| Objective:Ischemic stroke is a severe health condition with high mortality and disability rate.Kudiezi have shown to be effective in treatment of ischemic stroke in most clinical studies,although the exploration of its mechanism is not comprehensive.We therefore,via the MCAO/R model and neural cell lines model,undertake various system biology analyses,such as proteomics,high-throughput sequencing,bioinformatics,and network pharmacology,integrating different dimensions in vivo and in vitroto to explore a coherent account of the effects and mechanisms of Kudiezi in the treatment of ischemic stroke.Methods:(1)45 male SD rats were randomly divided into 15 in the sham-operated group and 30 in the model group.The modified thread plug method was used to establish the rat MCAO/R model.After successful modeling,the model group were randomly divided into 15 rats in model group and 15 in Kudiezi group.The dosage of Kudiezi was calculated based on the oral administration of 20g of Kudiezi for adults.After successful modeling,4 mL/kg Kudiezi extract was intraperitoneally injected into model animals every 3 hours.The sham group and model group were given intraperitoneal injection of equal amount of normal saline.After 24h,observed the neurological function score(mNSS),Ischemic infarction size(TTC staining),brain tissue morphological change(HE staining),neuronal cell apoptosis(TUNEL staining)of each group;(2)Proteomics was used to compare the protein expression profiles of cerebral ischemia penumbra in each group of animals,and verify by Western-blotting method;(3)Identify the components contained in Kudiezi and the components that cross the blood-brain barrier by UHPLC-Q-Exactive Orbitrap-MS.Analyze the target intersection of Kudiezi compnents and differential protein by network pharmacology method to identify the active cmponents.(4)PC 12 cells were cultured in DMEM F12K complete medium at 37?,5%CO2 incubator and treated with NGF for 3 days to stimulate PC 12 cell axon growth.And then 10 mM glutamate was used to treat the cells for 12 hours to establish the nerve excitotoxicity model,while Kudiezi and its active components were intervened for 3 hours.The cells after intervention were tested for cell viability by CCK-8 method.And the effect on glutamate levels in PC 12 cells also be detect.(5)After Kudiezi and its active ingredients intervene in the cells for 3 hours,the cells were collected and the cells were sequenced by RNA-Seq.Using bioinformatics to obtain signaling pathways mainly involved in regulation by Kudiezi and its active components.(6)Comprehensive analysis of in vivo proteomics results and in vitro transcriptomics results,and identifing core regulatory signaling pathways and it's downstream transcription factors.JASPAR is used to predict the binding sites of the above transcription factors in the promoter region of genes related to neurotransmitter transport.Intervene the above pathways with inhibitors,and verify the mechanism of transcriptional regulation by Kudiezi through the above pathways by RT-qPCR.Results:(1)This study found that the MCAO/R model had significant neurological impairment,and the neurological function score of the model animals was significantly reduced after treatment with Kudiezi.TTC staining found that MCAO/R model had obvious cerebral infarction,and the size of infarction in model animals was significantly reduced after Kudiezi treatment.Histological observation using HE staining revealed that there was significant tissue degeneration in the cerebral cortex and striatum of MCAO/R model animals.After Kudiezi treatment,the tissue degeneration was obvious getting better.Also found that the apoptosis of nerve cells in the Ischemia area of the model group and its surroundings increased by TUNEL staining;and after the treatment,the apoptosis of nerve cells decreased significantly.(2)Proteomics shows that the core network of differential proteins after Kudiezi intervention mainly involves the regulation of neurotransmitter levels.(3)A total of 81 components were found in Kudiezi by UHPLC-Q-Exactive Orbitrap-MS,9 of which could pass the blood-brain barrier.Among these nine components,the targets of Rutin,Ferulic acid and Adenosine had the highest agreement with the differential proteins regulated by Kudiezi.Therefore,we believe that Adenosine,Ferulic acid and Rutin are the active components of Kudiezi.(4)In the model of excitatory neurotransmitter toxicity stimulated with 10mM glutamate for 12h,the vitality of PC 12 cells was significantly inhibited;after intervention with Kudiezi and its active components,the cell viability caused by excessive glutamate significantly reduced.Calcium imaging showed that Kudiezi and its active components significantly inhibit the increase of intracellular calcium levels caused by glutamate;and after the treatment of Kudiezi and its active components,extracellular glutamate levels in PC12 cells were significantly reduced;And after the intervention of Kudiezi and adenosine,the level of glutamate in the cells was also significantly reduced.(5)We use bioinformatics method to analyse RNA-seq results.Comparing the enrichment analysis results of the differential genes in each experimental group,it was found that the pathways regulated by the differential genes in each experiment were overlapping and different.By overlapping the differential genes of PC 12 cells after the intervention of Kudiezi and its active components,a total of 277 core regulatory genes were obtained.The core regulatory genes are mainly involved in cAMP signaling pathway,PI3K-Akt signaling pathway,calcium signaling pathway,cGMP-PKG signaling pathway,axon guidance,D-glutamine and D-glutamic acid metabolism and many other signaling pathways.IPA analysis of core regulatory differential genes revealed that the canonical pathways involved in regulation include axon guidance,cAMP-mediated signaling,G protein-coupled receptor signaling,and eNOS signaling.(6)By analyzing the results of proteomics and RNA-seq,we discovered that Kudiezi and its active components can regulate cAMP signaling pathway,PI3K-Akt signaling pathway and other signaling pathways related to the regulation of neurotransmitter levels.Through analysis,it was found that there is cross-talk between the cAMP pathway and the PI3K pathway.Both pathways are involved in regulating Crebl and Nfkb1,and the cAMP pathway is also involved in regulating Nfatc4.They belong to transcription factors CREB,NF-?B and NFAT family.Using JASPAR to predict the binding site of transcription factors,it was found that Slc1a2 has both Creb1 and Nfkb1 binding sites in the promoter region of the gene;Vamp1 and Snap25 has Nfatc4 binding sites in the promoter region of the gene.Grin1 has both Nfkb1 and Nfatc4 binding sites in the gene promoter region.After blocking cAMP and PI3K pathways with inhibitors at the same time,the effects of Kudiezi and its active components on the above transcription factors disappeared,along with the disappearance of the regulation of genes related to neurotransmitter level regulation.Conclusion:The above research shows that Kudiezi plays an important role in treatment of MCAO/R model;the highlight of Kudiezi is regulating the neurotransmitters level in MCAO/R model,mostly associated with inhibition of nerve excitotoxicity.Furthermore the molecular mechanism is that Kudiezi and its components penetrated the blood-brain barrier simultaneously regulate the transcription factors of CREB,NF-?B and NFAT family via the cAMP pathway and PI3K pathway,and affect the transcriptional regulation of downstream genes including Vamp1,Snap25,Slc1a2 and Grin1.These genes inhibit exocytosis of vesicles,increase the transport of excitatory neurotransmitters and inhibit post-conduction of corresponding receptors,thereby exerting a neuroprotective effect by inhibiting neuronal excitotoxicity. |
PDF Full Text Request