Identification Of Chemical Material Basis Of Baihu-Jia-Guizhi Decoction And Investigation On The Functional Property Of This Formula Based On Network Target Analysis

Objectives:Baihu Guizhi Tang(BHGZT)originated from Zhang zhongjing’s classical prescription for arthralgia,which composes of cassia twig,gypsum,anemarrhena,processed licorice and japonica rice,with qingrexiehuo,tongluo analgesic effect.As a qingrexiehuo,tongluo analgesic prescription,it’s applied to rheumatoid arthritis,gouty arthritis,children cough and other urgent treatment,especially for heat bi.Most the researches of BHGZT have been only focusing on the chemical constituents of one single herbs in the prescription,and few studies focus on the chemical components of BHGZT.Although it is widely used in clinical practice,there are few studies on the relevant mechanism.This project used the UFLC-Triple-TOF-MS/MS to analyze BHGZT’s chemical constituents and the compounds which into the blood.Using network pharmacology research method,the drug target prediction,network analysis and experimental verification were combined to preliminarily discuss the cold property of BHGZT.To further study the cold property of BHGZT,we use the UFLC-Triple-TOF-MS/MS to analyze its chemical constituents and the compounds which into the blood,and then combine drug target prediction,network analysis and experimental verification using network pharmacological research methods.Method:1.The material basis research of BHGZT1.1 Chromatographic analysPhenomenex C18 was eluted with acetonitrile(containing 0.1%formic acid)and 0.1%formic acid solution as the mobile phase in a gradient mode.High-resolution triple quadrupole flight mass spectrometry was adopted to tests in both positive and negative modes.1.2 MS analysis methodIn peakview software,Formula Finder function was used to calculate the element composition of the compounds and IDA explore function was invoked to obtain secondary fragmentation ions of this mass number.The compounds were identified based on literature data and the reference substances.2.Serum pharmacochemistry of BHGZT2.1 Investigation the best timing for blood collectionAfter administering BHGZT by gavage to rats,blood was collected from orbit at0min,15min,30min,1h,2h,4h,8h,10h.The serum samples were processed with protein precipitation and tested as mentioned in method 1.1.2.2 Data processingThe mass spectra data of blank serum and drug-containing serum were simultaneously processed by Peakview software.Comparative analysis was used to determine whether the compound was origxinated from BHGZT.3.The pharmacodynamic experiment of BHGZT on RA3.1 Induction of CIA modelsMale SD rat were injectiond intradermally at the base of the tail.3.2 Groups and treatmentMale SD rat were divided into 6 groups with 8 rat per group,which were separately categorized into the normal control group,the Model group,the BHGZT 5.4mg/kg group,BHGZT 10.7mg/kg group,BHGZT 21.4mg/kg group.Treatment of BHGZT for 24 days via oral administration from the first day,and the rat were sacrificed on the 25th day of experiment.3.3 Severity assessment of arthritisArthritis severity was evaluated by arthritis score,arthritis incidence and body weight changes.hematoxylin eosin staining(HE)and micro computed tomography(micro-CT).4.Network target prediction and validation of BHGZT intervention in heat-RA4.1 Key putative targets of BHGZT acting on RA with the TCM heat pattern4.1.1 Data collection and organizationSearching and organization the compound information from the results of previous studies and Taiwan Database;PubMed and CNKI databases were used to searching Chinese and English literatures containing the keywords of "rheumatoid arthritis" and "heat syndrome",and extracted RA heat syndrome related genes with experimental evidence4.1.2 Prediction of candidate targets and screening of key targetsBy using the Chinese medicine target prediction system established byOur research group,candidate targets of BHGZT were obtained.Based on the String database,the interaction information between the appellate candidate target genes was extracted to construct an interaction network to screen the key candidate targets of BHGZT.4.1.3 Prediction BHGZT cold drug-related network target and construction of BHGZT key candidate target interaction network for RA heat syndrome-related gene;4.1.4 Biological significance interpretation of BHGZT cold drug-related pathways based on GO and KEGG data platforms.4.2 Vaildation of the regulatory effect of BHGZT on predicted targets4.2.1 In vivo animal experimental validationsThe expression levels of ADCY3、PPAR-y mRNA were detected by q-PCR.4.2.2 In vitro cell experimental validationsBHGZT were used for intervention in TNF-α induced human HFLS-RA cell model.The expression levels of IL-1,IL-6 and IL-8 mRNA were detected by q-PCR to preliminarily verified the network analysis mentioned above.Results:1.Analysis the material of BHGZTOn the basis of the mass fragmentation regularity of accurate molecular weight,35 compounds were identified in BHGZT including 16 steroid saponins,6 triterpenoid saponins,7 flavonoids,4 organic acids and 2 other compounds.Among them,10 components(timosaponin AⅢ,timosaponin Ⅰ,timosaponin BⅢ,timosaponin BⅡ,new mangiferin,mangiferin,liquiritin apioside,liquiritin,glycyrrhizic acid and cinnamic acid)were confirmed by comparing with reference substances,and the sources of the compound were identified.2.Analysis of serum drugs of BHGZT2.1 The mass spectrum of drugs-serum collecting at different time were campared.The results showed that the drugs-serum samples collecting 1h after drug administrationare most suitable for analysis.2.2 By comparing the fingerprints of blank serum,drugs-serum and BHGZT solution,a total of 25 components in blood were identified.14 flavonoids,7 anemones,3 organic acids and 1 triterpenoid saponins were detected in the negative mode.3.The pharmacodynamic experiment of BHGZT on RA3.1.1 The incidence rate in the model group was 100%,withthe arthritic symptoms,swelling,as well as bone erosion and cartilage damage.3.1.2 BHGZT could effectively decrease the incidence of CIA rats and alleviate the arthritic symptoms,swelling,as well as bone erosion and cartilage damage.4.Network target prediction and validation of BHGZT intervention in heat-RA4.1 Key putative targets of BHGZT acting on RA with the TCM heat pattern4.1.1 1312 candidate targets were predicted based on the constructed chemical library.4.1.2 The interaction information between candidate target genes of BHGZT was extracted,and the candidate target gene interaction network was constructed.Nodes with connection degree greater than 2 times of median were selected as the key candidate targets of BHGZT,which totaling 309.4.1.3 Heat RA-related genes-BHGZT candidate target gene interaction network was established and 79 key candidate targets for BHGZT intervention heat syndrome-RA were obtained through the screening of three topological characteristic values of connectivity,node interface and node tightness.4.1.4 The biological functional interpretation of 79 key candidate targets showed that BHGZT mainly acted on fat,sugar energy metabolism and inflammatory immunity.Three cytokines,il-1,il-6 and il-8,which are involved in both inflammatory immune regulation and energy metabolism regulation such as fat,were tested in vitro.4.2 Vaildation of the regulatory effect of BHGZT on predicted targets4.2.1 In vivo animal experimental validationsAnimal experiment showed that BHGZT decreased the expression of ADCY3、PPARγ mRNA.4.2.2 BHGZT regulation of cytokines IL-1,IL-6 and IL-8TNF-α-induction HFLS-RA cells inflammatory model was successfully established.Compared with the control group,TNF-α-induced HFLS-RA cells for 24 h significantly up-regulated the mRNA expression of IL-1,IL-6 and IL-8(P<0.01).BHGZT can effectively down-regulate the mRNA of IL-1,IL-6 and IL-8.BHGZT anti-RA by inhibiting the expression of cytokines.Conclusions:1.UFLC-Triple-TOF-MS/MS can be used to characterize the chemical composition of Baihu Guizhi Tang simply and quickly.Relevant researches provide references for the research of pharmacodynamic material basis,the quality control and the explanation of mechanism of action.2.Based on the research strategy of network pharmacology,by the prediction of drug candidate targets,the topological characteristics calculation of the network of disease-syndrome-related gene-drug candidate target interaction and the interpretation of the biological function of the target,the results show that the related pathways of BHGZT heat-RA mainly act on fat,sugar energy metabolism and inflammatory immunity.3.CIA model rats were successfully established.The incidence rate in the model group was 100%,and the joint lesions were obvious.It was confirmed that BHGZT could effectively delay the onset time of incidence,alleviate the arthritic symptoms,including red,swelling,as well as bone erosion and cartilage damage.4.According to in vivo experiments,the abnormal expression of ADCY3 and PPAR-γcould be regulated by BHGZT.Based on TNF-α-induced HFLS-RA cell model experiment,the experimental results showed that BHGZT could effectively inhibit the abnormal expression of IL-1,IL-6 and IL-8 mRNA,which were involved in both inflammatory immune response and energy metabolism,further illustrating the biological basis of BHGZT’s cold property.