Study On The Anti-inflammatory Pharmacodynamic Material Basis And Action Mechanism Of Huo Luo Xiao Ling Dan In Rheumatoid Arthritis

Huo Luo Xiao Ling Dan(HLXLD)is a traditional Chinese herbal formula consisting of 11 herbs,including Angelica sinensis(Oliv.)Diels.,Salvia miltiorrhiza Bge.,Boswellia carterii Birdw,Ligusticum chuanxiong S.H.Qiu.,Paeonia lactiflora Pall,Corydalis yanhusuo W.T.Wang.Cinnamomum cassia Presl.,etc.And this forluma is meaningful for the treatment of rheumatoid arthritis(RA)and related inflammatory anomalies.Research showed that arachidonic acid(AA)and its metabolites were closely related to inflammation of RA.The objective of this research was to clarify the effective material basic and active mechanism of HLXLD.1.Development of a UFLC-MS/MS method for the determination of eicosanoid metabolome in rat plasma and tissuesA UFLC-MS/MS method was developed for the simultaneous determination of a wide range of bioactive eicosanoids in rat plasma and tissues(knee joint,liver,kidney,spleen,and heart).The protein precipitation procedure was used to prepare the samples and AA-d8,PGE2-d4 and 15-HETE-d8 were taken as the internal standard.Analytes and internal standard were separated on a SHIM-PACK XR-ODS C18 column with 0.05%formic acid in water(pH adjusted with ammonia)and acetonitrile-methanol(20:80,v/v).Detection was performed on UFLC-MS/MS system with electrospray negative ionization(ESI-)and multiple reaction-monitoring mode.The calibration curves of the analytes were in good linearity(r>0.99)over the range of their respective concentration.Intra-and inter-day precision and accuracy of the analytes were well within acceptance criteria(15%).The absolute recoveries of analytes and IS were all more than 80%.The validated quantification method was satisfied according to the US-FDA document and other related guidelines with respect to limit of quantification,linearity,precision,accuracy,recovery,matrix effect and stability.2.Quantification of arachidonic acid and its metabolites by UFLC-MS/MS:application to identify the potential biomarkers of rheumatoid arthritis The changes of arachidonic acid metabolic profiles of the plasma and tissue(knee joint,liver,kidney,spleen and heart)of CFA-induced RA model rats had been evaluated.Compared with normal rats,AA,15-HETE,13-HODE,12-HETE,9-HODE,5-HETE,8-HETE,TXB2,PGD2,PGE2,PGF2α,LTE4,LTB4,LTC4,13-oxoODE,9-oxoODE,5-HpETE,and LTD4 in plasma from model rats had significant difference(P<0.05).In knee joint and spleen,the levels of AA,15-HETE,12-HETE,5-HETE,8-HETE,PGF2α,TXB2,LTE4,PGH2,PGD2,PGE2,LTB4,13-HODE,9-oxoODE and 13-oxoODE had significant difference(P<0.05).In liver and kidney,the levels of AA,15-HETE,12-HETE,LTD4,5-HETE,TXA2,PGI2,PGF2α,TXB2,PGD2,PGE2,PGF2α-EA,PGE1-EA had significant difference(P<0.05).AA,15-HETE,12-HETE,5-HETE,8-HETE,PGF2α,TXB2,5-HpETE,LTE4,PGE2,PGD2 and LTB4 in heart had significant difference(P<0.05).It revealed that RA fortified inducible expression of COX and LOX,as well as increased production of AA and eicosanoids.The method described here offers a useful tool for the evaluation of complex regulatory eicosanoids responses in vivo studies.3.The interventional effects of Huo Luo Xiao Ling Dan in arachidonic acid metabolomic on RA model ratsThe above proposed method was applied to determine the concentrations of arachidonic acid metabolic profiling in normal group,model group,HLXLD group and positive control group.From day 7 to day 28,the trajectory direction of HLXLD group and positive control groups gradually moved towards the initial space.On day 28,the trajectory direction of HLXLD group was superior to positive control groups,and positive control group-2 was more departing from normal group.Compared with model rats,the levels of AA,12-HETE,15-HETE,8-HETE,LTB4,PGE2,PGI2,PGD2,PGF2α,TXB2,etc in plasma from HLXLD group had significant difference(P<0.05).In methotrexate group,the concentrations of AA and metabolites mediated by COX(TXA2,PGE2,PGI2,PGD2,PGF2α,TXB2)were significantly retraced,while metabolites catalyzed by LOX had little modification or increased,and dexamethasone merely regulated the products metabolized from LOX(12-HETE,15-HETE,8-HETE,LTB4,etc).Using the potential biomarkers as a screening index,the results suggest that HLXLD can potentially reverse the process of RA by partially regulating AA metabolism through refrain the dual expression of cyclooxygenase(COX)and lipoxygenase(LOX).4.Identified parent compounds and metabolites from rats plasma after oral administration of HLXLD by UHPLC-ESI-Q-TOF-MSAn ultra high performance liquid chromatography coupled with electrospray ionization/quadrupole time-of-flight mass spectrometry(UHPLC-ESI-Q-TOF-MS)method in positive and negative mode was employed to identify parent compounds and metabolites from rat plasma after oral administration of HLXLD.A SHIM-PACK XR-ODS C18 column was used for the chromatographic separation.The mobile phases were 0.02%formic acid in acetonitrile and 0.02%aqueous formic acid.As a result,78 prototype components and 31 metabolites in plasma were found.The aromatization and hydration were the main metabolic pathways of lactones and tanshinones-related metabolites;demethylation and oxidation were the maj or metabolic pathways of alkaloid-related compounds;methylation and sulfation were the main metabolic pathways of phenolic acids-related metabolites.The results of this study provide references for further in vivo ADME studies and pharmacological efficacy research of HLXLD.5.The pharmacokinetics-pharmacodynamics of anti-inflammation active compounds in HLXLDThe plasma concentrations of 37 anti-inflammation active compounds in HLXLD were independent variables,and pharmacodynamics index that AA metabolic profiling were dependent variable.Pearson correlation analysis was performed on the plasma concentrations and pharmacodynamics.As a conclusion,23 maker compounds had correlation with arachidonic acid metabolic profiling,and among them,senkyunolide A,3-butylphthalide,bergapten,columbianetin,isoimperatorin,ferulic acid,glycyrrhetinic acid,11-keto-β-boswellic acid,α-boswellic acid,β-boswellic acid,acetyl-α-boswellic acid,and acetyl-β-boswellic acid had high correlation with AA metabolic profiling(r>0.8),which may be the main target components.Ligustilide,levistilide A,tanshinone ⅡA,osthole,columbianedin,imperatorin,notopterol,loganic acid,tetrahydropalmatine,dehydrocorydaline,and berberine had moderate correlation with AA metabolic profiling(0.5<r<0.8),which may be the secondary target component.13 maker compounds such as bergapten,columbianetin,imperatorin,α-boswellic acid,etc.had correlation with dual pathways(COX and LOX),which could explain the multicomponents and multitargets of HLXLD.The establish of pharmacokinetics-pharmacodynamics would elucidate the effective substance basis of HLXLD.