Novel detoxifier of spironolactone against triptolide-induced hepatotoxicity through inhibition of RPB1 degradation

Ethnopharmacological relevance: Triptolide is a major bioactive and toxic ingredient isolated from the traditional Chinese herb Tripterygium wilfordii (T. T. wilfordii) ) Hook F. It exhibits potent antitumor, immunosuppressive, and anti-inflammatory biological activities; however, its clinical application is hindered by severe systemic toxicity. Two preparations of T. wilfordii, , including T. wilfordii glycoside tablets and T. wilfordii tablets, containing triptolide, are commonly used in clinical practice. However, their adverse side effects, particularly hepatotoxicity, limit their safe use. Therefore, it is crucial to discover potent and specific detoxification medicines for triptolide. Aim of the study: This study aimed to investigate the detoxification effects and potential mechanism of action of spironolactone on triptolide-induced hepatotoxicity to provide a potential detoxifying strategy for triptolide, thereby promoting the safe applications of T. wilfordii preparations in clinical settings. Materials and methods: Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and crystal violet staining. Nuclear fragmentation was visualized using 4 ',6-diamidino-2- ' ,6-diamidino-2- phenylindole (DAPI) staining, and protein expression was analyzed by Western blotting. The inhibitory effect of spironolactone on triptolide-induced hepatotoxicity was evaluated by examining the effects of spironolactone on serum alanine aminotransferase and aspartate aminotransferase levels, as well as liver pathology in a mouse model of triptolide-induced acute hepatotoxicity. Furthermore, a survival assay was performed to investigate the effects of spironolactone on the survival rate of mice exposed to a lethal dose of triptolide. The effect of spironolactone on triptolide-induced global transcriptional repression was assessed through 5-ethynyl uridine staining. Results: Triptolide treatment decreased the cell viability, increased the nuclear fragmentation and the cleaved caspase-3 levels in both hepatoma cells and hepatocytes. It also increased the alanine aminotransferase and aspartate aminotransferase levels, induced the hepatocyte swelling and necrosis, and led to seven deaths out of 11 mice. The above effects could be mitigated by pretreatment with spironolactone. Additionally, molecular mechanism exploration unveiled that spironolactone inhibited triptolide-induced DNA-directed RNA polymerase II subunit RPB1 degradation, consequently increased the fluorescence intensity of 5-ethynyl uridine staining for nascent RNA. Conclusions: This study shows that spironolactone exhibits a potent detoxification role against triptolide hepatotoxicity, through inhibition of RPB1 degradation induced by triptolide and, in turn, retardation of global transcriptional inhibition in affected cells. These findings suggest a potential detoxification strategy for triptolide that may contribute to the safe use of T. wilfordii preparations.