Isolation And Identification Of Achyranthes Bidentata Active Peptide And Research On Its Biolofical Effects

Objective:The repair and functional reconstruction of injured nerve is a hot topic in biomedical field.It is urgent to develop new drugs which can promote neuronal growth and neuroprotection.Our lab used salting-out method to extract Achyranthes bidentata polypeptides(ABPP)from traditional Chinese medicine Radix Achyranthes Bidentatae Bl.The studies showed that ABPP has promising effects on promoting nerve regeneration and neuroprotection.In this study,we aimed to further separate and identify the active peptide monomer from ABPP under the guidance of activity and clarify its biological effects on promoting neuronal growth and neuroprotection on ischemic brain injury.Methods:Part ?:Isolation and identification of Achyranthes bidentata active peptide1.Salting-out method was used to extract ABPP from Achyranthes bidentata water extraction liquid.C18 reverse-phase column was used for gradient separation of ABPP to obtain secondary fractions.In vitro neuronal growth and oxygen-glucose deprivation(OGD)injury model of neurons were used to screening the active fractions isolated from ABPP.2.Biosphere C18 reverse-phase column and Q-TOF were used for further separation and identification of the screened active fraction,which were then screened through in vitro neuronal growth model to obtain the monomer active peptide.The amino acid sequence and secondary structure of monomer active peptide were characterized by Edman degradation and nuclear magnetic resonance technology.Part ?.Promotive effect of Achyranthes bidentata active peptide on hippocampal neuronal growth1.In vitro embryonic SD rats' hippocampal neurons were cultured to investigate the effects of Achyranthes bidentata active peptide(4 ng/ml,20 ng/ml,and 100 ng/ml)on neuronal growth.Growth index of hippocampal neurons was detected by real-time cell analysis system(RTCA).Immunocytochemical staining was used to observe the effects of Achyranthes bidentata active peptide on growth cone area,filamentous length,axon length,number of processes,number of branches,and synaptic density of hippocampal neurons.Growth speed of neurites was observed by living cell station.Western blot analysis was used to detect the expression level of postsynaptic density protein.2.Transcriptome sequencing was used to investigate the regulation of Achyranthes bidentata active peptide on cultured embryonic rat hippocampal neurons at different time points(0h,0.25h,1h,4h,and 12h).Bioinformatics techniques were used to analyze the hub genes involved in the regulation of hippocampal neuron growth.Part ?.Neuroprotective effect of Achyranthes bidentata active peptide on cerebral ischemia and reperfusion injury1.In vitro embryonic SD rats' cortical neurons were primary cultured and an OGD injury model was established.The protective effect of different concentration(8 ng/ml,40 ng/ml,and 200 ng/ml)of Achyranthes bidentata active peptide on OGD injured cortical neurons was detected by CCK-8.DCFH-DA,JC-1 and Hoechst staining were used to observe the protective effect of Achyranthes bidentata active peptide on ROS production,mitochondria dysfunction,and apoptosis of OGD insulted cortical neurons,respectively.Western blot analysis was used to detect the expression level of apoptosis-related proteins.2.In vivo cerebral ischemia and reperfusion model of rat was established by a monofilament middle cerebral artery occlusion(MCAO).The protective effects of different dose(0.1 mg/kg,0.5 mg/kg,and 1.0 mg/kg)of Achyranthes bidentata active peptide on cerebral infarction was observed through the measurement of cerebral infarct volume after TTC staining.SOD activity and MDA content in brain tissue were detected by xanthine oxidase and thiobarbital,respectively.Nissl staining was performed to observe the neurons integrity in ischemic penumbra.FITC-dextran perfusion was used to observe the effect of Achyranthes bidentata active peptide on vascular patency in the penumbra.Immunohistochemical staining was used to observe the effects of Achyranthes bidentata active peptide on fibrin and platelets deposition in the cerebral vascular endothelium and polymorphonuclear leukocytes infiltration in the penumbra.In situ zymography was used to evaluated the effect of Achyranthes bidentata active peptide on the activation of matrix metalloproteinases in ischemia penumbra.TUNEL and Western blot were performed to detect the effects of Achyranthes bidentata active peptide on apoptosis and the related proteins in the brain.Modified neurological severity score and Morris water maze test were performed to assess the effects of Achyranthes bidentata active peptide on long-term neurological deficits and learning and memory ability of MCAO rats,respectively.Results:Part ?:Isolation and identification of Achyranthes bidentata active peptide1.12 secondary fractions were obtained from ABPP by C18 reverse-phase chromatography,and the in vitro activity screening confirmed that the 11th fraction(named as ABPPk according to the order of elution)was the active one in promoting the outgrowth of neural processes and preventing the neuronal death induced by OGD.(China Invention Patent Number:201310108655.6)2.ABPPk was further separated by Biosphere C18 chromatography and Q-TOF to obtain two monomer peptides,referred to ABPPk1 and ABPPk2.In vitro neuronal growth activity screening showed that ABPPk2 had the neuroactive effects.Its precise molecular weight of ABPPk2 was 3416.46 Da.Edman degradation showed the sequence of amino acid as CXXXXXXCIPGXXXXCCXXVCVPIVTXXXXXXY.Stepwise derivation and NMR analysis showed the secondary structure of ABPPk2 has 3 pairs of disulfide bonds and reverse parallel folding structure.Part ?.Promotive effect of Achyranthes bidentata active peptide on hippocampal neuronal growth1.ABPPk2 can significantly promote the growth of hippocampal neurons as real time cell analysis detected in a dose-dependent manner.Immunocytochemical staining and Western blot analysis demonstrated that ABPPk2 can enlarge the growth cone area,prolong the axon length,promote the protuberance extension and synapse formation of hippocampal neurons,and upregulate the expression level of postsynaptic density protein.The observation from living cell station showed that ABPPk2 can accelerate the growth speed of neurites.2.Transcriptome sequencing and bioinformatics analysis of hippocampal neurons at different time points showed that ABPPk2 can significantly up-regulate the gene modules of transcription factors,neurogenesis and neuronal differentiation,and can induce the expression of differential genes.Thirteen hub genes were obtained by WGCNA-GO analysis,which were Satb2,Tmem2,Dlx1,Trim66,RGD1563615,Ctse,Rpap1,Ano1,Lhx8,Lgi1,Lrfn5,Ppplrlb and Vax1.The expression of one of them,Lhx8,was detected by RT-PCR,and the results were basically consistent with gene chip results.Part ?.Neuroprotective effect of Achyranthes bidentata active peptide on cerebral ischemia and reperfusion injury1.In vitro experiment results showed that ABPPk2 can reduce the decreased viability of OGD insulted neurons,reduce ROS production,reduce neuronal mitochondrial membrane potential reduction,and inhibit neuronal apoptosis.Western blot results showed that ABPPk2 upregulated the expression of anti-apoptosis protein(Bcl-2)and downregulated the expression of pro-apoptosis proteins(Bax,Cleaved caspase-3).ABPPk2 also inhibited the Cytochrome C and AIF released from mitochrondia to cytoplasm and nucleus,respectively.2.In vivo experiment results showed that ABPPk2 reduced infarct volume,reduced the MDA content and maintained SOD activity,improved the vascular patency of penumbra,reduced the neuronal death,reduced fibrin deposition and platelet aggregation in the vascular endothelium of penumbra,reduced the parenchymal neutrophil infiltration to parenchyma,and inhibited the activation of matrix metalloproteinases in a dose-dependent manner.Western blot analysis showed that ABPPk2 inhibited the expression of pro-apoptosis proteins and increased the expression of anti-apoptosis proteins.Modified neurological severity score and water maze test showed that high dose of ABPPk2 improved neurological deficits and learning and memory ability of MCAO rats for a long-term follow up.Conclusions:1.In this study,the monomer active peptide ABPPk2 with biological effects on promoting neuronal growth and neuroprotection was isolated and identified from Achyranthes bidentata.Its molecular weight was 3416.46 Da,and the amino acid sequence was CXXXXXXCIPGXXXXCCXXVCVPIVTXXXXXXY with a reverse parallel folding structure by 3 pairs of disulfide bonds.2.ABPPk2 could promote the neurite outgrowth and synapse formation of hippocampal neurons probably by regulating the expression of transcription factors,neurogenesis and neuronal differentiation related genes.3.ABPPk2 has neuroprotective effect on OGD insult rat cortical neurons and cerebral ischemia injury in rats.