| Longzuantongbi Granules(LZTBG)are compound Granules developed for active rheumatoid arthritis(RA)based on the characteristic of "Fawang" theory of Zhuang medicine.At present,it has been approved by Guangxi Drug Administration and use as an in-hospital preparation in the First Affiliated Hospital of Guangxi University of traditional Chinese medicine(Guangxi medicine preparation Zi M20190003000),with remarkable curative effect.Our laboratory has carried out evaluation research on the chemical composition,safety and effectiveness of LZTBG,but its regulatory effect on the body at the metabolic level is not clear yet.With the deepening of the study,according to the characteristics of"multi-component" and "multi-target" of LZTBG,further clarifying the pharmacodynamic substances of LZTBG and clarifying its action mechanism is not only the urgent demand for the clinical application of LZTBG,it is also the core research task of this subject.Based on UPLC-MS/MS technology,combined with pharmacodynamic experiments,characteristic molecular network analysis and metabolomics methods,this subject comprehensively characterized the chemical components of LZTBG;analyzed the drug-derived components in the plasma of LZTBG after single administration of normal rats,clarified the relationship between time concentration and dose concentration;a metabonomics study on the intervention effects of LZTBG on normal rats and RA rats was carried out,to explore the regulation effect of LZTBG on endogenous differential metabolites in plasma of normal rats and RA rats;further considering the effects of LZTBG on the metabolic regulation of RA rats during its efficacy,combined with intergroup trend discrimination and metabolic pathway network analysis,11 potential efficacy biomarkers related to efficacy and their targets of intervention were identified,and the correlation between their pharmacodynamic substances and potential efficacy biomarkers was elucidated.The research content of this subject mainly includes the following five parts:1.Chemical composition analysis of LZTBG based on UPLC-MS/MS technology and characteristic molecular networkThe ultra-performance liquid chromatography coupled with tandem mass spectrometry(UPLC-MS/MS)method was used to obtain the chromatographic and mass spectrometric data of LZTBG,and then quickly filtered and identified the chemical components of LZTBG by feature-based molecular networking(FBMN)analysis,and the target components were associated to generate the characteristic molecular network.Using this analysis strategy,235 compounds were identified and annotated,including 102 alkaloids(73 isoquinoline alkaloids,14 quinoline alkaloids and 15 other alkaloids),72 flavonoids(30 flavonoids and flavonols,15 dihydroflavones and dihydroflavonols,17 isoflavones,6 chalcones,4 anthocyanins and others),18 coumarins,13 lignans,6 phenylpropanoids,11 organic acid and 13 other types of compounds.Among them,25 compounds were confirmed by standard comparison analysis.2.Analysis of absorbed components in normal rat plasma after single administration of LZTBGBased on the self-constructed chemical composition information table of LZTBG,the prototype components were analyzed in plasma of normal rats after single administration of LZTBG by UPLC-MS/MS,and the time-concentration relationship was investigated.The results showed that 13 prototype components could be detected in plasma of normal rats after single administration of LZTBG,including 9 alkaloids,1 coumarin and 3 organic acids.Among them,the absorption rate of alkaloids into blood was slower,and the metabolism time in the body was longer.This results also provided evidence that the alkaloids may be the main pharmacophore components of LZTBG.3.Pharmacodynamic study of LZTBG on RA ratsThe collagen-induced arthritis(CIA)rat model was used in this study,intervention administration for 31 days.The body weight,incidence,arthritis index(AI),foot swelling,immune organs index,histopathological changes of knee joint and expression levels of cytokines TNF-α,IL-1β and IL-4 in serum were used as the indicators,the pharmacodynamics of LZTBG was systematically evaluated from whole to molecular level.The results showed that different doses of LZTBG had potential preventive and therapeutic effects,which could significantly reduce the incidence and AI score,improve synovial hyperplasia and inflammatory cell infiltration,reduce the destruction of bone and cartilage and pannus formation in the knee joint of RA rats,and reduce the expression levels of IL-1β and TNF-α.4.Metabonomics Study of LZTBG on RA ratsA metabolomic method based on UPLC-MS/MS technology was used to analyze the drug-derived components in the plasma of normal rats and RA rats after LZTBG administration,and the dose-concentration relationship was determined.Meanwhile,the regulation effect of LZTBG on endogenous differential metabolites in plasma of normal rats and RA rats was investigated.The results showed that 23 drug-induced components were identified in plasma of normal rats after LZTBG administration,main including 15 alkaloids and 5 coumarins.After LZTBG administration of RA rats,28 drug-derived components were found in plasma,main including 19 alkaloids,6 coumarins and 1 flavonoid.In conclusion,the absorption of LZTBG in rats with different physiological and pathological states was affected to a certain extent.Metabolomics analysis of Normal,NLH and NLL groups showed that LZTBG regulated 32 differential metabolites in plasma of normal rats,of which 26 were down-regulated and 6 were up-regulated;analysis the data of Model,LZTB_L and LZTB_H groups showed that LZTBG regulated 29 differential metabolites in plasma of RA rats,of which 22 were down-regulated and 7 were up-regulated.Further integrated and analyzed the data of Normal,Model and LZTB_L groups,combined with inter-group trend discrimination and metabolic pathway network analysis,found and identified 11 potential efficacy biomarkers related to drug efficacy,mainly involving nine disordered metabolic pathways,such as linoleic acid metabolism,α-linolenic acid metabolism,arachidonic acid metabolism,glycerophosphate metabolism,which provided an important basis for revealing the molecular mechanism of LZTBG intervention in RA rats.5.Correlation study on metabolic regulationIn this study,we compared the similarities and differences of drug-derived components in plasma of normal rats and RA rats administered with LZTBG,as well as the differences in the effects on endogenous metabolites after administration of LZTBG,combined with the analysis of time-concentration and dose-concentration relationship of the drug-derived components in blood,we identified the possible pharmacodynamic components of LZTBG and potential efficacy biomarkers.Furthermore,pearson correlation matrix analysis was used to analyze the correlation between potential efficacy biomarkers and pharmacodynamic components and inflammatory indicators.It was concluded that quinoline alkaloids were only positively correlated with lipid metabolites;isoquinoline alkaloids were not only positively correlated with lipid metabolites,but also negatively correlated with amino acid metabolites and butyric acid metabolites;coumarins were mainly negatively correlated with amino acid metabolites.It was speculated that body weight change was only positively correlated with lipid metabolites;IL-1β,TNF-α and paw swelling were mainly positively correlated with lipid metabolites and amino acid metabolites.AI scores were only negatively correlated with lipid metabolites.Histopathological scores were mainly negatively correlated with lipid metabolites,positively correlated with amino acid metabolites and butyric acid metabolites.This study elucidates the potential pharmacodynamic components of LZTBG and their molecular mechanisms of efficacy,and provides supporting evidence for the reliability of potential efficacy biomarkers associated with the efficacy of LZTBG. |
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