| Objective:The rise and development of metabolomics technology provides new research ideas for the modernization of traditional Chinese herbal compound prescription,and it has broad prospects for the application of metabolomics technology to the exploration of the mechanism of traditional Chinese herbal compound prescription The traditional Chinese herbal compound prescription is a scientific combination of traditional Chinese medicine theory and traditional Chinese medicine.It is also a bridge connecting basic research and clinical practice.Guizhi Decoction originated from the Eastern Han Dynasty and is known as "the ancestor of prescription" and "the leader of prescription ".It has treated the deficiency syndrome of the Guizhi Tang syndrome,and so far it has been tested by countless clinical diagnosis and treatment.Guizhi Decoction is known as "the ancestor of prescriptions" and "the leader of prescriptions",it originated from the Eastern Han Dynasty and used for the Guizhi Tang syndrome,So far,it has undergone numerous clinical diagnosis and treatment tests,w whether it is unilateral or its addition and subtraction,it is still widely used in clinical practice.However,the current research on the antipyretic effects of Guizhi Decoction mostly expounds unilaterally from the cytokines IL-1,TNF-?,and the central heating medium cAMP,PGE2,etc.,which cannot fully reflect the regulation of the body by drugs.Moreover,there are few studies on the compatibility mechanism of the drugs in Guizhi Decoction.Therefore,in order to further understand the mechanism of action of Guizhi Decoction,the antipyretic effect of Guizhi Decoction and its single drug was firstly based on pharmacodynamic experiments.The effect was evaluated,and then the metabolomics technology was used to further find the differential metabolic small molecules and related metabolic pathways related to fever of Guizhi Decoction and its single-drug,and the key targets were obtained through modular analysis.Taking metabolites as the starting point,the antipyretic compatibility and molecular mechanism of Guizhi Decoction were explored to provide new research ideas for further clarifying the mechanism of Guizhi Decoction.Methods:1.Study on Antipyretic Effect of Guizhi Decoction and Its Single DrugSixty-four SD rats were randomly divided into eight groups,blank group,model group,Guizhi Decoction group,Guizhi(Cinnamomi ramulus)group,Baishao(Paeoniae radix alba)group,Shengjiang(Zingiberis rhizoma recens)group,Gancao(Glycyrrhizae radix et rhizoma)group and Dazao(Jujubae fruitus)group,with 8 rats in each group for formal experiments,which were administered orally by gavage for 2 consecutive days before the formal experiment,and the rats in the blank group and the model group were given the same volume of distilled water.Before the experiment,the basal body temperature of each group of rats was measured,and the rat yeast heat model was replicated by injecting a yeast suspension into the back.The blank group was injected with the same volume of normal saline.After the rats were successfully modeled(the body temperature increased more than 0.8?),the rats in each group were administered once and repeated once after 1 hour.The rats'temperature was measured and recorded after 4h,5h,6h,7h and 8h of modeling.The changes were analyzed by using SPSS 23.0 statistical software.2.Metabolomics Study of Guizhi Decoction and Its Single DrugAfter the end of the pharmacodynamic experiment,blood was collected from the abdominal aorta,and the serum samples of each group were obtained after centrifugation.GC-MS technology was selected for metabolite detection.Proteins and other impurities were removed from the serum samples before detection and derivatized.GC-MSsolution version 2.5(SHIMADZU Corporation)was used to integrate the original map,and the detected metabolites were estimated and identified through the NIST database and other self-built libraries.At the same time,the HMDB and KEGG databases were used to search and confirm the endogenous metabolites.Data analysis was performed using Metaboanalyst 4.0 software,and pattern recognition analysis was performed for each administration group,blank group and model group,including PCA,PLS-DA and cluster analysis,to compare their differences and separations;based on the analysis of PLS-DA in the above,VIP tables of serum metabolites of rats in each administration group and model group were obtained,and the differential metabolites between each administration group and model group were screened out based on VIP value greater than 1 and manual integration calculation,and passed MetPA The plugin performs path analysis and selects a metabolic pathway with an Impact value greater than 0.10,indicating that the pathway may be closely related to the potential target pathway;using the MetScape plugin in Cytoscape to perform a network modular analysis of each group of differential metabolites,and using the average node degree value Threshold selection of relevant targets.Results:1.Result of antipyretic Effect of Guizhi Decoction and Its Single DrugThe temperature of the rats in the blank group fluctuated up and down in a small range based on the basal temperature.Compared with the blank group,the body temperature of the rats in the model group increased significantly after 4 h(p<0.01).The body temperature of rats in the Guizhi Decoction group was lower than that in the model group at various time points after the model was made for 5 hours,and there were significant differences(p<0.05)after the model was made for 6 and 7 hours.After administration,the rats in the Guizhi group had a certain downward trend after 6 hours of modeling,but there was no significant difference with the model group(p>0.05).The rats in the Baisao group were given 5 hours after modeling.The posterior body temperature was reduced,but there was no significant difference compared with the model group(p>0.05).The body temperature of the rats in the Dazao group at each time point was lower than that of the model group,and there was a significant difference(p<0.05 or p<0.01);there was no significant downward trend in body temperature in the Shengjiang group and Gancao group rats.2.Result of Metabolomics Study of Guizhi Decoction and Its Single DrugAccording to the 2D and 3D images of PCA and PLS-DA,it can be known that the sample points of the Guizhi Decoction group,Guizhi group,Baishao group,gancao group,and Dazao group can be separated from the blank group and model group.It is shown that each administration group can be separated from the blank group and the model group,and there is a certain aggregation trend within the group,which proves that there is a certain difference between the above administration groups and the model group.The Shengjiang group could not be separated from the model group in the three modes,and the two did not show significant differences,so no further analysis was performed.Based on pattern recognition analysis,a total of 13 different metabolites from the model group and the blank group were obtained,which were L-Lactic acid,Glycerol,Palmitic acid,Linoleic acid,D-Glucose,L-Serine,Stearic acid,Oxalic acid,L-Aspartic acid,Cholesterol,Urea,Arachidonic acid,and L-Threonine;compared with the model group,Guizhitang group obtained a total of 9 differential metabolites,respectively,Stearic acid,L-Lactic acid,Glycerol,Palmitic acid,Linoleic acid,D-Glucose,Urea,Arachidonic acid,L-Threonine;compared with the model group,Guizhi group obtained a total of 5 different metabolites,namely Arachidonic acid,Palmitic acid,Linoleic acid,Stearic acid,Cholesterol;Baiji group and Compared with the model group,a total of 7 differential metabolites were obtained,which were L-Lactic acid,Palmitic acid,Linoleic acid,L-Serine,Oxalic acid,Urea,Arachidonic acid;compared with the model group,a total of 5 Different metabolites:L-Lactic acid,Palmitic acid,D-Glucose,Urea,Arachidonic acid.Compared with the model group,six different metabolites were obtained.Is L-Lactic acid,Palmitic acid,D-Glucose,L-Serine,Oxalic acid,L-Threonine.Pathway analysis was performed using the differential metabolites obtained from the comparison between each administration group and the model group.It was found that there are 4 pathways related to the antipyretic effect of Guizhi Decoction,which are the Linoleic acid metabolism pathway,Arachidonic acid metabolism pathway,and starch and sucrose metabolism pathways and Glycerolipid metabolism pathways;there are 2 pathways related to the antipyretic activity of Guizhi,which are the Linoleic acid metabolism pathway and the Arachidonic acid metabolism pathway;4 pathways related to the antipyretic activity of Baishao,respectively Linoleic acid metabolism pathway,Arachidonic acid metabolism pathway,Glycine,serine and threonine metabolism pathways and Aminoacyl-tRNA biosynthesis pathways;there are two pathways related to Gancao antipyretic,respectively Starch and sucrose metabolism pathway and Arachidonic acid metabolism pathway;there are 3 pathways related to Dazao antipyretic,which are Starch and sucrose metabolism pathway,Glycine,serine and threonine metabolism pathway,and Aminoacyl-tRNA biosynthesis pathway.Through modular analysis,relevant groups of targets were screened out.There were 28 targets related to the antipyretic effect of Guizhi Decoction,of which 26 belonged to the cytochrome P450 family,and the remaining two targets were related to glycolysis.The targets related to antipyretic activity of Guizhi,Baishao and Gancao are the same as the 26 targets of Guizhi Decoction cytochrome P450;there are 14 targets related to antipyretic activity of Dazao.SHMT1 and SHMT2 are serine hydroxymethyltransferases,and other targets are related to carbohydrate metabolismConclusion:From the results of pharmacodynamics,Guizhi Decoction has a significant antipyretic effect on the rat fever model induced by yeast,Guizhi and Baishao have a tendency to reduce the body temperature of fever rats,Gancao and Shengjiang have no obvious antipyretic effect on dry yeast heat-induced rat model,and Dazao has a certain antipyretic effect,compared with other groups,it is closer to the blank group,but from the perspective of antipyretic,Dazao is not obvious compared with Guizhi Decoction.The advantage indicates that the antipyretic effect of Guizhi Decoction is better than that of each single-drug group.Comparing each group with the blank group at the same time,the results of the Guizhi decoction group and the blank group were closer,which further confirmed that Guizhi decoction had the best antipyretic effect on the body temperature of fever rats,this shows that in terms of antipyretic effect,the traditional Chinese medicine prescription Guizhi Decoction has more advantages than the single drug in its prescription.Based on pharmacodynamics,the molecular mechanism of antipyretic action of Guizhi Decoction and its single drug was explored using metabolomics technology.It was found that the mechanisms of antipyretic action of Guizhi Decoction on fever rats are mainly related to the Arachidonic acid metabolic pathway and Starch and sucrose metabolism pathway,and the main target is cytochrome P450.The antipyretic mechanisms of Guizhi,Baishao and Gancao are related to the Arachidonic acid metabolism pathway,and its target is the same as that of Guizhi decoction.In addition,the Gancao and Dazao mechanisms are also related to the Starch and sucrose pathways.In addition,the main pathways and targets related to antipyretic of Guizhi Decoction cover the main pathways and targets of each single-drug,further proving that Guizhi Decoction has more advantages in antipyretics than single-drugs,and the individual medicines in Guizhi Decoction have certain synergistic effects,and the specific mechanism needs further research.Through the study of this experiment,the molecular mechanism of Guizhi Decoction and its single drug on the model of rat fever caused by dry yeast was explored to a certain extent,and the reason for the antipyretic effect of Guizhi Decoction was explained from the perspective of small molecule metabolism and provided new ideas for follow-up research. |
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