High‐content analysis boosts identification of the initial cause of triptolide‐induced hepatotoxicity

Abstract Triptolide (TP) has been widely used in China for more than 40 years as an immunosuppressive agent. Recently, serious concerns have been raised over TP‐induced liver injury, though the real hepatotoxic mechanism is still unclear, particularly in terms of the initial cause. To our knowledge, this study is the first to screen systematically the mechanism of TP‐induced toxicity through a global cytotoxicity profile high‐content analysis using three independent cytotoxic assay panels with multiple endpoints of cytotoxicity, including cell loss, mitochondrial membrane potential, nuclear membrane permeability, manganese superoxide dismutase, phosphorylated gamma‐H2AX, light chain 3B, lysosome, reactive oxygen species and glutathione. We assessed nine parameters and four stress response pathway models by labeling nuclear factor erythroid 2‐related factor 2, activating transcription factor 6, hypoxia inducible factor 1α and nuclear factor κB and found that all testing parameters except glutathione and manganese superoxide dismutase showed concentration‐ and time‐dependent changes, as well as increased cell loss after TP treatment. Considering that RNA polymerase II is the molecular target of TP, we quantified transcription from inducible genes, bromodeoxyuridine incorporation, and expression from transiently transfected green fluorescence protein plasmids in HepG2 cells. The results show that inhibition of global transcription by TP took place at earlier times and at lower concentrations than those observed for cell death. Therefore, global transcriptional suppression and the cell dysfunction it drives play a central role in TP‐induced hepatotoxicity. This provides valuable information for the safe use of TP in the clinic.