| As a global public health problem,ischemic stroke endangers the health of human beings all over the world.With its high morbidity and high mortality,it has attracted wide attention from scientists.According to the World Health Organization,ischemic stroke is caused by the interruption of the blood supply to the brain generally because a blood vessel bursts or is blocked by a clot.This phenomenon cuts off the supply of oxygen and nutrients,thereby causing brain tissue damage.After ischemic stroke occurs,irreversible brain damage can cause a variety of physical disabilities,including dyskinesias,language disorders,changes in the level of consciousness,and impaired vision,etc.,which have a huge impact on the quality of life of stroke patients and even cause death.Due to the complex damage mechanism of ischemic stroke and the pathological process involving many processes,the existing drugs have the characteristics of narrow time window,high side effects,and unclear mechanism of action,which are not suitable for all types of stroke patients.Therefore,in view of the pathogenesis of ischemic stroke,looking for mild and universal treatment drugs with small side effects,high efficacy and clear mechanism of action has become the key to ischemic stroke drug research,and it is also a big challenge faced by scientists all over the world.Studies have found that Eleutherococcus senticosus(Rupr.& Maxim.)Maxim.(E.senticosus),as a traditional Chinese medicine,has immunomodulatory,anti-stress,anti-fatigue,anti-tumor effects,and has certain therapeutic effects on cardiovascular and cerebrovascular diseases.The theory of traditional Chinese medicine believes that E.senticosus can promote blood circulation,remove blood stasis,control blood pressure and dredge blood vessels.However,the medicinal value of E.senticosus root is gradually being valued by people,which leads to the gradual depletion of root resources.In recent years,E.senticosus leaves(ESL)have become an alternative resource for E.senticosus roots due to its chemical composition and medicinal effects similar to the roots with unique regenerative ability that the roots don’t have.Although studies have shown that ESL can also treat cardiovascular and cerebrovascular diseases,due to its complex chemical composition,which can act on multiple targets in the body,and it has disadvantages such as unclear medicinal substances and unclear mechanism of action.The research on the specific mechanism of its treatment of ischemic stroke has become more difficult and urgent.This thesis will utilize metabolomics research methods to explore and verify the overall mechanism of action of ESL in the treatment of ischemic stroke.The main research content includes the following aspects:1.Study on the serum lipidomics and neuroprotective effects of ESL in the treatment of ischemic strokeFirstly,the main active components of ESL,the 40% ethanol elution fraction was prepared and analyzed based on the UPLC method.The results showed that the 40%ethanol eluted part of ESL mainly contained organic acids,flavonoids and glycosides.Secondly,a rat ischemic stroke disease model was established and the 40% ESL ethanol eluted part was used for four weeks of administration treatment.Lipid abnormalities,nerve damage,oxidative stress and inflammatory response are the four main aspects of stroke onset.The research work in this chapter focuses on the above four aspects.This study established a lipidomics research strategy that was developed by UPLC-Q-TOF/MS analysis.The quantification of neurotransmitters in serum and brain tissue of rats was performed using the UPLC-TQ/MS.Also,we have quantified oxidative stress and inflammatory reaction by measuring the contents of SOD,MDA,TNF-α,IL-6 and IL-10 via the ELISA kits for serum and brain tissue.According to UPLC-Q-TOF/MS-based lipidomics analysis,27 lipidomics biomarkers were identified in this study,including PC,PE,SM and TG,which were distributed in various lipid metabolic pathways,including glycerophospholipid,linoleic acid,alpha-linolenic acid,glycerolipid,sphinglipid and arachidonic acid metabolism pathways.The results showed that by reversing the changes in lipid content caused by the disease,ESL has the therapeutic effect on ischemic stroke.10 neurotransmitters in brain tissue and serum of rats were quantitatively studied by UPLC-TQ/MS in the course of nerve injury during ischemic stroke.The results showed that ischemic stroke can lead to the excessive release of excitatory amino acids such as Glu and Asp,resulting in neurotoxicity.It can also increase the amounts of GABA,5-HT,NE,E,DA and tau,reduce the contents of inhibitory neurotransmitter Gly and neuroinflammation regulator,Ach.ESL could adjust the levels of the above neurotransmitters in brain tissue and serum to normal levels,indicating that it can play a central nervous system protective effect by regulating the neurotransmitter contents of ischemic stroke rats.Finally,the results of ELISA showed that ESL can treat the ischemic stroke to a certain extent in reducing the oxidative and inflammatory damage.Therefore,ESL may play a therapeutic role in the treatment of ischemic stroke in different ways.This research preliminary revealed the mechanism of ESL in the treatment of ischemic stroke,and provided support for the further application of ESL.2.Fecal metabolomics study of ESL in the treatment of ischemic stroke and its effect on microbiota-gut-brain axisBi-communication system of microbiota-gut-brain axis is associated with the occurrence and prognosis of ischemic stroke and has gradually attracted wide attention.UPLC-Q-TOF/MS-based untargeted fecal metabolomics method combined with UPLC-TQ/MS-based fecal bile acid targeted metabolomics analysis method and16 S r RNA fecal microbiota sequencing were used to explore the therapeutic effect of microbiota-gut-brain axis on ischemic stroke by ESL.Firstly,we have suggested a UPLC-Q-TOF/MS-based untargeted fecal metabolomics strategy combined with multivariate statistical analysis to screen potential biomarkers with significant differences and conducted pathway analysis.Therefore,40 biomarkers involved mainly in arachidonic acid metabolism,biosynthesis of unsaturated fatty acids,bile acid biosynthesis,and sphingolipid metabolism pathways were identified.Nearly all X of the potential biomarkers had significant changes after ischemic stroke,while ESL can regulate concentrations of them to return to the normal state.Secondly,the UPLC-TQ/MS-based targeted metabolomics method quantitatively analyzed 13 bile acids in fecal samples.When ischemic stroke occurs,rat fecal bile acid metabolism is disordered.ESL can adjust the contents of bile acids above and make them closer to the normal levels.Further more,ischemic stroke triggered gut microbial disturbances by enriching pathogens and reducing probiotics,which could be accommodated by ESL.The above results provide a basis for the regulation of ESL on the microbiota-gut-brain axis of rats with ischemic stroke,and lay a foundation for the study of the mechanism of ESL for the treatment of ischemic stroke.3.The mechanism verification of ESL in the treatment of ischemic stroke by regulating probioticsThe probiotics that can be regulated by ESL were selected to feed rats with ischemic stroke,and it was verified that the increase in the level of probiotics after administration of ESL was an important factor reflecting its therapeutic effect.Firstly,Lactobacillus reuteri(L.reuteri)and Clostridium butyricum(C.butyricum)were cultured and bacterial solutions were prepared to feed rats with ischemic stroke.After four weeks of feeding,the microbiota composition of rat feces was detected.The results showed that after the two kinds of probiotics were fed,the composition and content of the feces microbiota of stroke rats changed significantly,which was more similar to that of the sham-operated group and was different from the model group.Secondly,the quantitative method based on UPLC-TQ/MS was used to detect the changes of neurotransmitters in the brain tissue of rats with ischemic stroke fed with probiotics.The results showed that after the administration of the two probiotics,the contents of neurotransmitters with neurotoxicity decreased,while the contents of neurotransmitters with neuromodulation effect were significantly increased,which were closer to that of healthy rats.Finally,the contents of various inflammatory factors IL-1β,IL-6,IL-10 and TNF-α in rat serum and brain tissue and the contents of MDA,SOD,COX-2 and MAO in brain tissue were detected by ELISA.When ischemic stroke occurred,the contents of IL-1β,IL-6,TNF-α,MDA,COX-2 and MAO increased significantly,while the contents of IL-10 and SOD decreased significantly.After two kinds of probiotics were fed,the levels of the above indicators in stroke rats can be adjusted to normal levels,indicating that both probiotics can have a certain protective effect on the body by reducing inflammation and oxidative stress damage.This study verified that ESL can protect the body by influencing the metabolism of the microbiota-gut-brain axis,increasing the abundance of probiotics in the body,and regulating the nerve damage,inflammatory response and oxidative stress damage caused by stroke.4.Urine metabolomics study of ESL in the treatment of ischemic strokeUrine samples can accurately and sensitively reflect the metabolic changes of the body,which is the most important biological sample in metabolomics research.UPLC-Q-TOF/MS-based untargeted urine metabolomics method was used to study and verify the mechanism of ESL in the treatment of ischemic stroke.The urinary metabolomics strategy was developed by combining UPLC-Q-TOF/MS with multivariate data analysis.The gene-enzyme-biomarker network was built by Cytoscape 3.6.0 on the basis of the potential biomarkers filtered out via urinary metabolomic analysis.Then,the potential target enzymes of ESL in the treatment of ischemic stroke were selected for further validation analysis via the ELISA kits.A total of 42 biomarkers associated with ischemic stroke have been filtered out,among which 38 species can be adjusted by ESL.These biomarkers were involved in several metabolic pathways,including taurine and hypotaurine,arachidonic acid,cysteine and methionine,steroid hormone biosynthesis,tryptophan,and tyrosine metabolism pathways.The gene–enzyme–biomarker network was built,and three predicted target proteins,including cyclooxygenase-2(COX-2),monoamine oxidase(MAO),and nitric oxide synthase(NOS)were selected as the potential target enzymes for ESL in ischemic stroke treatment.The contents of the above three enzymes in the serum and brain tissue of rats treated with ESL were similar to those of healthy ones.The results of this experiment will help to further understand the pathogenesis of ischemic stroke,XII and provide scientific basis for the research on the potential therapeutic mechanism of ESL.5.CIL LC-MS-based urinary metabolomics for the investigation on the mechanism of action of ESL against ischemic stroke in ratsThe high-efficiency chemical isotope labeling(CIL)LC-MS metabolomics method is to use reagents targeting specific functional groups to label biological samples,so that all metabolites with the same functional group can generate corresponding derivative metabolites.While improving the overall metabolomics coverage,isotopes are introduced into labeled metabolites,and metabolites derived from heavy isotope reagents are used as internal standards for light isotope reagent-derived metabolites,thereby improving the qualitative and quantitative accuracy of metabolites.In this study,the CIL LC-MS-based urinary metabolomics was used to analyze the amine/phenol sub-metabolome and the carboxyl sub-metabolome.The results show that ESL can regulate metabolic disorders after ischemic stroke through multiple pathways in the body.Compared with the results of traditional urine metabolomics studies,CIL LC-MS method has screened out more potential biomarkers and covered more metabolic pathways in the body,resulting in broader coverage and more accurate quantitative metabolomics results.At the same time,more metabolites with significant differences were matched in each pathway to clarify the significant changes and mechanisms of upstream compounds and downstream products in the pathway,so as to make the research on each pathway more comprehensive.Finally,the mechanism of ESL in the treatment of ischemic stroke was systematically elaborated from the perspectives of neuroprotection,regulation of energy metabolism,inhibition of inflammation injury,and antagonism against oxidative stress.Thus,the mechanism of action of ESL in treating ischemic stroke can be more comprehensively explained.In conclusion,this thesis first systematically studied the mechanism of ESL in the treatment of ischemic stroke in rats by metabolomic methods based on a variety of biological samples including rat serum,feces,urine and brain tissue.Combined untargeted metabolomics research on the urine and feces samples with the targeted metabolomics study of serum lipids,neurotransmitters and fecal bile acids,it is clear that ESL can regulate multiple metabolic pathways in rats with ischemic stroke,and has the neuroprotective effect as well as the effect of regulating the lipid disorder.Secondly,through the analysis of the microbiota,it is preliminarily concluded that ESL can exert the therapeutic effect of ischemic stroke by regulating the microbiota-gut-brain axis through two-way communication system.It is speculated that the possible mechanism is to increase the intestine probiotics content thus has a certain regulatory effect on the body.Finally,using a metabolomics method based on CIL LC-MS,a comprehensive analysis of the amine/phenol sub-metabolome and carboxyl sub-metabolome in the body was carried out,and the therapeutic mechanism of ESL on ischemic stroke was systematically explained.This study provides a theoretical basis for further elucidating the pharmacodynamic material basis and mechanism of action of ESL in the treatment of ischemic stroke,and also laid the foundation for the development of new drugs for the treatment of ischemic stroke with ESL.Finally,it will open up a new strategy for the systematic study of the mechanism of action of traditional Chinese medicine. |
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