| Background:Gout is a crystal-associated arthropathy caused by urate deposition,which is related directly with hyperuricemia caused by disorder of purine metabolism and/or decrease of uric acid excretion.Gout can be complicated by kidney disease and even renal damage,and also associated with hyperlipidemia,hypertension,diabetes and so on.Arthrosis damage often occurs in gout patients,which seriously reduces the quality of life.<2016 guidebook for gout in China>suggested that nonsteroidal antiinflammatory drugs and colchicine should be used for acute attack of gout,while it is recommended to reduce uric acid for patients with frequent episodes of acute gout arthritis or chronic gouty arthritis or gouty arthritis.The commonly used medicine were NSAIDs,Colchicine,Benzbromarone,Allopurinol and Febuxostat,but most of them have limitations in application because of their side effects.In our study,more than 100 kinds of Chinese medicine have anti-gout efficacy.Properties of the medicine are widely distributed and efficacy’s significant.However,some components,effective components or compound decoction of anti-gout medicine may cause liver injury,which may lead to risk of adverse drug reactions during the treatment,such as Saikosaponin D,PNS Panax Notoginseng Saponins in saponins,colchicine and arecoline in alkaloids.The aqueous extract of Curcumae rhizoma,Chebulae fructusand Olibanum can also cause the same risk as well as volatile oil,aqueous extract,and alcohol extract of Euodiae fructus.Experimental studies on commonly used traditional Chinese medicine such as Dioscoreae nipponicae rhizoma,Curcumae rhizoma;Stephaniae tetrandrae radix,Chebulae fructus,Gentianae radix et rhizoma and Sophorae flavescentis radix have shown the liver injury they caused.The commonly used prescription Da Chaihu Decoction,in which Bupleuri radix is the most important herb,may cause autoimmune hepatitis during its application.The characteristic of anti-gout treatment,which the period of treatment is long and drug combination is commonly used,further increased the risk of liver safety.Therefore,the mechanism research of liver injury against gout Chinese medicine can prevent the occurrence of adverse reactions,reduce safety risks and conduct safety warning.Therefore,from the point of the anti-gout traditional Chinese medicine,this study explors the mechanism of liver injury components in anti-gout Chinese medicine by arranging the components,screening the core targets and key pathways.The research aims at offering reference for the safe and rational drug use of gout and the methodology of the herb-induced liver injury.Objective:1.To screen important liver injury components,core targets and key pathways of anti-gout Chinese medicine by constructing "component-target-pathway" network,which lays the foundation for further exploring its mechanism.2.Molecular biological methods are used to verify the activation of important liver injury components to the core targets,and to explore the mechanism based on the construction of network results,which provides experimental evidence for preventing liver injury caused by anti-gout Chinese medicine.Method and ContentIn this paper,computer simulation method and biological methods are applied to study the mechanism of liver injury components in anti-gout Chinese medicine.1.Literature reviewIn the literature review part,we mainly discuss research status of herb-induced liver injury,application of computer simulation methods in safety research of traditional Chinese Medicine and research status of anti-gout Chinese medicine.2.Experiental researchChapter One Exploring of liver injury components,targets and pathways of anti-gout Chinese medicineFirstly,we reviewed and gathered the literatures of anti-gout Chinese medicines,including relevant experimental studies,clinical trial reports and case reports,in case of exploring 32 kinds of potential hepatic damage components of anti-gout medicine.Secondly,core targets and initial pathways were explored based on analysis of "component-target-pathway" network,which was constructed by dissecting networks of component-associated protein-protein interaction as well as liver injury-associated protein-protein interaction.Secondly,based on significant injury components and core target,we dug important liver injury components according to binding free energy obtained by molecule docking,and analyzed the patterns between components and target.Through Chapter One,the significant liver injury components(Dioscin,Baicalin,Saikosaponin D,Tetrandrine,Rutaecarpine,and Evodiamine)and core target(p38 protein)of anti-gout medicine were obtained,which provided the basis for the follow-up biological experiments.Chapter Two Research on the core target of liver injury components in anti-gout Chinese medicineFirstly,different doses of significant liver injury components were used to L-02 hepatocytes,and the changes of alanine aminotransferase,aspartate aminotransferase,and lactate dehydrogenase as well as p-p38/p38 expression were observed.Secondly,inhibitor SB203580 interfered with L-02 cells induced by components to observes the changes of related enzyme activities and p-p38/p38 expression.Through Chapter Two,the significant liver injury components can increase the activity of alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase,and increase the expression of p-p38/p38.The results verified the computer simulations experient and explained mechanism that the significant liver injury components of anti-gout medicine lead to liver injury by activation of p38.Rusult1.There were a total of 171 kinds of Chinese medicine and 32 kinds of liver injury components of anti-gout Chinese medicine gained from document mining.2.Using nentwork pharmacology,153 important targets for liver injury and 4 core targets were identified,namely MAPK14(p38),NOS2,PPARG,and TNF,and they were mutually upstream and downstream.In this experiment,p38 protein ranked first,and the literature search considered it to be a more critical target.The screening of key pathways involved viruses,cancers,MAPK signaling pathways,etc,but viruses and cancer-related pathways are inconsistent with the purpose of this study,so follow-up will not be discussed.The sequencing of MAPK signaling pathway was the key pathway for liver injury of anti-gout Chinese medicine because it was more advanced and the results of the screening of the core target were also supported.3.Using p38 protein as a receptor and 32 potential hepatic injury components as ligands,six significants ligands were screened by molecular docking methods based on binding free energy order,which were named Dioscin,Baicalin,Saikosaponin D,Tetrandrine,Rutaecarpine,and Evodiamine.4.L-02 liver cells was used to investigate the liver injury and mechanism of significant liver injury components of anti-gout Chinese medicine.Each component can increase the activity of alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase in cell culture medium and increased p-p38/p38 expression.Inhibitor SB203580 can correct the above process.ConclusionSignificant liver injury components,such as Dioscin,Baicalin,Saikosaponin D,Tetrandrine,Rutaecarpine,and Evodiamine of anti-gout Chinese medicine may cause L-02 cell viobility decreased while cell membrane permeability and p-p38/p38 expression were increased.The above results suggested that the mechanism of liver injury caused by anti-gout Chinese medicine may be related to the activation of p38. |
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