| Simiao Yong'an Decoction has been used as a classic prescription for the treatment of gangrene in ancient times.It has definite therapeutic effects on various types of cardiovascular diseases,such as atherosclerosis,venous thrombosis,and vasculitis,diabetes and chronic complications,rheumatic immunity,and tumors.Simiao Yong'an Decoction is precise and proper,but the interaction of various components and the interaction of various factors make the material basis and mechanism of the compound effective have not been clarified.Inflammation is the pathogenic factor and initial cause of many diseases.It is closely related to the occurrence and development of many diseases.Literature reports and previous studies of our research group showed that Simiao Yong'an Decoction may play a variety of pharmacological effects and inhibit inflammatory response,but the pharmacodynamic material basis,related targets and how it plays its anti-inflammatory effect have not been clarified.Therefore,this experiment analyzed and identified the components absorbed into the blood of healthy SD rats after they were given Simiao Yong'an Decoction by gavage based on the UHPLC-LTQ-Orbitrap,and provided a basis for the study on the pharmacodynamic substance basis of this compound.Screened the potential components of Simiao Yong'an Decoction with anti-inflammatory activity and predict its anti-inflammatory mechanism through network pharmacology.Furthermore,combined with the blood components and literature reports,the in vivo metabolism of luteolin,kaempferol and isoliquiritigenin was studied to lay a foundation for clarifying the pharmacodynamic material basis and mechanism of Simiao Yong'an Decoction.Based on the monocyte-macrophage inflammatory model,the safe drug concentration of Simiao Yong'an decoction and its three components was screened.Cell administration and detection were conducted to preliminarily explore the mechanism of anti-inflammatory effect in vitro from the changes of phosphorylated ERK1/2 expression and inflammatory factors.To explore the research mode of "chemical composition-network pharmacology-metabolism research-efficacy verification",and to provide a quick and multi-dimensional research strategy for the basic research of traditional Chinese medicine efficacy substances.The specific content of the research is as follows:1 Analysis of Simiao Yong'an Decoction absorbed into the blood componentIn this experiment,healthy SD rats were randomly divided into two groups:the administration group was given Simiao Yong'an Decoction by gavage;the blank group was given distilled water of equal volume by gavage.Rats in each group were given gavage twice a day for 7 days.Blood was collected from abdominal aorta one hour after the last administration.Samples were processed to obtain drug-containing serum and blank serum.UHPLC-LTQ-Orbitrap was used to analyze and identify the components absorbed blood components of Simiao Yong'an Decoction in rats.The Waters AQUITY UPLC BEH C18 column(2.1 mm × 100 mm,1.7 ?m)was used in the experiment.The mobile phases(A)and(B)were 0.1%formic acid aqueous solution and acetonitrile,respectively,with gradient elution,and the flow rate was 0.3 mL·min-1.High-resolution mass spectrometry used a thermal spray ion source and the detection mode was negative ion.Combining the mass spectrum information,standard quality spectrum information and related literature of the components,a total of 21 components were obtained.It was identified 7 prototype components and 14 metabolic components.Four components failed to speculate the structure-due to insufficient comprehensive information of multi-level mass spectrometry fragmentation fragments.This experiment laid the foundation for the research on the substance basis of Simiao Yong'an Decoction.2 Research on the anti-inflammatory mechanism of Simiao Yong'an Decoction based on network pharmacology and molecular dockingIn this chapter,based on the idea of network pharmacology,the chemical components of Simiao Yong'an Decoction were retrieved through the TCMSP database.A total of 128 active components were selected based on the reported active components,blood components and ADME parameters,and a total of 247 drug targets were matched.Based on GeneCards database and OMIM database,1507 inflammatory targets were screened.A network of target genes related to the main active components of Simiao Yong'an Decoction and target genes related to inflammation were constructed,and a total of 145 intersection targets were obtained.GO gene enrichment analysis and KEGG pathway enrichment analysis were performed.The molecular docking study of 6 major active ingredients including quercetin and 4 major proteins including STAT3 was conducted to predict the anti-inflammatory effect mechanism of Simiao Yong'an Decoction.Simiao Yong'an Decoction mainly acted on STAT3,AKT1,MAPK and other key targets through quercetin,luteolin,kaempferol and isoliquiritigenin to regulate various biological functions and signaling pathways Effect to achieve anti-inflammatory effects.This showed that Simiao Yong'an Decoction exerts synergistic effects through multiple ingredients,multiple targets and multiple pathways.3 Metabolism analysis of luteolin,isoliquiritigenin and kaempferol in ratsThirty two healthy male SD rats were randomly divided into four groups.The three groups were given three kinds of control suspension of 20 mg·mL-1,and the blank group was given equal volume distilled water.Four groups of rats were orbitally bled at 10 min,30 min,1 h,1.5 h,2 h,4 h and 6 h after gavage.After the sample processing,the blood samples at various time points were combined to obtain blank plasma and drug-containing plasma.The urine and feces of rats from 0 to 24 h were collected,and samples of urine and feces were obtained after sample treatment.The original components and metabolites of blood,urine and feces of rats were analyzed and identified by UHPLC-LTQ-Orbitrap technology.The Waters AQUITY UPLC BEH C18 column(2.1 mm × 100 mm,1.7 ?m)was used.The mobile phase was a gradient elution of 0.1%aqueous formic acid(A)and acetonitrile(B),with a flow rate of 0.3 mL·min-1.High-resolution mass spectrometry used a thermal spray ion source and the detection mode was negative ion.According to the literature reports,the mass spectrometry of the standard and the fragmentation information of the compounds,the relevant components were analyzed.After luteolin administration,10 components were identified in plasma,11 components were identified in urine and 3 components were identified in feces.After isoliquiritigenin administration,6 components were identified in plasma and 1 3 components were identified in urine.After kaempferol administration,8 components were identified in plasma,3 components were identified in urine and 1 component was identified in feces.The metabolic pathways of the three components in rats were similar,and most of the obtained components were phase ? metabolites except the prototype components.4 Establishment of monocyte-macrophage inflammatory model and screening of drug concentrationIn this chapter,a monocyte-macrophage inflammatory model was successfully established by PMA induced with 100 ng·mL-1 combined with LPS stimulation of 1 ?g·mL-1.Luteolin,isoliquiritigenin and kaempferol which have high degree and good binding potential with protein,were screened in combination with the blood components of rats,the results of network pharmacology and relevant literature reports.The cell drug concentration ranges of Simiao Yong'an Decoction and three monomers were determined by CCK-8 experiment.Simiao Yong'an Decoction had no obvious toxicity to cells in the range of 0?500 ?g·mL-1,luteolin in the range of 0?2.5 ?g·mL-1,kaempferol in the range of 0?5 ?g·mL-1,and isoliquiritigenin in the range of 0?7.5 ?g·mL-1.Therefore,this concentration range was selected as the safe concentration for the experimental cell administration.5 A preliminary study on the mechanism of Simiao Yong'an Decoction and its active ingredients in the anti-inflammatory effectIn this chapter,based on the mononuclear macrophage-macrophage inflammatory model,the anti-inflammatory effect mechanism of Simiao Yong'an Decoction and the monomer components luteolin,kaempferol and isoliquiritigenin were preliminarily explored from the expression of phosphorylated ERK1/2 and the changes of inflammatory factors.It was concluded that luteolin and kaempferol could inhibit LPS-induced THP-1 macrophage inflammation to a certain extent,and the mechanism might be related to the inhibition of ERK1/2 phosphorylated protein level and the reduction of the expression of inflammatory cytokines.Simiao Yong'an Decoction and isoliquiritigenin did not show a direct effect on inhibiting the phosphorylated protein level of ERK1/2 and improving the inflammatory response of macrophages. |
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