| Objectives: Our previous studies indicated that roasting was able to reduce the toxicity and enhance the efficacy of Tripterygium wilfordii(TW)in adjuvant arthritis,but the mechanisms were not clear.This paper explored the mechanisms of roasted TW on toxicity attenation and efficacy improvement based on metabolomics and succinate/vascular endothelial growth factor(VEGF)signaling pathway.This study improved the effectiveness and safety of clinical application of TW,provided a demonstration for the study of the processing mechanism of toxic Chinese medicine,as well as novel insights into the toxicity mechanism of toxic drugs and the pathogenesis of rheumatoid arthritis(RA).Methods:(1)Total extract,roasted total extract,total alkaloids,roasted total alkaloids,celastrol,roasted celastrol,and 1-hydroxy-2,5,8-trimethyl-9-fluorenone were administered continuously for 3 days.The severity of liver and kidney injury was assessed by comparing the histopathological changes and serum levels of alanine transaminase(ALT),aspartate transaminase(AST),creatinine(Cr)and blood urea nitrogen(BUN).Based on urine metabolomics,the metabolic changes induced by TW and its roasted products were analyzed to explain the impact of altered metabolic pathways on toxicity in vivo.(2)A collagen-induced arthritis(CIA)rat model was used,and dexamethasone acetate,total extract,roasted total extract,total alkaloids,roasted total alkaloids,celastrol,roasted celastrol,and1-hydroxy-2,5,8-trimethyl-9-fluorenone were treated for continuous 20 days.The effects of roasting before and after on paw swelling,arthritis score,serum levels of interleukin-1β(IL-1β),and pathological sections of the synovial tissue of CIA rats were observed.Urine metabolomics techniques were used to characterize the metabolic differences and explore the mechanisms of efficacy improvement.(3)A CIA rat model was used,and dexamethasone acetate,total extract,roasted total extract,non-alkaloids parts,non-roasted alkaloids parts,1-hydroxy-2,5,8-trimethyl-9-fluorenone,celastrol,and roasted celastrol were treated for 20 consecutive days.The efficacy was evaluated by serum levels of IL-1β and histopathological changes in the synovial membrane.The activity of succinate dehydrogenase(SDH)and the content of succinate and fumarate in synovial membrane were detected to analyze the source of succinate accumulation.In order to further explore the mechanisms of efficacy improvement,the expression of VEGFA and platelet endothelial cell adhesion molecule-1(CD31)in synovial tissue was measured by immunohistochemistry.Results:(1)Sixteen toxicity biomarkers were identified between control group and total extract group.Twelve toxicity biomarkers were identified between control group and total alkaloids group.Eight toxicity biomarkers were identified between control group and celastrol group.These metabolites mainly involved in seven metabolic pathways including sphingolipid metabolism,taurine and hypotaurine metabolism,tryptophan metabolism,glycerophospholipid metabolism,fatty acid biosynthesis,pentose and glucuronate interconversions,and steroid hormone biosynthesis.After roasting,the toxicity of total extract,total alkaloids and celastrol were relieved as evidenced by ameliorative serum parameters and pathological changes in hepatic and renal tissues.Furthermore,roasting regulated the levels of fourteen candidate biomarkers in the total extract group,ten potential biomarkers in the total alkaloids group and seven candidate biomarkers in the celastrol group to normal levels.These metabolites were also involved in the seven metabolic pathways mentioned above.(2)The paw swelling,arthritis score,serum levels of IL-1β in CIA rats were significantly reduced in total extract,roasted total extract,total alkaloids,roasted total alkaloids,celastrol,roasted celastrol and1-hydroxy-2,5,8-trimethyl-9-fluorenone groups.Synovial hyperplasia and inflammatory cell infiltration were significantly improved in the total extract,roasted total extract and total alkaloids groups.The roasted total extract was obviously more effective than the total extract,and the total alkaloids was obviously more effective than the roasted total alkaloids,while the anti-RA effects of celastrol,roasted celastrol and 1-hydroxy-2,5,8-trimethyl-9-fluorenone were not significantly different.Fifteen RA-related biomarkers were identified from the model group,six of which were associated with enhanced efficacy,involving valine,leucine and isoleucine biosynthesis,tryptophan metabolism,energy metabolism,and arachidonic acid metabolic pathways.(3)The paw swelling,arthritis score,serum levels of IL-1β,and succinic acid content in synovial membrane were significantly decreased in total extract,roasted total extract,non-alkaloids parts,non-roasted alkaloids parts,triptolide,and triptonide groups.The expression of CD31 and VEGFA in synovial membranes was inhibited in total extract,roasted total extract,non-alkaloids parts,non-roasted alkaloids parts and triptolide groups.After roasting,the anti-RA efficacy,inhibition of succinic acid accumulation,and inhibition of CD31 and VEGFA expression of total extract and non-alkaloids parts were significantly enhanced.The SDH activity of the model rats was significantly decreased and the fumarate content was significantly increased compared with normal rats.The SDH activity and fumarate content were not significantly different from those of the model rats after treatment with each drug.Conclusions:(1)Roasting reduced hepatotoxicity and nephrotoxicity induced by TW.The reduction of celastrol and total alkaloids played an important role in the detoxification of roasting on TW.Roasting may ameliorate TW-induced metabolic disorders in sphingolipid metabolism,taurine and hypotaurine metabolism,tryptophan metabolism,glycerophospholipid metabolism,fatty acid biosynthesis,pentose and glucuronate interconversions,and steroid hormone biosynthesis.(2)After roasting,the therapeutic effect of TW on CIA rats was significantly improved.Roasting may enhance efficacy by modulating valine,leucine and isoleucine biosynthesis,tryptophan metabolism,energy metabolism,and arachidonic acid metabolism.(3)TW modulated the succinate/VEGF signaling pathway to improve RA.Roasting enhanced the anti-RA effect by regulating this signaling pathway.The inhibition of succinate accumulation by TW was not related to the SDH activity. |
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