Exploring The Molecular Basis Of Cold-hot Nature Of TCM Based On Systems Pharmacology Model

Objective:The system pharmacology method was introduced into the theoretical research on cold-hot nature of traditional Chinese medicine(TCM).In order to find the molecular basis of cold-hot nature,we need to find the key targets of herbs with cold and hot nature by network building and topological analysis,and study the related diseases of these targets,as well as GO analysis and KEGG analysis.On this basis,the vivo experiment and molecular biology experiments were conducted to analyze the results of multi-level and multi-angle research.In conclusion,the ultimate purpose is to provide a new method for further study of the essence of cold-hot nature.Methods:Part one.The systems pharmacological research of cold-hot nature of TCM1.1 Choosing herbs with cold-hot natureThere are three principles to choose herbs with cold-hot nature.First,there are commonly used clinical drugs.Second,these herbs have clear curative effects and can be supported by its related books and literatures.Third,these herbal natures have already been confirmed by relevant studies or reports.Hence,we finally selected 10 kinds herbs with cold nature and 10 kinds herbs with hot nature,these herbs with cold nature include Phellodendri Chinrnsis Cortex,Sophorae Flavescentis Radix,Gentianae Radix Et Rhozima,Coptidis Rhizoma,Scutellariae Radix,Radix Puerariae,Houttuyniae Herba,Gardeniae Fructus,Paeoniae Radix Alba,Radix Rhei Et Rhizome.And these herbs with hot nature include Angelicae Sinensis Radix,Panax Notoginseng(Burk.)F.H.Chen Ex C.Chow,Cinnanmomi Cortex,Aconiti Lateralis Radix Praeparata,Ephedra Herba,Evodiae Fructus,Alpiniae Officinarum Rhizome,A.Dahurica(Fisch.)Benth.Et Hook,Piperis Fructus,Curcumaelongae Rhizoma.1.2 Constructing composition database and screening active ingredientsDatabases such as TcmSP,TCMID,TCM Database@Taiwan,and NCBI Pubchem were queried to collect all the chemical components contained in drugs with cold-hot nature.Then,all the chemical components were standardized to build composition database.In the end,we used different computational models to predict the compounds' parameters,and used OB,DL and HL to screen out the active ingredients.1.3 Predicting potential targetsA SysDT drug-target predictive model was used to predict the potential targets of all active ingredients.And targets' information from many databases such as SEA,STITCH,TTD,and HIT were integrated.Finally,all potential targets were uniformly standardized by UniProt database.1.4 Component-target network construction and topological analysisThe component-target network of drugs with cold and hot nature were constructed through Cytoscape software.And the entire network was analyzed by Network Analyzer plug-in.So,the results of the topological parameters can be outputted.1.5 Screening key targetsThrough the degree centrality algorithm,all key targets were screened out in the network.On this basis,degree centrality was used as a core indicator,and closeness centrality and betweenness centrality were used as auxiliary indicators to further evaluate and analyze these key targets and find the targets that can reflect the essence of cold-hot nature of TCM.1.6 Disease correlation analysisAll disease information related to key targets was collected in TcmSP databse.ICD 10 code was then used to reclassify these diseases to obtain the frequency of occurrence of each type of disease.1.7 GO analysis and KEGG analysisBased on GO and KEGG databases,ClueGO plug-in was used for the enrichment analysis of biological processes and metabolic pathways of key targets.Then the most important target was selected for vivo experiment and molecular biological experiments.Part two.Experimental verification of key target of herbs with cold natureSection one.Antipyretic effect studies1.1 The establishment of the yeast-induced hot modelThe 20%yeast suspension prepared in ad-vance was heated to 37? and injected subcutaneously in the back of mice in the model group and each drug group.Meanwhile,mice in the blank group were injected subcutaneously with the equal volume of saline.1.2 Grouping and administration230 Kunming mice(20±2 g)were randomly divided into 23 groups with 10 mice per group.Every group includes half male and half female.The categories were as follows:blank group,model group,aspirin group,and high and low dose groups of Phellodendri Chinrnsis Cortex,Sophorae Flavescentis Radix,Gentianae Radix Et Rhozima;Coptidis Rhizoma,Scutellariae Radix,Radix Pu.erariae,Houttuyniae Herba,Gardeniae Fructus,Paeonitae Radix Alba,Radix Rhei Et Rhizome.Each mouse in drug group was orally administered 0.3 mL per day.At the same time,each mouse in the blank group and the model group was given the same volume of distilled water.1.3 Temperature detectionThe mice were adapted for feeding for 3 days,then were administered intragastrically at 9:00 am daily.After the second day of administration,fasting for 12 hours was allowed.At the third morning,the body temperature was measured once per hour for three times.The average body temperature was taken as the basic body temperature,and body temperature data floated above 0.5? were excluded.According to its weight,the mice were injected with the yeast suspension in the back and administered intragastrically.And the body temperature was measured once per hour for 6 times.Using SPSS software to process and analyze these data.Section two.Gene expression level studiesThe gene expression of PTGS2 in the hypothalamus of each mice was detected by qPCR.Then the data was analyzed and processed by SPSS software.Section three.Protein expression level studiesThe protein expression of PTGS2 in the hypothalamus of each mice was detected by Western Blot.The bands were processed and analyzed by Image J and SPSS software.Results:Part one.Results of systems pharmacological research(1)Ten kinds of herbs with cold nature contained 134 active ingredients.Meanwhile,ten kinds of herbs with hot nature contained 249 active ingredients in total.Among them,the ratio between the number of active ingredients and all compounds in each herb is about 10%to 20%.At the same time,more than one active ingredient was contained in more than one herb.(2)There were 20 key targets in the ingredient-target network of herbs with cold nature,among which the most important targets are PTGS2,PTGS1,and SCN5A.There were 36 key targets in the ingredient-target network of herbs with hot nature,among which the most important trgets are GABRA1,PTGS2,and PTGS1.(3)There were 123 potential diseases related to herbs with cold nature,among which 120 diseases had ICD 10 code.Then these diseases were divided into 14 categories.The top three disease categories were tumors,circulatory diseases,and nervous system diseases.Herbs with cold nature also showed some therapeutic effects in prostate cancer,breast cancer,cardiovascular disease and epilepsy.In addition,a total of 137 potential diseases were related to herbs with hot nature.It included 126 diseases with ICD 10 code,and could be divided into 13 categories.The top three disease categories were tumors,nerve system diseases,mental and behavioral disorders.Besides,drug with hot nature showed potential therapeutic effects in Alzheimer's disease,pain,cognitive disorders,anxiety symptoms,Parkinson's disease and other mental diseases.(4)The main biological processes of herbs with cold nature were positive regulation of cyclase activity,regulation of muscle contraction,regulation of smooth muscle contraction,and maternal process involved in female pregnancy.The main metabolic pathways were estrogen signaling pathway,regualtion of lipolysis in adipocytes,salivary secretion.The main biological processes of herbs with hot nature were negative regulation of synaptic transmission,gamma-aminobutyric acid signaling pathway,monoamine transport,and regulation of smooth muscle contraction.The main metabolic pathways were neuroactive ligand-receptor interaction,tyrosine metabolism,taste transduction and salivary secretion.Part two.Experimental verification resultsSection one.Antipyretic effect study resultsThe study results showed that mice injected with yeast suspension on the back produced a fever reaction,increased energy metabolism,and a significant increase in body temperature.Most of herbs with cold nature can weaken the fever reaction in yeast-induced mice and reduce energy metabolism and body temperature.At the same time,there is a certain dose-dependence between herbs with cold nature and cooling effect,and cooling effect is enhanced with increased drug doses.These drugs include Phellodendri Chinrnsis Cortex,Sophorae Flavescentis Radix,Gentianae Radix Et Rhozima,Coptidis Rhizoma,Gardeniae Fructus.However,Scutellariae Radix,Radix Puerariae,Houttuyniae Herba not only can not reduce the body temperature of yeast-induced mice,but also enhance energy metabolism and body temperature.Section two.Gene expression level study resultsqPCR assay showed that the expression of PTGS2 gene in the hypothalamus increased significantly in the mice injected with yeast suspension.Compared with the model group,herbs with cold nature decreased the PTGS2 gene expression in the hypothalamus of yeast-induced mice.There are 9 in 10 kinds of herbs with cold nature reduced the level of PTGS2 gene expression after intragastric administration.Section three.Protein expression level study resultsWestern Blot assay showed that the expression of PTGS2 protein in the hypothalamus of mice injected with yeast suspension was significantly increased.In addition,herbs with cold nature can reduce the level of PTGS2 protein expression.When yeast-induced mice were given a certain dose of herbs with cold nature,the PTGS2 protein expression in most drug groups decreased significantly.Conclusion:Based on the research model of systems pharmacology,this study sought to find the active ingredients,key targets and potential diseases of herbs with cold and hot nature through data collection,network construction and topology analysis,as well as metabolic pathway analysis.Based on the above results,the vivo experiment and molecular biology experiments were conducted for further study.The results showed that the fever reaction and the energy metabolism of the body can be reduced by regulating PTGS2 target,which may be an important molecular basis for cold nature of TCM.This study provides an important reference for further revealing the essence of cold-hot nature,and also provides new research approach for the theoretical research of cold-hot nature.