| ObjectiveStroke is a high incidences disease in the middle-aged and elderly population with high disability and mortality.Although it can be treated with tissue plasminogen activator(t-PA),t-PA has only a short treatment window(3～4.5h)with side effect of hemorrhagic transformation,neurotoxicity and increasing mortality.Therefore,it is important to find more effective treatment drugs and strategies.Network pharmacology is a new subjuet of modern new drug research paradigm,emphasizing the dynamic regulation of "network target" in biological networks and pathways with the characteristics of integrity,systemicity and dynamics,which coincide with the theories of Tratitional Chinese Medicine(TCM)of" holistic view of TCM and syndrome differentiation".Therefore,network pharmacology approach is a powerful strategy and method for the modernization of TCM formula,which also can be applied to deeply investigate the biological connotation of TCM theories "Jun-Chen-Zuo-Shi","Yi-Bing-Tong-Zhi".Danggui-Shaoyao-San(DSS),as a famous Chinese herbal formula,was created by Zhongjing Zhang in the Eastern Han Dynasty and comprisies of Angelica Sinensis Radix(ASR),Paeoniae Radix Alba(PRA),Chuanxiong Rhizoma(CR),Atractylodis Macrocephalae Rhizoma(AMR),Poria(P),and Ali smatis Rhizoma(AR).DSS has the effect of nourishing blood,promoting blood circulation,removing blood stasis and chasing phlegm,which primary for the treatment of gynecological diseases.Since the 1980s to date,it has shown potential therapeutic effects in the treatment of stroke.In this thesis,we focuses on investigation of the mechanism of action of DSS in the treatment of ischemic stroke.On the one hand,we used the network pharmacology method to clarify the biological connotation of DSS s"Jun-Chen-Zuo-Shi" and "Yi-Bing-Tong-Zhi" from the molecular perspective,as well as the mechanisms of DSS treatment of ischemic stroke from the holistic view.On the other hand,combinaing the connotation of DSS in the treatment of diseases explored by network pharmacology with the original meaning of DSS,based on the model of Middle Cerebral Artery Occlusion(MCAO)in rats with ischemic stroke,investegation the difference in the overall effect of DSS aqueous extract and ethanol extract on acute phase of cerebral ischemia-reperfusion injury.Furthermore,explore the potential mechanisms of ethanol extract of DSS on cerebral ischemia-reperfusion injury,and finally high performance liquid chromatography-ultraviolet(HPLC-UV)method was used to explore the main active ingredients in the ethanol extract of Danggui Shaoyao Powder.MethodsExperiment 1:Exploring the therapeutic mechansisms of Danggui-Shaoyao-San in the treatment of ischemic stroke by using network pharmacology approachFirstly,the chemical composition database of DSS is constructed through authoritative Chinese herbal medicine database and text mining method.Secondly,absorption(A),distribution(D),metabolism(M),excretion(E)are used as parameters to screen the active components in DSS.Thirdly,using random forest and support vector machine algorithms predicted the targets of obtained active components.Following by analysis the synergistic mechanism,molecular function and biological process of the targets involved in DSS using InteractiVenn website,GluGO plug-in,respectively.Then,interaction networks aomong active components,targets,pathways,and diseases are constructed.Following by visualizing this networks constructed above using Cytoscape software.Lastly,network analysis was performed to comprehensively explore the associated biological process as well as therapeutic mechanisms contained in DSS.Experiment 2:Investigation the mechanisms of ethanol extract of DSS on MCAO model ratsHerbs in DSS of ASR,PRA,CR,AMR,P,AR are mixed in a ratio of 3:16:4:8:4:8,and some of them were used for aqueous extract preparation:a suitable amount of DSS material was soaked in 8 times of distilled water for 1 hour.After that,the filtrate was collected,and the filter residue was decocted with 6 volumes of distilled water for another 1 hour.The filtrate was collected again and the twice filtrates was mixed thoroughly to lyophilize and store for usage.Ethanol extract of DSS preparation:another suitable amount of DSS material was soaked in 10 times of 70%ethanol overnight.Then,extracted using ultrasonically 3 times(1 hour/time),and the three extracts were combined and lyophilized for use.Male SD rats of 270-290g were used to bulled the MCAO rat model with reference to the Longa’ s method.There were 5 groups in the experiment(not all experimental animals were used in each experiment),namely,sham operation group(SHAM),model group(MCAO),DSS aqueous extract group(MCAO+DSS/W),DSS ethanol extract group(MCAO+DSS/E or MCAO+DSS),and SIRT1 specific inhibitor EX527 treatment groug(MCAO+EX527+DSS)with 6 rats in DSS/W and MCAO+EX527+DSS groups,while 11-12 rats in other grougs.Rats in each group were given corresponding treatment at 30 minutes before reperfusion:SHAM and MCAO groups were given an equal volume of double distilled water,which DSS/W,DSS/E and MCAO+EX527+DSS experimental groups were given 12g/kg of DSS aqueous or ethanol extract treatment.Rats were assessed for neurological function prior to sacrifice,with reference to the modifined Neurological Severity Score(mNSS)method.Brain tissue was taken at 24 hours after operation.The infarct size of brain tissue was estimated by TTC staining and this brai n tissue were further used for related proteins analysis.The brain edema of rat brain tissue was evaluated by Garcia’ s method(only used in the experiment of different effects of different extracts of DSS on brain edema in MCAO model rats).The other brain tissue was prepared as frozen tissue section for distinct studies.TUNEL staining was applied to detect the apoptosis in brain cortex and striatum.3NT was observed by immunofluorescence,and the reactive oxygen species in brain tissue was investigated by HET and SOX1 probes.Western Blot(WB)approaches were used to detect the expression of Caspase3,Ceaved-caspase3,Bcl2 and Bax protein for investigating the death of neurons.The expression of P47phox,P67phox,iNOS and SIRT1 protein was also tested by WB method to observe the oxidative stress in brain tissueExperiment 3:Determination of main active ingredients in ethanol extract of Danggui Shaoyao Powder by HPLC-UVHigh-performance liquid Chromatography(HPLC)was applied to determine the major ingredients in DSS ethanol extrat.The Agilent column(Eclipse Plus C18,4.6×250mm,5μm),acetonitrile(A)and 0.05%phosphoric acid(B)served as mobile phase for gradient washing.Mobile phase A:B=95%-88%:5%-12%(0-15 min),Mobile phase A in 88%for isocratic elution at 15-25 min.Mobile phase A changed from 88%to 75%at 25-60 min.Mobile phase A changed from 75%to 45%at 60-80min.Mobile phase A changed from 45%to 30%at 80-100min.Mobile phase A changed from 30%to 5%at 100-120min.Mobile phase A in 5%isocratic elutionat at 120-130min.Mobile phase A changed from 5%to 95%at 130-138 min.mobile phase A in 95%isocratic elution at 138-145 min.The flow rate was 1 ml/min,the column temperature was 25℃,the injection volume was 5 ul,and the detection wavelength was 230 nm.A method for the analysis of paeoniflorin,alibiflorin and ferulic acid in DSS was established.After verification the linear relationship,precision,repeatability,stability,and recovery rate of the method,then the content of paeoniflorin,alibiflorin and ferulic acid in DSS were determined.ResultsExperiment 1:Exploring the therapeutic mechansisms of Danggui-Shaoyao-San in the treatment of ischemic stroke by using network pharmacology approachThis experiment used network pharmacology to explore the mechanism of action of DSS in the treatment of ischemic stroke.The results show that DSS as a TCM formula has the features of"multicomponent-multitarget-multipathway":52 bioactivity ingredients in DSS can act on 248 protein targets.Among these targets,25(10.37%)targets are the common target of PRA,CR and ASR,and CR,PRA has 58(24.07%),40(16.60%)specific targets,respectively,indicating that they play a major role in the treatment of diseases.Although there are only two specific targets in ASR,other targets involved in ASR mainly coincide with CR and PRA(56 and 25,respectively),so that ASR can synergistically enhance the therapeutic effects of PRA and CR with these common targets,while CR also has a synergistic effect on PRA.Other herbs in DSS play a secondary role for treating diseases.The principle of "Yi-Bing-Tong-Zhi" of DSS means that DSS has therapeutic effects on stroke,is that DSS has therapeutic effects on stroke,neurodegenerative diseases,respiratory diseases,genitourinary diseases,immune system diseases,etc.,for that these diseases have the same pathophysiology protein and molecular mechanisms.Sperifically,DSS participates in the treatment of ischemic stroke through a variety of pathways,including regulation of glutamate excitotoxicity,mitochondrial and endoplasmic reticulum dysfunction,and neuropathic syndrome and other pathological pathways.Experiment 2:Investigation the mechanisms of ethanol extract of DSS on MCAO model ratsThe MCAO model rats were used to explore the protective effect and mechanism of DSS extract on acute cerebral ischemia-reperfusion injury.In the terms of neurological deficits,compared with MCAO model group rats,after the rats were treated DSS aqueous extract(DSS/W,12g/kg)by gavage,the mNSS score of the rats was not decreased,while the DSS ethanol extract(DSS/E,12g/kg)treatment group was significantly reduced(P<0.05).In the terms of the infarct size of rat brains,compared with the MCAO model rats,the infarct size of the DSS/W group was not decreased,while the DSS/E group was was significantly reduced(P<0.05).As for the cerebral edema,compared with the MCAO model rats,neither the aquous extract nor ethanol extract of DSS did not have a therapeutic effect on cerebral edema.It can be concluded that the therapeutic effect of DSS ethanol extract on MCAO model rats is more effective than that of water extract.Therefore,in the in-depth mechanism research,we only use DSS ethanol extract to continue to explore the mechanism of action on MCAO rats.The results showed that compared with the SHAM group,DSS could significantly reduce the number of apoptotic neurons in the cerebral cortex and striatum and significantly reduce the expression of Bax protein after DSS ethanol extract(DSS,12g/kg,P<0.05),up-regulated the expression of Bcl2 protein and down-regulated the expression of Cleaved-caspase3/caspase3 protein.At the molecular mechanism level,compared with the MCAO model rats,the reactive oxygen species 0 and the content of 3NT was significantly decreased in the brain tissue after DSS treated at 30 min before reperfusion.The expression of P46phox,P67phox and iNOS protein in brain tissue was also down-regulated,but SIRT1 protein expression was significantly up-regulated.When SIRT1 specific inhibitor EX527 was used,the therapeutic effect of DSS on MCAO model rats was disappeared.Experiment 3:Determination of main active ingredients in ethanol extract of Danggui Shaoyao Powder by HPLC-UVIn this experiment,HPLC-UV method was used to determine the main active ingredients in the ethanol extract of DSS.The results show that the method system has good applicability and the linear relationship of the reference products is good.The contents of paeoniflorin,albiflorin and ferulic acid were determined by HPLC to be 39.7512 ug/mg,5.3411 ug/mg and 0.8221 ug/mg,respectively.ConclusionResearch conclusion based on network pharmacology1.The specific connotation of DSS formula"multi-component-multi-target-multi-pathway" mainly involves:DSS has 52 active components,which can act on 248 protein targets.From the molecular persperctive,the biological connotation of "Jun-Chen-Zuo-Shi:is that DSS simultaneously act on various diseases that they share the common pathogenic target and molecular mechanisms.2.DSS has a therapeutic effect on ischemic stroke mainly via regulating glutamate exitotoxicity,mitochondria and endoplasm dysfunction and neuritis syndrome.In summary,the advantage of applying network pharmacology to TCM formula research is that it can not only grasp the complexity of the formula macroscopically,but also explore the internal mechanism of complexity from a microscopic point of view.One large and one small,one move and one quiet,can make TCM prescription and the involved in theory of TCM are moving from ambiguity to concreteness,which makes new content into TCM and also provides new ideas and methods for disease treatment.Research conclusion based on MCAO rat model and HPLC-UV method1.The DSS ethanol extract was more effective than the aqueous extract in the MCAO model rats with cerebral ischemia-reperfusion injury.2.The protective effect of DSS ethanol extract on acute cerebral ischemia-reperfusion injury is mainly through anti-oxidative stress,and its mechanism is SIRTl-dependent.3.The HPLC-UV method is simple,rapid,accurate and reproducible.It can be used to detect the content of paeoniflorin,paeoniflorin and ferulic acid,the main active components in the DSS compound.In summary,the therapeutic effect of DSS ethanol extract on the acute phase of cerebral ischemia-reperfusion injury is better than that of water extract,which can inhibit neuronal apoptosis through anti-oxidative stress in a SIRTl-dependent manner,decreased the area of cerebral infarction,which leading to alleviate the neurological deficit of MCAO model rats.The content of paeoniflorin,albiflorin and ferulic acid in the ethanol extract of DSS was 39.7412ug/mg,5.3411ug/mg,0.8221 ug/mg,respectively. |
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