| Acute cerebral infarction is a category of apoplexy in traditional Chinese medicine science.The pathogenesis of acute cerebral infarction is complicate,and fire-toxicity is considered to be one of the important pathogenesis of acute cerebral infarction.The formation of fire-toxicity is closely related to heat and blood stasis.Based on this,the combination use of two kinds of Chinese medicine injection on clearing heat and activating blood may play a synergistic role in treating acute cerebral infarction.However,due to the characteristics of multi-component,multi-target and multi-process of Chinese medicine injection,it is difficult to reveal the mechanism of combination use of two kinds of Chinese medicine injection by the traditional method of "reductionism" in a systematic and comprehensive way.Metabonomics provides a good platform for Chinese medicine research in the prevention and treatment of acute cerebral infarction.Metabonomics is a significant part of system biology.It could deeply elucidate the pathophysiological mechanism of the disease from a whole point of view.By observing the changes of the endogenous metabolite before and after the treatment of Chinese herbs or after the combination use of the two kinds of Chinese medicine injection,the mechanism of the two kinds of Chinese medicine injection is clarified.In this study,Kudiezi injection(KDZ)and Xueshuantong injection(XST)was selected,as the first line clinical treatment for acute cerebral infarction.KDZ was partial to the clearing heat and detoxification of the collaterals.The effect of XST was activating blood circulation and removing blood stasis.The synergistic effect mechanism of the combination use of KDZ and XST was observed from the level of the metabolite.ObjectiveAn application model based on metabonomics pattern recognition method was established to find out for potential biomarkers in acute cerebral infarction patients with fire-toxicity syndrome.Through the overall and dynamic changes of endogenous metabolites,the synergistic mechanism of the combination use of KDZ and XST in the treatment of acute cerebral infarction patients with fire-toxicity syndrome was evaluated.The function and regulation mechanism of the combination use of KDZ and XST in treating the acute cerebral infarction patients with fire-toxicity syndrome were also revealed.The model of rat middle cerebral artery occlusion was used to verify the mechanism of treating acute cerebral infarction with fire-toxicity syndrome from the whole(human,rat)and molecular level in order to provide scientific basis for its clinical treatment.MethodsPart one:15 cases of acute cerebral infarction with fire-toxicity syndrome within 48h were selected,and 16 non-stroke patients were selected as control group.Draw the venous blood on an empty stomach of the two groups without any treatment.The blood samples were centrifuged to obtain serum for measurement of endogenous metabolites.Liquid chromatography-mass spectrometry was used to detect the difference in serum metabolites of the two groups.Multivariate statistical analysis and partial least square method-discriminant analysis were used to analyze the variability of metabolic profile data.With variable projection importance>1,combing with T test P<0.05,the significant difference metabolites were found.The metabolites can be identified by retrieving the online database,and metabolites pathway is further screened to find the key pathway that has the highest correlation with the metabolite.Part two:60 cases of acute cerebral infarction with fire-toxicity syndrome within 48h were randomly divided into 4 groups:KDZ group(KDZ injection+basic western medicine treatment),XST(XST injection+basic western medicine treatment),KDZ+XST group(KDZ injection+XST injection+basic western medicine treatment),15 cases in each group and were treated for 14 days.The venous blood was drawn on the empty stomach,and the serum was centrifuged ater the treatment.The same metabolomics test method was adopted to observe the serum differential metabolites of the three Chinese medicine injection groups and the basic treatment group.The three Chinese medicine groups compare with the treatment control group in order to find the signal pathway related to the difference of metabolites in the KDZ,XST,KDZ+XST groups,especial the KDZ+XST group.Part three:Sprague Dawley male rats were operated by the the middle cerebral artery occlusion model with the modified thread embolus method.The rats were randomly divided into 5 groups:Sham group,Model group,KDZ group,XST group and KDZ+XST group.The ischemia was 1.5 hours,and the reperfusion was 72 hours.The degree of neurological deficiency was evaluated by Garcia JH score.The volume of cerebral infarction was measured by TTC staining.The water content of brain in rats was also measured.The three test is to observe the effect of three Chinese medicine injection in treating MCAO rats.The expression of mTOR-ERR alpha-GLS protein was detected by Western blot.Results①There was obvious difference between the stroke group and control group in PCA analysis.Compared with the control group,the levels of phosphatidyl choline,phosphatidyl ethanolamine,serine and ornithine in the acute cerebral infarction patients with fire-toxicity syndrome were decreased,while the levels of lysophosphatidylcholine,seroamide,sphingomyelin,aspartic acid,galactoceramide and taurine were increased in the acute cerebral infarction patients with fire-toxicity syndrome.②There was a significant difference between the KDZ+XST group and treatment control group.Compared with the treatment control group,the levels of phosphatidyl choline,glucosanamide and glutamine in the serum of patients of the KDZ+XST group increased,and the lysophosphatidyl alcohol,amines,sphingomyelin,taurine,aspartic acid and glutamic acid in the serum of patients of the KDZ+XST group were decreased.The combination use of KDZ and XST could increase the level of phosphatidyl choline and reduce the the levels of lysophosphatidyl alcohol,ceramide,sphingomyelin,aspartic acid and taurine,which were gradually restored to the normal level.After the treatment of KDZ,the level of phosphatidyl choline was increased,and the level of phosphatidyl ethanolamine and lysophosphatidylcholine was reduced.After the treatment of XST,the level of phosphatidyl choline,ornithine were increased,and the level of lysophosphatidylcholine and lysophosphatidylethanolamine was reduced.③Compared with the ischemic 1.5h reperfusion 72h model group,the combination use of KDZ and XST could improve the degree of neurological deficiency(p<0.05),reduce the volume of cerebral infarction and the brain water content(p<0.01),and increase the expression of mTOR,ERR alpha and GLS protein(p<0.05～0.01)in the ischemic side cortex.After the treatment of KDZ or XST,it could also improve the degree of neurological deficiency(p<0.05),reduce the volume of cerebral infarction and the brain water content(p<0.05～0.01),and increase the expression of mTOR,ERR alpha and GLS protein(p<0.05～0.01)in the ischemic side cortex.Compared with the KDZ group,the KDZ+XST group could increase the expression of ERR alpha and GLS protein(p<0.05).Conclusion①There are several metabolic pathways in the acute cerebral infarction patients with fire-toxicity syndrome,including glycerol phospholipid metabolism(phosphatidylcholine,phosphatidyl ethanolamine,lysophosphatidylcholine),sphingolipid metabolism(ceramide,sphingomyelin),aspartate and glutamic acid metabolism(aspartate acid)and taurine metabolism(taurine).②The combination use of KDZ and XST may play a role in the treatment of acute cerebral infarction patients with fire-toxicity syndrome by regulating the metabolism of glycerol phospholipid metabolism(phosphatidyl choline,lysophosphatidyl ethanolamine),sphingolipid metabolism(ceramide,sphingomyelin),taurine metabolism(taurine),aspartic acid and aspartate and glutamic acid metabolism(aspartate acid).③The combination use of KDZ and XST may play a protective role by regulating the metabolic pathway of glutamine and glutamate by the mTOR-ERR alpha-GLS signaling pathway. |
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