| BackgroundStroke is equivalent to "apoplexy" in Traditional Chinese Medicine,with "three high"(high incidence,high morbidity and high mortality)characteristics.It can be divided into ischemic stroke and hemorrhagic stroke in pathogenesis.Ischemic stroke is the majority in China.Improving prevention and treatment of stroke and "three high" is a hot and difficult issue of international research.The pathological process of ischemic stroke is a cascade enzymatic reaction of multi-factor,multi-link and multi-pathway damage with complex molecular network mechanism.Stroke have different syndromes:wind,fire,phlegm,blood stasis,deficiency.Blood stasis is the core of apoplexy and therapies activating blood circulation is common treatment of ischemic stroke.Research showed HuoxueHuayu recipe can improve blood supply in the ischemic region,which is an initial process in ischemic cascade reaction.The rapeutic purpose of ischemic stroke treatment is not only to improve blood supply in ischemic zone,but also block "waterfall cascade molecular chain" of cerebral ischemia.It is important to reveal the role of HuoxueHuayu recipe in the cascade reaction and the molecular network mechanism of anti-cerebral ischemia,which is helpful to elaborate mechanism of HuoxueHuayu recipe and provide theoretical basis for prevention and treatment of cerebral ischemia.Based on this,the study chose HuoxueHuayu recipe-Danhong recipe(Danhong injection)with good curative effect as starting point.The effects of Danhong injection in different aspects of pathological cascade reaction of cerebral ischemia were investigated.Combined with proteomics and metabolomics technologies,the comprehensive study on characteristics and mechanism of Danhong injection in anti-cerebral ischemia was conducted to interpret the role of HuoxueHuayu recipein prevention and treatment of cerebral ischemia on molecular level.The research was supported by National Natural Science Foundation of China(No.81330086)project.ContentThis study includes two parts:literature review and experimental study.Literature research:We reviewed the pathological mechanism,signal transduction pathways related,clinical effects,pharmacological effects and related mechanisms of Danhong injectionin cerebral ischemia to provide reference for later experiments.Experimental research:On the basis of compatibility of Salviae Miltiorrhizae Radix et and Rhizoma Carthami Flos and,the "dose effect"and "time effect" of Danhong injection on cerebral ischemian,the effects of Danhong injection in different aspects of pathological cascade reaction of cerebral ischemia were investigated.The mechanism of Danhong injection in anti-cerebral ischemia was explained inprotein and amino acid level for prevention and treatment of cerebral ischemia.The experiments consist of three parts:1.Compatibility of Salviae Miltiorrhizae Radix et and Rhizoma Carthami Flos based on data-miningIn this study,formula containing Salviae Miltiorrhizae Radix et and Rhizoma Carthami Flos in database of Dictionary of Chinese Medicine Prescription(DCMP)and National Standard for Chinese Patenr Drugs(NSCPD)were extracted using Traditional Chinese Medicine Inheritance Support System(TCMISS).Composition rules were analyzed with data mining methods such asassociation rules.Improved mutual information method and complex system entropy helped in better understanding the compatibility principles and indications of Danhong injection and provided theoretical support for following experimental studies.2.Anti-cerebral ischemia effect of Danhong injection based on pathological response of cerebral ischemiaFirstly,the model of cerebral ischemia was induced with different doses of Danhong injection for selecting the best dose.We observed the effect of Danhong injection on cerebral ischemia in 12h and 24h.Secondly,the protective effects of Danhong injection on pathological cascade reactions of cerebral ischemiain neuronal apoptosis and blood-brain barrier.We reinvestigated to clarify its characteristics in cerebral ischemiaand lay foundation for later mechanism research.3.Anti-cerebral ischemia mechanism of Danhong injection based on omicsBased on proteomics technology,identified the effects of Danhong injection on protein level in hippocampus of rats with cerebral ischemia by mining data,screening changes of enriched proteins and pathways and verification.At the same time,based on metabolomics technology,the effects of Danhong injection on amino acid metabolism in cortex of rats with cerebral ischemia were also analyzed to comprehensively explain its mechanismin anti-cerebral ischemia.Results1.Study on the compatibility of Salviae Miltiorrhizae Radix et and Rhizoma Carthami Flos based on data-miningThere were 39 prescriptions in DCMP,74 Chinese traditional medicines in NSCPD.Salviae Miltiorrhizae Radix et,Rhizoma Carthami Flos are often combined with herbs promoting blood circulation and activating qi,most of which were distributed to heart and liver channel.Their main disease area is chest bi-syndrome,angina pectoris,palpitation,stroke,blood stasis,pain syndrome,gynecological disease.2.Study on the anti-cerebral ischemia effect of Danhong injection based on pathological response of cerebral ischemia(1)Dose-effect and time-effect study of Danhong injection on anti-cerebral ischemiaThe effects of Danhong injection of different doses(1.44,0.72,0.36,0.18mL/kg)on cerebral ischemia in rats showed 0.72mL/kg best improved the neurological function and the volume of cerebral infarction in rats with cerebral ischemia.There was dose effect relationship in 0.72 mL/kg,0.36 mL/kg and 0.18 mL/kg dose,effect of 0.72mL/kg Danhong injection on cerebral ischemia in 24h was better than that of 12h.(2)Effects of Danhong injection on blood-brain barrier permeability in rat with cerebral ischemiaDanhong injection improved the neurological function score and infarct volume in rats with cerebral ischemia,reduced the degree of cerebral edema and breakdown of blood brain barrier and adjusted the content of ?-actinin protein in brain and heart tissue under cerebral ischemia.The results suggested that changes of a-actinin protein might be one of the molecular bases in the Danhong injection treatment of brain and heart disease.(3)Effects of Danhong injection on neuron apoptosis in rat of cerebral ischemiaNeuronal apoptosis appeared in hippocampus of rats after cerebral ischemia,ischemia induced expression of Apaf-1 and its downstream molecules Caspase-9 and Caspase-3,thus reduced Bcl-2/Bax ratio.Danhong injection could significantly inhibit the activation of Apaf-1,Caspase-9 and Caspase-3,the expression of Bax,Bcl-2.Bcl-2/Bax ratio reversed.The results showed that Danhong injection could inhibit the expression of Apaf-1 in hippocampus of rats with cerebral ischemia and the activity of Caspase signaling pathway,which played a role in anti-apoptosis and finally slowed the expansion of Caspase mediated apoptosis in the penumbra of the ischemic core.3.Study on the anti-cerebral ischemia mechanism of Danhong injection based on omics(1)Study on the anti-cerebral ischemia mechanism of Danhong injection based on target metabolomicsThe amino acids(Val,Ile,Leu,Lys)and neurotransmitter amino acids(Gln,Glu,Gly,Ser,Tau,Tyr,Phe)in rats with cerebral ischemia significantly changed.Danhong injection could selectively regulate the abnormal amino acids which provide energy in the brain,inhibit Glu content and excitotoxicity amplification of Gln,Gly,and Ser.It suggested Danhong injection could improve brain energy metabolism and release/synthesis of neurotransmitter amino acids,thus play a role in brain protection.(2)Study on the anti-cerebral ischemia mechanism of Danhong injection based on proteomics technologyUsing proteomics experiments,the molecular mechanism of Danhong injection against cerebral ischemia was studied.12 proteins were significantly correlated with the effect of Danhong injection on cerebral ischemia,mainly concentrated in the WNT signaling pathway,AMPK signaling pathway and MAPK signaling pathway.Founded that glycogen synthase kinase-3?(Glycogen synthase kinase-3?,Gsk-3?)and serine/threonine protein kinase(RAC-alpha serine/threonine-protein,kinase,Akt1)were core regulatory proteins of Danhong injection against cerebral ischemia through protein interaction network analysis.Through verification experiment,11 proteins in western blot and mass spectrometry,except Ctbp2,had the same trend.Gsk-3? protein inhibitor experiments also showed that Danhong injection played a role in candidate protein expression in the hippocampus of cerebral ischemic injury.In conclusion,this study preliminarily revealed the characteristic of Danhong injection on cerebral ischemia:the protein network,relative signal pathways and amino acids,thus systematically explained the mechanism of HuoxueHuayu recipe(Danhong injection)on cerebral ischemia.In addition,the obtained protein network might be the core unit of pathological mechanism of cerebral ischemia,providing reference and basis for further understanding and preventing of cerebral ischemia.The innovation points of the study1.It is showed that the effect of Danhong injection on cerebral ischemia isrelated to energy metabolism improvement,regulation of amino acid metabolism,inhibition of neuronal apoptosis and protection of blood brain barrier;2.It found 12 related protein networks in anti-cerebral ischemia effect of Danhong injection,including three signal pathways:WNT,AMPK and MAPK. |
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