| Objective:To clarify the clinical efficacy of Xingnao Jianshen prescription for acute cerebral infarction(experimental phlegm-heat and fu-organism),chemical components in the compound,and to explore the anti-hypoxic mechanism in vitro to provide objective evidence and guidance for clinical medication.Methods:The first chapter is to enroll clinical patients with ischemic stroke(experimental phlegm-heat and fu-organs).Non-randomized controlled trials are used to evaluate NIHSS,BI,mRS and traditional Chinese medicine syndromes(internal wind,phlegm-dampness,internal fire,and blood Blood stasis)points and changes in the curative effect of constip ation.To clarify the relevant conclusions about clinical efficacy evaluation of Xingnao Jianshen prescription.The second chapter is the application of the analysis method of Network pharmacology to construct the target set of "drug-component-disease"and biological information prediction,and explore the possible mechanism pathways and potential targets of Xingnao Jianshen prescription in ischemic stroke.The third chapter is the application of HPLC-MS/MS method to analyze the full spectrum of Xingnaojianshen prescription to clarify the chemical components contained in the prescription.The fourth chapter is the use of PC 12 cells to simulate the morphology and function of nerve cells in vitro,and conduct chemical hypoxia modeling.After group intervention,use the proteomics 4D label free test to clarify the in vitro anti-hypoxia mechanism of the Xingnao Jianshen prescription.Results:1.Clinical results.By comparing the changes before and after treatment of NIHSS,BI,and mRS in the experimental group and the control group,it can be shown that the intervention method of combining Xingnao Jianshen prescription is better than the basic treatment of western medicine,which does have clinical effects.Ability of daily living and recovery of disabled function;through the changes of the two groups of patients’ TCM syndrome elements,it can be shown that the combined intervention method of Xingnao Jianshen prescription is better than the basic treatment of Western medicine recommended by the guide,and it is significantly improved TCM syndrome,but there is no significant difference in the efficacy of improving internal wind syndrome;the first defecation time of the experimental group is longer than the control,but the total effective rate of constipation is higher.2.Network pharmacology prediction results.In this study,the drug components of Xingnao Jianshen prescription and the targets of acute cerebral infarction were analyzed for interaction.After KEGG bioinformatics analysis,it was found that the main regulatory pathways are cellular Apoptosis、HIF-1 signaling pathway,etc,,which aspects closely related to ischemic cerebrovascular disease.At the same time,target proteins such as NF-kBp65,Bcl-2,and Caspase 3 have also been determined,and they have been verified by Westem blotting experiments,and they can be determined as targets of drug effect.point.3.Full spectrum analysis results.There are 398 kinds of ingredients in Xingnao Jianshen prescription,293 kinds of positive ion modes,64 kinds(including those that cannot be classified temporarily);105 kinds of negative ion modes,24 kinds.Among them,the intersection of positive and negative ions found that the types of ingredients are consistent with flavonoids,which are also the ingredients with higher relative concentration expression in the compound.There are 46 chemical components of flavonoids.The highly prominent concentrations are flavonoid ficusine and quercetin-3,4’-di-O-glucoside,etc.4.Experimental results of anti-hypoxia mechanism in vitro.The viability of PC 12 cells decreased after hypoxia,after pre-incubation with Xingnao Jianshen prescription on the basis of modeling,it can significantly increase the viability of cells damaged by hypoxia.The results of proteomics found that the anti-hypoxia mechanism of Xingnao Jianshen prescription is mainly enriched in cell apoptosis and regulation of various cell metabolisms.The specific mechanism pathways are enriched in PPAP,PI3K/AKT,HTF-1,Steroid biosynthesis,Metabolic pathways,etc.The protein of interest was determined according to the correlation strength and the multiple of difference,and the protein verification of PRMwas carried out.It was found that the change trend of Perilipin,CoA synthase and other proteins was consistent with the omics,indicating that Xingnao Jianshen prescription does have an anti-hypoxia mechanism.According to the combination of antibodies and antigen recognition after the processing of each group of PC 12 cells,it can be found that the intervention of Xingnao Jianshen prescription can make cell samples undergo trimethylation,acetylation,lactation and ubiquitination.Conclusion:The conclusion of the clinical study is that Xingnao Jianshen prescription has a clinical therapeutic effect on ischemic stroke(experimental phlegm-heat-fu-organs),and the combined application can be more effective than the control.The conclusion of network pharmacology is that Xingnao Jianshen prescription does have a regulatory effect in this disease,and it has been verified by western to regulate apoptosis protein.The conclusion of LC/MS is that there are many chemical components in Xingnao Jianshen prescription,and it is very possible that the active ingredients are flavonoids.Of course,this needs to be verified by experiments.The conclusion of in vitro experiments is that Xingnao Jianshen prescription has anti-hypoxia effect on PC 12.The main mechanism pathways are enriched in apoptosis,regulation of cell glycolipid and product metabolism,etc.In addition,it was discovered that the level of histone modification may have a regulatory effect on protein groups such as trimethylation,acetylation,lactation,and ubiquitination. |
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