteoporosis Mechanism And Metabolomics Of Polysaccharide From Portulaca Oleracea L.

Posted on:2024-06-09Degree:MasterType:Thesis
Country:ChinaCandidate:Y H FuFull Text:PDF
GTID:2544307112986709Subject:Drug analysis
Abstract/Summary:PDF Full Text Request
Objective:Osteoporosis is a metabolic bone disease closely related to oxidative stress.Therefore,a diet rich in antioxidants and changes in antioxidant lifestyles may increase bone density and reduce the risk of brittle fractures.Portulaca oleracea L.is an edible plant,which is used as a natural antioxidant in folk medicine.This study aims to screen and characterize the protective effect of polysaccharides from Portulaca oleracea L.(POP)against H2O2-stimulated osteoblast apoptosis in vivo and in vitro,and to elucidate its anti-osteoporosis mechanism.Methods:The enzymes viscozyme,celluclast,α-amylase,andβ-glucanase were used to extract POPs.Step gradient alcohol precipitation was performed to obtain POPs.Hydrogen peroxide(H2O2)was selected as an osteoporotic inducer.A MTT assay was carried out to detect cell viability in H2O2-stimulated MC3T3 cells.Determination of intracellular ROS levels in MC3T3 cells was performed using a DCFH-DA probe.FT-IR and HPLC-GPC method was used to analysis structural characterization of POP.The monosaccharide composition was determined via 1-phenyl-3-methyl-5-pyrazolone(PMP)derivatization.The protective effect of POP on oxidative stress induced apoptosis was evaluated by Hoechst 33342,Acridine Orange/Ethidium Bromide(AO/EB)Staining and flow cytometry analysis.Western blot was preformed to detect the protein expression of Nrf2,Keap1,HO-1,NQO1,Capase3,Bcl2,Bax and cytochrome C.The zebrafish was selected as the in vivo model.Fluorescent probes,DCFH-DA and diphenyl-1-pyrenylphosphine,were used to detect ROS production and lipid peroxidation,respectively.Visualization of bone development in zebrafish was analyzed by calcein-containing staining.In addition,the untargeted zebrafish metabolomics based on ultra-high performance liquid chromatography coupled with quadrupole-orbitrap high-resolution mass spectrometry(UPLC-Q-Orbitrap-HRMS)analysis combined with multivariate statistical analyses was used to investigate the potential anti-osteoporotic mechanism of POP.Results:Among four enzyme-assisted extractions,cellulase-assisted extraction had the highest yield.The first participating fraction of viscozyme extract POP(VPOP1)exhibited the highest antioxidant activity.VPOP1(Mw=7.6 k Da)with strong anti-osteoporosis activity was screened as an acidic heteropolysaccharide comprising mannose.VPOP1 treatment not only increased cell viability but also reduced ROS production in H2O2-induced MC3T3 cells.Staining and flow cytometry results show that VPOP1regulated the apoptosis of MC3T3 cells in a dose-dependent manner.The Western blot analysis results suggested that the level of Nrf2,Keap1 and Bcl-2 decreased significantly and those of HO-1,NQO1,caspase-3,Bax,and cytochrome C increased after the treatment with H2O2.Meanwhile,VPOP reversed the effect of H2O2.The in vivo experiment results showed that treatment of the 72hpf zebrafish embryos with VPOP1 inhibited ROS production and lipid peroxidation in a concentration-dependent manner,and could promote the growth of 9dpf zebrafish embryonic bone.The metabolomics analysis showed that compared with the model group,the levels of 28 potential biomarkers were gradually restored to normal after the treatment with VPOP.Among these biomarkers,leukotriene D4,11,12,15-trihydroxyeicosatrienoic acid,leukotriene A4,L-dopa,and L-tyrosine can be used as important biomarkers and therapeutic targets.Pathway analysis revealed that arachidonic acid metabolism(ACM),tyrosine metabolism(TM),phenylalanine,tyrosine and tryptophan biosynthesis(PTTB),and sphingolipid metabolism(SM)were the major intervened pathways.Conclusion:Among the 14 fractions,VPOP1 is an acidic heteropolysaccharide containing mannose,which has strong anti-osteoporosis activity.VPOP1 protected osteoblasts from oxidative damage in vivo and in vitro by regulating the Nrf2-Keap1 pathway.The antioxidant activity of VPOP can inhibit the apoptosis of MC3T3 cells by regulating the mitochondria-dependent pathways.Furthermore,the anti-osteoporosis effect of VPOP has been reliably confirmed by the metabolomics approach.VPOP can prevent osteoporosis by interfering with ACM,TM,PTTB,and SM.Further studies are needed to evaluate the efficacy of leukotriene D4,11,12,15-trihydroxyeicosatrienoic acid,leukotriene A4,L-dopa,and L-tyrosine as clinical biomarkers for the prediction of osteoporosis and their effect on bone health as dietary supplements.Hence,low-Mw VPOP1 from POL can be developed as a potential anti-osteoporotic functional food.
Keywords/Search Tags:Portulaca oleracea L. polysaccharide, Osteoporosis, Oxidative stress, Zebrafish, Metabolomics
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