Abstract
BackgroundTriptolide (TP), an active constituent of Tripterygium wilfordii, possesses numerous pharmacological activities. However, its effects on cytochrome P450 enzymes (CYP450s) in rats remain unexplored.MethodsIn this study, the effects of triptolide on the six main CYP450 isoforms (1A2, 2C9, 2C19, 2D6, 2E1, and 3A) were investigated both in vivo and in vitro. We monitored the body weight, survival proportions, liver index, changes in pathology, and biochemical index upon TP administration, in vivo. Using a cocktail probe of CYP450 isoform-specific substrates and their metabolites, we then carried out in vitro enzymatic studies in liver microsomal incubation systems via ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Finally, we verified our results at the messenger ribonucleic acid (mRNA) and protein level through quantitative real-time polymerase chain reaction (RT-qPCR), western blotting, and immunohistochemical detection.ResultsThe in vivo toxicity study confirmed that Sprague-Dawley (SD) rats exhibited dose-dependent hepatotoxicity after intragastric administration of TP [200, 400, and 600 μg/(kg.day)] for 28 days. In case of the CYP450 isoforms 3A, 2C9, 2C19, and 2E1, the in vitro metabolic study demonstrated a decrease in the substrate metabolic rate, metabolite production rate, and Vmax, with an increase in the Km value, compared with that observed in the control group. Additionally, a TP dose-dependent decrease in the mRNA levels was observed in the four major isoforms of CYP3A subfamily (3A1/3A23, 3A2, 3A9, and 3A62) and CYP2C9. A similar effect was also observed with respect to the protein levels of CYP2C19 and CYP2E1.ConclusionsThis study suggests that TP can cause hepatotoxicity by reducing the substrate affinity, activity, and expression at the transcriptional and protein levels of the CYP450 isoforms 3A, 2C9, 2C19, and 2E1. TP also has the potential to cause pharmacokinetic drug interactions when co-administered with drugs metabolized by these four isoforms. However, further clinical studies are needed to evaluate the significance of this interaction.
Highlights
Triptolide (TP), an active constituent of Tripterygium wilfordii, possesses numerous pharmacological activities
Chromatography-mass behavior and specificity study Since our initial efforts were focused on monitoring the changes in cytochrome P450 enzymes (CYP450s) content upon oral administration of TP, we explored whether the six main CYP450 isoformspecific substrates and their metabolites were suitable for use in cocktail incubations
Verification in messenger ribonucleic acid (mRNA) and protein level Effect on mRNA expression of CYP3A and CYP2C9 After oral administration of either the vehicle or TP (200, 400 or 600 μg/(kg.day)) for 28 days, the mRNA levels of many CYP3A subfamily isoforms and CYP2C9 in TPtreated groups showed a significant decrease (P < 0.05 or P < 0.01) in comparison with that observed in the control group (Fig. 8a)
Summary
Triptolide (TP), an active constituent of Tripterygium wilfordii, possesses numerous pharmacological activities. Its effects on cytochrome P450 enzymes (CYP450s) in rats remain unexplored. Triptolide (TP) (Fig. 1a) is a major bioactive diterpenoid isolated from the traditional Chinese medicinal herb Tripterygium wilfordii Hook F (TWHF). It shows promising anti-inflammatory, immunomodulatory, antiproliferative, proapoptotic, and neuroprotective activities [1,2,3,4,5,6]. Its clinical application is limited owing to acute and chronic side effects induced in multiple organs. The mechanism underlying to TP-induced liver injury is caused by many reasons. In L-02 cells, TP decreased mitochondrial membrane potential and Bcl-2, promoted the release of
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