| ObjectiveTo evaluate the neuroprotective effect of nuciferine on MCAO-rat and OGD-PC12,and further explore the potential mechanism from the perspective of G3BP1-mediated stress granules.Methods1.Literature researchLiterature search for this research retrieved from literature databases and search engines such as CNKI,Pub Med,Web of Science,Google and using nuciferine,stroke,ischemia,stress granule,G3BP1,neuroprotective/neuroprotection,MCAO,OGD and TCM as keywords.This research has systematically reviewed and analyzed the pathological process of cerebral ischemia,the mechanism of traditional Chinese Medicine,the animal model of stroke,the drug development of neuroprotective agents,the formation and depolymerization of stress granules and their function,the physicochemical properties and pharmacological mechanism of nuciferine,which expected to provide ideas and reference for the experimental design and development of this study.2.Neuroprotective effect of nuciferine on cerebral ischemia in ratsSD rats weighted 230-260 g were chosen and those rats were randomly divided into sham group,model group,EGB761 group,low(10 mg/kg)/middle(20 mg/kg)/high(40 mg/kg)dose of nuciferine group.Cerebral ischemia was induced by MCAO operation in all rats except sham group.On the pharmacodynamic level,Zea-longa fivelevel scoring method was used to evaluate the behavioral function of rats,infarct volume was evaluated by TTC staining,edema rate was evaluated by weighing the left and right brains,and death within one week after MCAO was investigated to evaluate the effect of nuciferine on survival rate.Morphologically,hematoxylin eosin(HE)staining was used to evaluate the pathological damage of brain tissue,neuron damage in brain tissue was evaluated by Nissl staining and Neun immunohistochemistry,and cell apoptosis in brain tissue was evaluated by Tunel and Cleaved Caspase-3immunohistochemistry.In addition,enzyme-linked immunosorbent assay(ELISA)kit or immunohistochemical method was also used in this study.Inflammatory and oxidative stress indexes such as TNF-α,IL-1β,NF-kB p65,total antioxidant capacity(T-AOC),GSH/GSSG,superoxide dismutase(SOD)and malondialdehyde(MDA)in serum and brain tissue were investigated to evaluate the anti-inflammatory and antioxidant capacity of nuciferine.3.Study on the characteristics of anticerebral ischemia effect of nuciferineIn this study,serum metabolomics and TMNP strategy were used to study the characteristics of anticerebral ischemia effect of nuciferine.In the metabolomics section,the rats were divided into 6 groups.Blood samples were collected from abdominal aorta24 hours after MCAO,and serum samples were collected by centrifugation for NMR detection after pretreatment.The detection method was 1D CPMG-presat mode,baseline correction and phase adjustment were carried out by MestReNova software,and the original data were normalized by area normalization method.The normalized data were further analyzed by principal component analysis(PCA)and Orthogonal Partial Least Squares-Discriminant Analysis(OPLS-DA)with SIMCA-P software,and the differential metabolites were screened by SPSS software.Chenomx NMR Suite database,Human Metabolome Database,KEEG,Metabo Analyst,Met Scape and Cytoscape were used to further identify differential metabolites and build metabolic networks.According to the results of network construction,the key enzymes regulating these differential metabolites were obtained,and the expression of these key enzymes at the mRNA level was investigated by PCR.In the TMNP section,based on the rat model of MCAO,all animals were divided into sham group,model group and the nucifeine(40 mg/kg)group.The cerebral penumbra tissues were taken 24 h after MCAO,mRNA was extracted and transcriptomic sequencing was performed.The TMNP strategy was adopted in this study.Firstly,the gene expression profiles information on 5 dimensions of tissue,cell,pathological process,biological process and signaling pathway was extracted and established from UCSC Xena database,Cellmarker database,Medical Subject Headings(MeSH)combined with Pub Med literature database,Gene Ontology(GO)database,Kyoto Encyclopedia of Genes and Genomes(KEGG)database.Then,the similarity between the transcription data induced by nuciferine and the gene expression profiles on the 5 dimensions established above was calculated by TMNP,and the pharmacological characteristics of nuciferine could be calculated and predicted.4.Study on the target of anticerebral ischemia of nuciferineThis part of study covers the following five parts: target prediction based on TMNP,molecular Docking,cellular thermal shift assay(CETSA),drug affinity responsive target stability(DARTS)and bio-layer interferometry(BLI).The target prediction based on TMNP was based on Connectivity Map database,the gene tag of the target was constructed,and then obtained the possible target information of nuciferine through correlation calculations.In the docking part,the structure of nuciferine and G3BP1 were download from database,and after pretreatment of their structural formula,the autodock software was adopted for molecular docking.According to the CESTA and DARTS experimental scheme,the total protein of the cell was extracted first,and a certain concentration of nuciferine(30/60 μM)was added.After incubation,gradient heating(37,45,53,61,69,77,85℃)was carried out in PCR instrument or a certain concentration of pronase was added to pyrolyze or enzymolize the protein.The effect of nuciferine on thermal tolerance and enzymatic tolerance of G3BP1 was evaluated by western blot.In the part of BLI experiment,biomolecular interaction instrument was used to obtain the binding and dissociation information of nuciferine with G3BP1 and its NTF2 domain,and the data was analyzed through Forte Bio Data Analysis 9.0 to obtain the kinetic parameters of nuciferine binding with G3BP1 protein.5.Study on the formation of stress granules by nuciferineIn this study,MCAO rat model and oxygen-glucose deprived PC12 cell model was used respectively.At the animal level,immunohistochemistry was used to evaluate the formation of the stress granules in the ischemic brain tissue by using G3BP1 and TIA1 as the marker protein.Based on immunofluorescence method,the co-localization of astrocytes marker GFAP,glial cells marker Iba-1,neuronal cell marker Neun and G3BP1 was assessed in order to evaluate the formation of stress granules in which cells.Furthermore,the co-localization intensity of G3BP1 and cell markers of the nuciferine group and the model group was also evaluated.At the cellular level,the safe dose of nuciferine was first investigated by MTT method,and on this basis,the protective effect of nuciferine on oxygen-glucose deprived PC12 cells was evaluated by MMT method and Calcein/PI double staining.Furthermore,si RNA interference technique was used to investigate the effect of nuciferine on cell protection after G3BP1 gene silencing.Finally,the aggregation of G3BP1 was investigated in different groups of neuronal cells by immunofluorescence method,which in order to evaluate the effect of nuciferine on stress granules at the cellular level.Results1.Literature researchExtensive and in-depth literature studies have found that ischemic stroke poses a serious threat to human health and there has an urgent need for innovative neuroprotective drugs.G3BP1 mediated stress granules are a kind of subcellular structure formed in cytoplasm under adverse environmental stimuli,and play an important role in the tumor and viral infection.In recent years,study found that the formation of stress granules can inhibit the apoptosis of neuronal cells in the cerebral ischemia,and also have important regulating effect on neurodegenerative diseases,which provide new ideas and perspectives for the development of innovative neuroprotective drugs.Nuciferine has obvious anti-cerebral ischemia effect,including anti-inflammatory,antioxidant,vasodilating,blocking L-glutamic acid,etc.However,it is not clear whether the anti-cerebral ischemia effect of nuciferine is mediated by stress granules.2.Neuroprotective effect of nuciferine on cerebral ischemia in ratsCompared with the sham group,the model group showed obvious neurobehavioral disorders and severe cerebral edema.TTC staining results showed obvious white ischemic area in brain tissue.HE pathological results showed that the ischemic tissue had obvious pathological damage,indicating that the animal modeling was successfully established.Compared with the model group,the neurobehavioral scores,cerebral infarction volume and edema rate of the nuciferine group(40 mg/kg)were significantly decreased(p<0.01).In addition,nuciferine(40 mg/kg)can prolong the survival time of ischemic rats,reduced ischemic brain pathological damage and decreased Tunel and Cleaved Caspase-3 positive cell numbers(p<0.01),increased the number of nissl and Neun positive cells(p<0.01),decreased the contents of MDA,IL-1β and TNF-α in brain tissue or serum(p<0.05 or p< 0.01),decreased the number of positive cells of IL-1βand NF-kB p65 in brain tissue(p<0.05 or p<0.01)and increased total antioxidant capacity(T-AOC),GSH/GSS ratio and SOD activity(p<0.05 or p<0.01).These results suggested that nuciferine has an obvious neuroprotective effect after cerebral ischemia,which may be related to anti-inflammatory,antioxidant and anti-apoptosis.3.Study on the characteristics of anticerebral ischemia effect of nuciferineBased on the study of serum metabolomics,39 endogenous small molecule metabolites were detected in serum.The results of PCA analysis showed that the clustering of each group was well separated,and the nuciferine group was located between the vehicle group and the sham group,suggesting that nuciferine has the effect of regulating the endogenous small molecule disorder caused by ischemia.After further analysis,a total of 19 differential metabolites were identified,and compared with the vehicle group,15 metabolites including arginine,isoleucine,glutamate,ornithine,histidine,N,N-dimethylglycine,betaine,glycine,glutamine,glucose,glycerol,creatine,serine,allantoin and tyrosine increased significantly after the treatment of nuciferine.Four metabolites including lactate,lysine,threonine and choline were significantly reduced.These differential metabolites can be enriched into 9 core metabolic pathways and are associated with 15 key metabolic enzymes.Through the comprehensive analysis of the differential metabolites,metabolic pathways and metabolic enzymes,it was found that the regulation of nuciferine on endogenous small molecule metabolites was mainly related to the effects of excitatory toxicity,inflammation,oxidative stress and other biological and pathological processes after cerebral ischemia.Forthurmore,TMNP strategy was used to establish the specific gene expression profiles of 31 tissues,2431 cells,437 pathological processes,5864 biological processes and 345 signaling pathways.The cluster heat map and PCA results of transcriptome data showed that there were little differences in each group,but significant differences between groups.The results of differential gene analysis showed that the expression of667 genes was reversed,and these differential genes could be significantly enriched into 25 signaling pathways and 52 biological processes.Including mRNA surveillance pathway,response to endogenous stimulus and other stress-related pathways,divalent metal ion transport,calcium ion transport and other metal ion transport processes.The correlation analysis based on TMNP showed that nuciferine had a good reverse effect on brain and spleen at tissue level,astrocytes and neural progenitor cells at cell level.At the same time,nuciferine can also act on immune and inflammatory processes such as inflammatory response,necrosis,response to cytokine,and synaptic related processes such as serotonergic synapse,axon guidance,synaptic signaling,and vascular remodeling such as blood vessel morphogenesis,angiogenesis,blood vessel development,etc.The results of TMNP research suggest that nuciferine may play a neuroprotective role after cerebral ischemia by regulating multiple immune and inflammatory biological processes mediated by central brain or peripheral spleen.4.Study on the target of anticerebral ischemia of nuciferineIn this study,the specific gene expression profiles of 4540 gene targets were constructed through the TMNP strategy,and the potential targets of nuciferine were focused on G3BP1 with the highest score through correlation calculations.And then,molecular docking technology was used to find that the binding energy of nuciferine and G3BP1 was-9.4 kcal/mol.The CETSA study showed that the band signal of G3BP1 protein decreased significantly with the increase of temperature.Compared with the negative control group,the band signal of nuciferine(60μM)pre-incubation group was significantly higher when the temperature was above 61℃(p<0.05 or p<0.01).DARTS experiment also found that compared with pronase group,the protein band signal was significantly enhanced after preincubation with nuciferine(30/60 μM)(p<0.05 or p< 0.01).The results of BLI experiment showed that nuciferine had good binding ability with G3BP1 and NTF2 domain.These results suggest that nuciferine may bind to the NTF2 domain of G3BP1 protein to further exert biological functions.5.Study on the formation of stress granules by nuciferineImmunohistochemical results of ischemic penumbra in rats showed that the number of TIA1 and G3BP1 positive cells increased significantly in the model group(p<0.01).compared with model group,the positive numbers of TIA1 and G3BP1 in nuciferine(40 mg/kg)group were further increased(p<0.01).Compared with TIA1,the signal variation trend of G3BP1 in each group was much more obvious.Immunofluorescence co-localization experiment showed that G3BP1 was mainly colocalized with neuronal cell marker Neun,and the number of cells with fluorescence co-localization was higher in nuciferine(40 mg/kg)group than in the model group.Cell experiments showed that nuciferine(60 μM)significantly increased the number of OGD-induced PC12 cells containing stress granules(p<0.01)and cell activity(p< 0.01).However,MTT results showed that the protective effect of nuciferine on OGD-PC12 cells was significantly inhibited after si RNA-G3BP1(p<0.05).The results of this study suggest that stress granules are mainly formed in neuronal cells after cerebral ischemia,and nuciferine can increase the number of stress granules in brain tissue of MCAO rats and OGD-PC12 cells.In vitro cell experiments also confirm that its neuroprotective effect is mediated by stress granules.ConclusionNuciferine showed an obvious anti-cerebral ischemic effect,including improving the neurobehavioral function,reducing the volume of cerebral infarction,reducing the rate of cerebral edema,and alleviating the pathological injury of ischemic brain tissue,and this effect was associated with the increase of G3BP1-mediated stress granules in neuronal cells,intervening in immune and inflammatory reactions and regulating oxidative stress state after ischemia. |
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