| Tripterygium glycosides tablets are the main clinical preparation of traditional Chinese medicine Tripterygium wilfordii Hook.f.,commonly used to treat rheumatoid arthritis and autoimmune diseases,with remarkable effects.However,its hepatotoxicity is also reported most frequently.Although there have been many studies,our research group has also found the effective/toxic substance signal of tripterygium glycosides tablets in collagen-induced arthritis(CIA)rats in the early stage,but its hepatotoxicity and effective substance basis have not yet been clarified.Objective: Integrating metabolomics,network analysis,identification of absorbed components and metabolites in vivo,cellular efficacy/toxicity screening,and molecular docking to reveal the efficacy/toxicity substances and mechanism of tripterygium glycosides tablets.Methods: UHPLC-Q-TOF-MS metabolomics was used to find the differential metabolites in serum,liver,and spleen of rats with CIA treated with tripterygium glycosides tablets,respectively.The differential metabolites were identified by online databases(HMDB and LIPID MAPS).FC values and ROC curves were used to screen key differential metabolites in serum associated with tripterygium glycosides tablets.The online software Metabo Analyst was used to perform pathway analysis searching for important metabolic pathways of tripterygium glycosides tablets.Network analysis was conducted to construct of "compound-reaction-enzyme-gene" networks for differential metabolites in serum,liver,and spleen,respectively,and find differential metabolites-related genes.The differential metabolite related genes in serum were intersected with liver and spleen differential metabolite related genes,respectively,to screen the efficacy genes and hepatotoxicity genes of tripterygium glycosides tablets.The toxicity of the 19 components in tripterygium glycosides tablets were studied separately using normal rat liver cells to find hepatotoxic components.Cell proliferation assay was used to examine the safe concentration interval of19 components on RAW264.7 cells.Based on the LPS-induced in vitro inflammation model of RAW264.7 cells,the effects of each component on NO,IL-6 and TNF-α secreted by RAW264.7 cells were investigated at the maximum safe dosing concentration to screen the anti-inflammatory components in tripterygium glycosides tablets.The cleavage pattern of the compounds in tripterygium glycosides tablets was analyzed.The absorbed components and metabolites in the serum of CIA rats taking tripterygium glycosides tablets were identified using mass spectrometric information of the standard and compounds.The in vivo absorbed components were intersected with the in vitro hepatotoxic components and anti-inflammatory active components,respectively,to find the effective and toxic substances in tripterygium glycosides tablets.The screened effective components and hepatotoxic components were docked with the corresponding proteins of the effective genes and hepatotoxic genes using Auto Dock Vina,respectively,to screen the key targets for the efficacy and hepatotoxicity of tripterygium glycosides tablets.Results: 23,142 and 16 differential metabolites were identified in serum,liver,and spleen,respectively.The differential metabolites in serum were mainly related to the glycerophospholipid metabolic pathway,and four key differential metabolites were screened,namely Lyso PC(18:0),Lyso PA(20:4),PS(O-20:0/17:1),and Lyso PA(18:2).73,306,and 25 genes associated with differential metabolites were found in serum,liver,and spleen,respectively,by network analysis.A total of 23 genes related to the efficacy of tripterygium glycosides tablets and 46 genes related to the hepatotoxicity of tripterygium glycosides tablets were finally obtained by taking the intersection.4 hepatotoxic components and 16 anti-inflammatory active components of tripterygium glycosides tablets were found by in vitro cellular assays.16 prototype components and 70 metabolites were identified in the serum of CIA rats.Among them,the compound peritassine A was not identified as a prototype in serum,but four metabolites of peritassine A were identified using the standard.Therefore,a total of 17 absorbed components of tripterygium glycosides tablets were identified in CIA rats.The in vivo absorbed components were intersected with the hepatotoxic and effective components screened in vitro,respectively.Ultimately,a total of 9 effective components were obtained,namely neotripterifordin,triptophenolide,celastrol,demethylzeylasteral,peritassine A,wilfortrine,wilfordine,evonimine,and wilforgine;2 hepatotoxic components were obtained,namely celastrol and demethylzeylasteral.9 efficacy components docked with 23 efficacy targets with a minimum binding energy of-11 kcal/mol and a maximum of-4.7 kcal/mol.The target PLA2G4D、LCAT、PLD1、PLD2 and AASDHPPT can strongly bind with 5or more components(binding energy less than-7 kcal/mol).2 hepatotoxic components docked to 46 hepatotoxic targets with a minimum binding energy of-10.6 kcal/mol and a maximum of-6.2 kcal/mol.The receptors with docking binding energy less than-9 kcal/mol with celastrol are PLA2G4 D,PLA2G4A,PLD1,and PLD2,while the receptors with docking binding energy less than-9 kcal/mol with demethylzeylasteral are PLA2G4 D,PLD2,LCAT,and B4GALT1.Conclusions: Integrating various experimental methods,this study found that the efficacy of tripterygium glycosides tablets was mainly related to neotripterifordin,triptophenolide,celastrol,demethylzeylasteral,peritassine A,wilfortrine,wilfordine,evonimine,and wilforgine;the hepatotoxicity of tripterygium glycosides tablets was mainly related to celastrol and demethylzeylasteral.Tripterygium glycosides tablets in CIA rats mainly affect the glycerophospholipid metabolic pathway,regulating the metabolites Lyso PC(18:0),Lyso PA(20:4),PS(O-20:0/17:1)and Lyso PA(18:2).Its efficacy is mainly related to the regulation of the targets PLA2G4 D,LCAT,PLD1,PLD2,and AASDHPPT.Its hepatotoxicity was mainly associated with the regulation of the targets PLA2G4 D,PLA2G4A,PLD1,PLD2,LCAT,and B4 GALT. |
