| ObjectiveIschemia/reperfusion injury is the major cause of mortality following circulatory shock,organ transplantation,myocardial and cerebral ischemia/reperfusion.Such injury remains a challenging unsolved problem.Data from a randomized clinical trial revealed that antioxidants failed to alleviate cardiovascular diseases,indicating that a more complicated system than oxidative injury per se drives hypoxia/re-oxygenation(H/R)-induced injury.Therefore,it is essential to explore new strategies to protect against H/R-induced injury through different mechanisms.Oligosaccharides extracted from M.officinalis have been reported to participate in various biological activities,including anti-oxidant,anti-stress and anti-fatigue functions,immunomodulation and protection from bone loss and degeneration,enhancing reproductive functions when treating infertility,and promoting angiogenesis in a chicken embryo-angiogenesis assay.However,no evidence has clearly indicated the main component of its function,as well as mechanism and target protein.In this study,the structural properties of oligosaccharides extracted from M.officinalis roots identified them as being inulins.In addition,we found that purified inulin 5 play a protective role on the vascular endothelium undergoing H/R-induced injury.Furthermore,the underlying mechanism and target protein were investigated through microarray analysis and magnetic beads binding inulin 5.Methods2.1.Extraction and separtion of M.officinalis oligosaccharidesThe dried roots of M.officinalis(10.0 g)were crushed into homogeneous particles,passed through a 40-mesh sieve,and extracted with 70%ethanol(200 ml)for 60 min at 90℃ in a round-bottomed flask.After recycling the ethanol vapor,a concentrated solution was obtained,which was diluted in distilled water and sequentially extracted in ether,ethyl acetate,and n-butanol.The ether-,ethyl acetate-,and n-butanol-soluble fractions were discarded,and then oligosaccharides of M.officinalis were obtained in the water-soluble fraction.Approximately 10g of the water-soluble fraction of M.officinalis was dissolved in 80 ml H2O and freeze-dried.Then,8.3 g of the freeze-dried fraction was dissolved in 100 ml of pyridine and 100 ml of acetic anhydride,and stirred at 0℃ for one day.The solvent was evaporated under reduced pressure at room temperature.CHCl3 was added to the residue and washing was performed with saturated solutions of aqueous sodium bicarbonate and NaCl.After drying with magnesium sulfate,the CHCl3 was distilled off under reduced pressure.Fractions were collected by silica gel column chromatography(developing solvent:toluene:ethyl acetate=2:1→1:1→1:2→1:3→1:5).Finally,each acetylated monomer was isolated.The molecular weight of purified each acetylated monomer was estimated by MALDI TOF-MS and the structure of the acetylated monomer was identified by 1H NMR and COSY(Medicinal Chemistry Pharmaceuticals,Co.,Ltd,Japan,shown in the Supplementary material).Monomers per-Ac were respectively dissolved in methanol(5 ml)and sodium methoxide(MeONa,18 mg)for 2.5 h at room temperature,after which Dowex 50W×2H+resin was added.After Dowex filtration,the solvent was evaporated under reduced pressure.The residue was dissolved in H2O and was desalted using a Bio-Gel P-2 gel-filtration column.After freeze drying,monomers were acquired by performing deacetylation,as described above.The purity of monomer was more than 98%analyzed by high performance liquid chromatography(HPLC).2.3.Analysis by MALDI-TOF-MS and NMR spectroscopyThe water-soluble fraction of M.officinalis prepared at a concentration of 2 mg/ml with H2O,mixed with matrix 2,5-dihydroxybenzoic acid sodium salt,and spotted(0.5 μl)on the target plate.The key components were analyzed by MALDI-TOF-MS(AB SCIEX TOF/TOF 5800,positive mode).Extract samples derived from radix of Morindae Officinalis contain a series of fructan after estimated by MALDI TOF-MS.It has been reported that the plant fructan has various structures.Therefore,1H NMR and COSY were used to determine the structure based on spectral comparison with standared samples.2.4.DPPH radical-scavenging assayInulin 4-7 at varying concentrations(0.31,0.62,1.25,2.5,5,10,20,40,and 80 mM;100 μl)or vitamin C(0.015,0.03,0.06,0.125,0.25,0.5,1.0,2.0,and 4.0 mM;100 μl)were added to 100 μl of 0.1 mM DPPH solution in ethanol,followed by shaking and a further 30-min incubation in the dark before measuring the absorbance at 517 nm in a microplate reader.The percent radical-scavenging activity was calculated using the following formula:Radical-scavenging activity(%)=([Ac-As]/Ac)×100%,where Ac is the absorbance of the control and As is the absorbance of the test compound.Each assay was repeated 3 times.2.5.H/R-induced injury modelHUVECs were cultured in RPMI1640 containing 10%fetal bovine serum in a humidified incubator with 5%CO2 at 37℃.At passage 3,HUVECs(4×104 cells/ml,100 μl)were seeded into a 96-well plate(4×103 cells/well)and cultured for 12 h in RPMI 1640 without fetal bovine serum to promote synchronization.Cells in the normoxic group were incubated under the normoxia condition and were treated with various concentrations of vitamin C,or Inulin 4-7 for 44 h.In the H/R group,HUVECs were treated with Na2S2O4 at a concentration of 5 mM in hypoxia solution(0.9 mM Na2HPO4,6.0 mM NaHCO3,1.8 mM CaCl2,1.2 mM MgSO4,40 mM sodium lactate,10 mM HEPES,98.5 mM NaCl,10.0 mM KCl;pH=6.8),after which they were transferred into a hypoxic incubator in a humidified atmosphere equilibrated with 94%N2,1%O2,and 5%CO2 for 1 h(to promote hypoxia).Subsequently,the hypoxia solution was replaced with RPMI1640 containing 1%fetal bovine serum and various concentrations of vitamin C(positive-control group),or Inulin 4-7,after which the cells were transferred to a humidified incubator with 5%CO2 at 37℃(reoxygenation).HUVECs were randomized into the following groups(n=5,per group):the normoxic group(N),the N+vitamin C group(0.06,0.12,0.25,0.5,1.0,and 2.0 mM),the N+Inulin 4-7 group(2.34,4.68,9.38,18.75,37.5,75,and 150 μM),the H/R group,the H/R+ vitamin C group(0.06,0.12,0.25,0.5,1.0,and 2.0 mM),and the H/R+Inulin 4-7 group(2.34,4.68,9.38,18.75,37.5,75,and 150 μM).2.6.Cell Counting Kit-8(CCK-8)assaysAfter treatment with vitamin C or Inulin 4-7 for 44 h,cell proliferation was determined using the Cell Counting Kit-8 according to the manufacturer’s instructions.Then,WST-8,5 mg/ml,10 μl,was added to each well followed by a 2-h incubation to allow the formation of orange formazan crystals.The absorbance at a wavelength of 450 nm was determined using a 96-well microplate reader.Each assay was repeated 5 times.Cell viability%=([As-Ac]/Ac)×100%2.7.Tube-formation assays with HUVECsThe formation of capillary-like structures was assessed in a 24-well plate using a 290-μl growth factor-reduced Matrigel.Early-passage(passage 1-3)HUVECs(6 x 104 cells/ml;300 μl)cultured in normal oxygen for 19 h,or HUVECs injured by H/R(hypoxia for 1 h and reoxygenation for 18 h)and treated with or without vitamin C(2 mM),Inulin 5(30 μM),or Inulin 5(30 μM)+LY294002(PI3K inhibitor,1 pM),were re-suspended and seeded on the top of the Matrigel.After 18 h at 37℃,images were taken,and the total tube lengths and branching points were quantified using Image-Pro plus software,version 5.1.Each assay was repeated 3 times under similar conditions.2.8.Affymetrix Human Transcriptome Array(HTA)2.0The Affymetrix HTA 2.0 contained approximately 67,000 transcript clusters and 573,000 probe-selection regions.Total RNA was isolated from HUVECs in the normoxic,H/R and H/R+Inulin 5(30 μM)groups(n=3)using the TRIzol Reagent following the manufacturer’s instructions.The RNA integrity number was determined by inspecting the RNA integrity with Agilent Bioanalyzer 2100 RNA.The RNA-integrity mumber>7 was considered to be of sufficient quality for microarray experiments.Qualified total RNA was further purified using the RNeasy Micro Kit and the RNase-Free DNase Set.The gene-expression profile and alternative splicing events(analysis of variance[ANOVA],P<0.05,fold change>1.5)were analyzed by Shanghai Biotechnology Corporation.The Database for Annotation,Visualization and Integrated Discovery(DAVID)(http://david.abcc.ncifcrf.gov)was used to analyze related Kyoto Encyclopedia of Genes and Genomes(KEGG)functional pathways.2.9.Quantitative reverse transcription-polymerase chain reaction(RT-qPCR)RT-qPCR was used to confirm expression of cyclin E2(CCNE2),cyclin dependent kinase 6(CDK6),cyclin dependent kinase inhibitor 1A(CDKN1A/p21),and tumor protein p53(TP53/p53)mRNA in synchronized HUVECs after 1-h hypoxia and 3-h reoxygenation treatments,with or without Inulin 5(30 μM).Total RNA was prepared using the TRIzol Reagent,and total RNA(1 μg)was reverse-transcribed using a PrimeScriptTM RT Reagent Kit with gDNA Eraser,according to the manufacturer’s protocol.cDNA was diluted 1:3 with H2O and used for real-time PCR.Real-time PCR was performed using the SYBR Premix Ex TaqⅡMix Kit and an ABI 7500 system with the following primers for human p21,p53,CCNE2,CDK6,and GAPDH(a reference gene):CDKN1A/p21:forward 5’-catgtggacctgtgtcactgtcttgta-3’and reverse 5’-gaagatcagccggcgtttg-3’;TP53/p53:forward 5’-tcgagatgttccgagagctgaat-3’ and reverse 5 5’-gtctgagtcaggcccttctgtctt-3’;CCNE2:forward 5’-tgggaactttgtcctgtaacaatca-3’ and reverse 5’-cacaaggcagcagca gtcagta-3’;CDK6:forward 5’-gtgaccagcagcagcggacaaataa-3’ and reverse 5’-agcaaga cttcgggtgctctgta-3;’,and GAPDH:forward 5’-gcaccgtcaaggctgagaac-3’ and reverse 5’-tggtgaagacgccagtgga-3’.Cycle threshold(Ct)values were acquired.The relative expression levels of p53,p21,CCNE2,and CDK6 mRNA in cells were calculated using the 2-△△Ct method,as follows:△Ct=Ct of target gene-Ct of reference gene(GAPDH);△△Ct=the mean value of(△Ct of target gene in experimental sample-△Ct of target gene in reference sample)±standard deviation.The relative initial levels of each template were calculated as the mean value of(2-△△Ct)±standard deviation.To ensure accuracy,the experiments were performed in triplicate.2.10.Cell cycle analysisSynchronized HUVECs were divided into the following eight groups:(i)normal,(ii)normal+Inulin 5(30 μM),(ⅲ)N+Inulin 5(30μM)+LY294002(PI3K inhibitor,1 pM),(iv)N+LY294002(1 μM),(v)hypoxia 1 h+re-oxygenation 18 h(H/R),(ⅵ)H/R+Inulin 5(30 μM),(ⅶ)H/R+Inulin 5(30 μM)+LY294002(1 μM),and(ⅷ)H/R+LY294002(1 μM).Cells in each group were harvested and washed with PBS and fixed overnight at 4℃ in 75%ethanol.After being treated with RNase A and propidium iodide solution for 15 min in the dark,the cell cycle distribution in each group was determined by flow cytometry,and the results were analyzed using ModFit LT 5.0 software.2.11 Western blot analysisPrior to protein extraction,hypoxic cells were re-oxygenated for 0.5,1.5,3,or 6.HUVECs were lysed in ice-cold Radio-Immuno-precipitation Assay Lysis and Extraction Buffer.Equal amounts of proteins(50 μg)were separated by 12%sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes.After blocking in 5%nonfat milk,the membranes were incubated overnight at 4℃ with antibodies against p-Akt(1:2000),Akt(1:2000),GAPDH(1:2,000),anti-eNOS(1:700),and anti-p-eNOS(Ser1177)(1:700);washed with Tris-buffered saline(TBS)containing 0.05%Tween-20(TBST);and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 2 h at 37℃.Immunoreactive bands were detected using the chemiluminescence gel imaging system.2.12 Confocal microscopy analysisHUVECs were inoculated into a special dish for laser confocal microscopy.When the cells were 80%full,the model of hypoxia/reoxygenation injury was created.At the same time as reoxygenation,FITC and FITC-Inulin5 were added in groups,37℃.Incubate for 12 h.After fixation,washing,ER-Tracker Red and DAPI staining,the cells were observed under a laser confocal microscopy and photographed.2.13 Inulin-5 binding magnetic beads analysisHUVECs were lysed with NP40 lysate and the total protein concentration was approximately 10 mg/ml.Took 0.5 mg of inulin-5 binding magnetic beads and negative control magnetic beads,added 200 μl of 100 mM KCl buffer,mixed by pipetting,4℃,washed 3 times,Added 200 μl of total protein solution after centrifugation,mixed by pipetting.The binding reaction was carried out at 4℃ for 4 hours on a magnet holder on shaker.After magnetic separation,200 μl of 100 mM KCl buffer was washed 3 times.Then,30 μl of 1 M KCl buffer was added and mixed by pipetting,standed in 4℃ for 5 minutes.After magnetic separation,SDS-PAGE electrophoresis was carried out.2.14 SDS-PAGE electrophoresis,silver staining and protein profilingAfter SDS-PAGE electrophoresis,the gel was subjected to silver staining by washing,fixing,eluting,washing,sensitizing,silver staining,water washing,developing and terminating.The gel strips were subjected to desilvering,dehydration,reduction and alkylation.Trypsinized the protein,extracted the peptide,and concentrated to about 15μl with a freeze dryer.Then Easy-n LC1000 Orbitrap Fusion Protein Spectrometer was used to analyze.2.15 Transfection of HUVECs with SiRNA-Annexin A2Inoculated 2×105 cells/well into 6-well plates,and the HUVECs density should be about 50%.Mixed 5μl 120μM siRNA stock solution and 120μl 1×ribo FECTTM CP buffer,and incubated for 5min at room temperature.Added 12 μl of riboFECTTM CP transfection reagent,mixed by pipetting,and incubated for 0-15 min at room temperature.Added riboFECTTM CP mixture to 1863μl serum-free medium(6-well plate per well),mixed;culture at 37℃ for 48h.Knockdown efficiency was verifed by RT-PCR and Western-blot.2.16 Detection the effect of inulin-5 on the proliferation and CDK6 in H/R HUVECs after knockdown Annexin A2.Inoculated 2×103 cells/well into 96-well plates or 2×0105 cells/well into 6-well plates,and divided into seven groups,N,H/R,H/R+Inulin5,H/R+SiRNA-NC,H/R+SiRNA-NC+Inulin5,H/R+SiRNA-Annexin A2,H/R+SiRNA-Annexin A2+Inulin5.After transfected 48h,HUVECs were made hypoxia/reoxygenation model,according to the above group to give inulin 5(dissolved in 1%serum medium)or 1%serum medium.Each group was cultured for 42 hour to be detected by CCK-8 or 2 hour for performing RT-qPCR.2.17 Surface plasmon resonance analysis of affinity between Inulin 5 and Annexin A2The Annexin A2 protein was expressed and purified.The Biacore biomolecular interaction instrument coated the Annexin A2 protein on the CM5 chip to detect the affinity between Inulin 5 and Annexin A2.2.18 Molecular docking analysis of between Inulin 5 and Annexin A2 or Inulin 5 and Annexin A2-S100A10Maestro software was used to predict the binding sites of Inulin5(CAS:59432-60-9)to Annexin A2(PDB ID:1W7B)or Annexin A2-S100A10(PDB ID:5LPU).Statistical analysesThe experimental data were analyzed by 1-way ANOVA and Student’s t-test.The results were expressed as the mean ± standard error and 95%confidence interval.P-values<0.05 were considered statistically significant.Statistical tests were conducted using SPSS software,version 19.0 and GraphPad Prism 5 software.Results3.1.Degrees of polymerization and the molecular weights of oligosaccharides in M.officinalisThe degrees of polymerization and the molecular weights of oligosaccharides were determined by MALDI-TOF-MS.In the 500-3000 region of the mass/charge(M/Z)spectrum,the most abundant ion adducts[M+Na]+were identified at m/z 527,689,851,1013,1175,1337,1499,1661,1823 and 1985.So the molecular weight of these oligosaccharides were 504,666,828,990,1152,1314,1476,1638,1800,1872 respectively.The ratio of the area of each oligosaccharide was determined relative to that measured for the[M+Na]+spectral peak area for m/z=527(Inulin 3),which was defined as 1.3.2.Structures of oligosaccharides in M.officinalisThe NMR data revealed that the oligosaccharide composed of fructose unit chains(linked by P-(2-1)-D-fructosyl-fructose bonds)of various lengths,and terminated by an a-D glucopyranosoyl bond.The structures were a-D-Glcp-(1→2)-[beta-D-Fruf-(2→1)-beta-D-Fruf](n)-(2→1)-beta-D-Fruf,which are characteristic of inulin-type oligosaccharides.3.3.DPPH radical-scavenging activity of Inulin 4-7 from M.officinalisInulins 4-7 exerted dose-dependent radical-scavenging activities.The IC50 values of Inulins 4-7 were 13.81 mM(95%confidence interval:12.63-15.09,n=3),15.08 mM(95%confidence interval:13.95-16.31,n=3),18.21 mM(95%confidence interval:16.31-20.35,n=3),and 18.72 mM(95%confidence interval:16.76-20.91,n=3),respectively.The potency and efficiency decreased with increasing degrees of polymerization.3.4.Inulin 4-7 promoted cell survival and proliferation of HUVECs with and without H/RInulin 4 and Inulin 5 significantly enhanced HUVEC viability compared with the H/R control group,in dose-dependent manners.The EC50 values for Inulin 4 and Inulin 5 were 12.65 μM(95%confidence interval:7.14-22.43 μ,n=5)and 29.97μM(95%confidence interval:9.15-98.14 μM,n=5),respectively.Inulin 6 and Inulin 7 showed similar effects but to a lesser extent,compared with Inulin 4 and Inulin 5.Moreover,Inulin 5 significantly promoted proliferation under normoxia conditions,and the EC50 for Inulin 5 under normoxia conditions was 12.57 μM(95%confidence interval:7.97-19.84 μM,n=5).The EC50 value for the positive control vitamin C was 0.60 mM(95%confidence interval:0.53-0.69 mM,n=5).3.5.Inulin 5 promoted tube formation of HUVECs with or without H/R-injuredLittle tube formation was observed when H/R-injured HUVECs were cultured in Matrigel.While treatment with Inulin 5 significantly stimulated the capillary network formation of endothelial cells(P<0.05),including increasing areas covered by HUVECs and network lengths.Furthermore,LY294002(PI3K inhibitor,1 μM)inhibited the effect of Inulin 5.3.6.Experimental results of Affymetrix Human Transcriptome Array(HTA)2.0The microarray datasets of the Affymetrix HTA 2.0(n=3)were submitted to the Gene Expression Omnibus Database under GEO accession number GSE103770(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103770).Univariate analysis revealed that 517 genes(including 19 up-regulated genes and 498 down-regulated genes)showed differential expression with more than 1.5-fold difference between the H/R-injury and normal groups.In addition,649 differentially expressed genes(including 641 up-regulated genes and 8 down-regulated genes)were observed between Inulin 5-treated(30 μM;12 h)and untreated H/R cells.The expression of 330 genes decreased after H/R injury,but markedly increased after a 12-h Inulin 5 treatment,as illustrated in the heatmap..3.7.Biological pathways and functional-enrichment analysisWe performed an enrichment analysis using the DAVID database to highlight the most relevant biological pathways influenced by H/R injury and Inulin 5.KEGG pathway analysis showed that down-regulated genes in HUVECs after H/R injury were enriched in terms of processes such as DNA replication,mismatch repair,homologous recombination,cell cycle,oocyte meiosis,apoptosis,cancer and P53,as well as the neurotrophin-signaling pathway,and that these genes were mostly up-regulated after Inulin 5 administration.Moreover,Inulin 5 treatment up-regulated other genes associated with nucleotide excision repair,progesterone-mediated oocyte maturation,ubiquitin mediated proteolysis,vascular smooth muscle contraction,long-term potentiation,and insulin pathways,as well as signal-transduction pathways such as the mitogen-activated protein kinase(MAPK),mammalian target of rapamycin(mTOR),phosphatidylinositol,inositol phosphate metabolism,and Wnt pathways.3.8.Inulin 5 modulated the mRNA expression of cell cycle-related genes in HUVECsCCNE2 and CDK6 have been shown to promote the G1/S cell cycle transition,whereas p21 and p53 mediated cell cycle arrest in G1 phase in response to various stress stimuli.Inulin 5 upregulated the mRNA-expression levels of CCNE2 and CDK6(P<0.01,n=6),while downregulating the mRNA-expression levels of p53 and p21(P<0.01,n=6),compared to H/R control cells.3.9.Inulin 5 Induced Cell Cycle Transition of HUVECs after H/R TreatmentThe impact of Inulin 5 on cell cycle progression was assessed by flow cytometry.Inulin 5 treatment enhanced the percentage of cells in S and G2/M phase compared to the H/R group(P<0.01,n=3),and LY294002(PI3K inhibitor,1 μM)inhibited the effect of Inulin 5.3.10.Inulin 5 regulated the PI3K-Akt-eNOS signaling pathwayThe phosphorylation on Ser1177 of eNOS and total eNOS expression were decreased in response to H/R treatment(P<0.05,n=3),in agreement with previously research.However,Inulin 5 significantly increased total eNOS expression,eNOS Ser1177 phosphorylation,and the p-eNOS/eNOS ratio in HUVECs undergoing H/R(P<0.05,n=3).In parallel,Akt Ser473 phosphorylation significantly decreased during H/R(P<0.05,n=3),whereas total Akt expression did not change when cells underwent H/R or Inulin 5 treatment(P>0.05,n=3).However,Inulin 5 significantly increased Akt Ser473 phosphorylation after H/R(P<0.05,n=3).LY294002 inhibited the effect of Inulin 5.3.11.FITC-Inulin5 located in the cytoplasm or on the membrane of HUVECsUnder the confocal microscope,FITC-Inulin5 was mainly located in the cytoplasm or on the membrane.FITC-Inulin5(the green fluorescent)was co-localized with the endoplasmic reticulum(red)but not with the DAPI stained nuclei(blue).3.12.Inulin 5 binding magnetic beads pulled down target proteinsThe bandage of 35-40KD by protein mass spectrometry was most likely to be Annexin a2.The bandage of 40-55KD was identified as the elongation factor EF-1a.3.13.Si-RNA Annexin a2-3 knocked down the expression of Annexin A2 in HUVECs after transfection.Compared with the SiRNA negative control,transfection of Si-RNA Annexin a2-3 into HUVECs in serum-free medium significantly knocked down Annexin A2-3,and knockdown efficiency about 77.3±3.4%(P<0.01,n=3).In the whole culture medium of 10%fetal bovine serum,Si-RNA Annexin a2-3 could also knock down Annexin a2,but the knockdown efficiency is 38.4±6.4%(P<0.01,n=3).According to the experimental results,Si-RNA Annexin a2-3 was selected to transfect HUVECs in serum-free medium for subsequent experiments.3.14 Inulin 5 losed protective effect on H/R HUVECs after knockdown Annexin A2 by SiRNAAfter Annexin A2 knocked down,inulin 5 losed its protective effect on H/R HUVECs cells(P>0.05,n=6).While,compared H/R with H/R+Inulin5 group,or compared H/R+SiRNA-NC with H/R+SiRNA-NC+Inulin5 group,inulin 5 protected HUVECs from H/R injury(P<0.01,n=6).3.15 Inulin 5 losed promoted effect on CDK6 in H/R HUVECs after knockdown Annexin A2 by SiRNAAfter Si-RNA knocked down Annexin A2,inulin 5 could not promote the expression of CDK6 in H/R HUVECs.There was no significant difference between the H/R+Si-Annexin A2 group and the H/R+si-Annexin A2+Inulin 5 group(P>0.05,n=9).While,compared H/R with H/R+Inulin5 group,or compared H/R+SiRNA-NC with H/R+SiRNA-NC+Inulin5 group,inulin 5 promoted intracellular CDK6 in H/R HUVECs(P<0.01,n=9).3.16 Surface plasmon resonance detected affinity between Inulin 5 and Annexin A2The binding ability between molecules was shown as resonance unit(RU),which reflected the corresponding change in the refractive index of the optical signal.The value of RU between Inulin5 and Annexin A2 increased with the concentration of Inulin 5.ka=27.12,KD=37.5μM.3.17 Molecular docking analysis of Inulin 5 and Annexin A2 or between Inulin 5 and Annexin A2-S100A10Molecular docking showed that Inulin 5 formed hydrogen bond and hydrophobic interactions with Annexin A2 or Annexin A2-S100A10.ConclusionThe novel findings of this study are as follows.(ⅰ)Morinda oligosaccharides are composed of inulin oligosaccharides with a degree of polymerization of 3-13,and can appropriately remove nitrogen free radicals.(ⅱ)Inulin 5 protects hypoxic and reoxygenated HUVECs by promoting cell cycle progression and tubule formation through activating the PI3K-Akt-eNOS signaling pathway.(ⅲ)Annexin A2 is one of the target proteins of inulin 5 protecting human umbilical vein endothelial cells. |
PDF Full Text Request