Studies On Screening And Mechanism Of Action Of Traditional Chinese Medicine For Treating Gout

Gout is a common inflammatory arthritis associated with the dysfunction of purine metabolism and caused by sodium urate deposition in and around the joints.With dietary and lifestyle changes,the incidence of the disease has increased annually.Gout attack can cause a number of important complications such as common renal disease,cardiovascular disease and metabolic syndromes.Therefore,gout is a serious threat to human health.At present,safe,effective,and economical treatment of gout drugs are less.Thus,in recent years,the development of new anti-gout drugs with high efficacy are eagerly needed,for which,natural resources are research hotspot.More and more Traditional Chinese Medicine(TCM)is used to treat gout disease,but the mechanism of action of drugs is unclear.In the study,the TCM of treating gout is screened at the molecular level,the cellular level and the animal level.Moreover,the metabolomics method was applied to study the mechanism of TCM treatment of gout.The main results are summarized as follows:1.Screening of the anti-gout traditional herbs from TCMs using both in vitro and vivo modelsMonosodium urate(MSU)induced inflammation and xanthine oxidase(XO)activity abnormalities led to increased uric acid production are the two major pathogenesis of gout.Therefore,the cell model was established by stimulating human umbilical vein endothelial cell(HUVEC)using monosodium urate(MSU)solution in vitro and an ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-TQ/MS)method for the detection of uric acid were established.The crude extracts from 33 TCMs were screened at the cellular and molecular levels.The results indicated that the extracts of Ginkgo biloba L.leaves,Salvia miltiorrhiza Bge.,Rheum palmatum L.,Polygonum cuspidatum Sieb.et Zucc.,Selaginella tamariscina(Beauv.)Spring.and Paeonia veitchii Lynch.seemed to be more effective on the anti-inflammation and inhibiting XO activity.In this study,the rat model of acute gout with hyperuricemia was established by intraperitoneal injection of xanthine and oxonic acid potassium salt and articular injection of MSU.The joint swelling,the expression of IL-1? in serum and the pathological changes in joints and kidneys were assessed.The results showed that the crude extracts of Ginkgo biloba L.leaves,Salvia miltiorrhiza Bge.,Rheum palmatum L.,Polygonum cuspidatum Sieb.et Zucc.,Selaginella tamariscina(Beauv.)Spring.and Paeonia veitchii Lynch.could improve the above changes when compared with model group,including the joint swelling was reduced,the level of serum IL-1? decreased and the pathological changes of renal and synovial tissue were ameliorated.2.Metabolomic studies of six TCMs in the in vitro model of acute goutThe crude extracts of Ginkgo biloba L.leaves,Salvia miltiorrhiza Bge.,Rheum palmatum L.,Polygonum cuspidatum Sieb.et Zucc.,Selaginella tamariscina(Beauv.)Spring.and Paeonia veitchii Lynch.were selected to study the mechanism of TCM in acute gout model in vitro by MSU-induced.Potential biomarkers were identified by ultra-performance liquid chromatography-electrospray ionization quadruple time-of-flight/mass spectrometry(UPLC-ESI-QTOF/MS)coupled with orthogonal projection to latent structure squares-discriminant analysis(OPLS-DA)approaches between the model group and the control group.The results showed that the 16 potential endogenous biomarkers were identified and the primarily metabolism pathways involved glutathione metabolism,purine metabolism,pyrimidine metabolism,leucine metabolism and cysteine metabolism.The crude extracts have different reverse effects on regulation on the disorder of intracellular biological small molecules by MSU-induced.Ginkgo biloba L.leaves could reverse more potential biomarkers.3.The mechanism of action of six TCMs in the model of acute gout with hyperuricemia ratsIn this study,the endogenous metabolites were identified from urinary samples by the UPLC-ESI-QTOF/MS technique combined with principal component analysis(PCA)and OPLS-DA method in the control group,the model group and the drug-treated group.A total of 27 potential biomarkers were identified which associated with acute gout.These potential biomarkers are mainly involved in tryptophan metabolism,purine metabolism,pyrimidine metabolism,tyrosine metabolism and fatty acid metabolism.Six TCM could play the role of treatment of acute gout and hyperuricemia from different metabolic pathways.(a)Study on the mechanism of the extract of Ginkgo biloba L.leave in acute gout with hyperuricemia rats.The study successfully identified 18 potential biomarkers mainly including in tryptophan metabolism,purine metabolism,pyrimidine metabolism,lysine degradation and fatty acid metabolism.(b)Study on the mechanism of Selaginella tamariscina(Beauv.)Spring.in acute gout with hyperuricemia rats.The study successfully identified 18 potential biomarkers mainly including in tryptophan metabolism,lysine degradation,tyrosine metabolism,pyrimidine metabolism and purine metabolism.(c)Study on the mechanism of Polygonum cuspidatum Sieb.et Zucc.in acute gout with hyperuricemia rats.A total of 21 potential biomarkers were identified.These biomarkers are mainly involved in tryptophan metabolism,TCA cycle,phenylalanine metabolism,lysine degradation and taurine metabolism.Study on the mechanism of Rheum palmatum L.in acute gout with hyperuricemia rats.18 potential biomarkers were identified.The main metabolic pathways were tryptophan metabolism,TCA cycle,phenylalanine metabolism and lysine degradation.Polygonum cuspidatum Sieb.et Zucc.and Rheum palmatum L.are rich in anthraquinone compounds.There are 10 potential biomarkers that could reverse by them.(d)Study on the mechanism of Salvia miltiorrhiza Bge.in acute gout with hyperuricemia rats.A total of 16 potential biomarkers were identified.The main metabolic pathways including tryptophan metabolism,TCA cycle,tyrosine metabolism,purine metabolism,pyrimidine metabolism and phenylalanine metabolism.(e)Study on the mechanism of Paeonia veitchii Lynch.in acute gout with hyperuricemia rats.A total of 14 potential biomarkers were identified.The main metabolic pathways are tryptophan metabolism,TCA cycle,phenylalanine metabolism,fatty acid metabolism and lysine degradation.4.Quantitative analysis of major potential biomarkers in urine samples of acute gout with hyperuricemia ratsQuantitative analysis of major biomarkers was performed on an ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-TQ/MS)combined with multiple reaction monitoring(MRM).There are 10 potential biomarkers were analyzed,including cytosine,indole-3-carboxylic acid,tyrosine,3-hydroxyanthranilic acid,orotic acid,allantoin,uric acid,suberic acid,sebacic acid and citric acid in the urine of the control group,the model group and the drug-treated group.The method is sensitive,fast,reproducible and highly accurate by investigating inter-day precision,inter-day accuracy,intra-day precision,intra-day accuracy,matrix effect and sample recovery.The results showed that the change of biomarkers were consistent with the change of potential biomarkers.And the potential biomarkers screened in Chapter 4 were reliable.In a word,the results of this study provide a new direction for the development of new TCM for treating gout.And the discovery of potential biomarkers provides a theoretical basis for the clinical diagnosis of gout in the development and progression.