Impairment of triptolide on liver mitochondria in isolated liver mitochondria and HL7702 cell line

To observe the impairing effects of triptolide on liver mitochondria in isolated rat-liver mitochondria and human normal liver HL7702 cell line. Rat-liver mitochondria were isolated from adult female Sprague-Dawley (SD) rats. Liver mitochondria were incubated with 0, 1.25, 2.5, 5 and 10 mu mol/L triptolide for detecting mitochondrial swelling and with 0, 2.5, 5 and 10 mu mol/L triptolide for mitochondrial permeability transition pore (MPTP) activity. Mitochondrial swelling was estimated by measuring the apparent absorbance change during 600 s in the mitochondrial suspensions at 520 nm with a mitochondrial swelling examining kit. The effect of triptolide on MPTP was determined with a fluorescence detection kit by detecting the fluorescence intensity at an excitation wavelength of 488 nm emitted at 527 nm. Human normal liver HL7702 cells were treated without or with 0.02, 0.1 and 0.5 mu mol/L triptolide for 24 h for analyzing mitochondrial transmembrane potential (Delta psi m) and reactive oxygen species (ROS). Delta psi m was measured using the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). ROS was measured using fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH-DA). The cells were harvested and dyed with JC-1 and DCFH-DA, and analyzed by flow cytometry, respectively. Incubation of isolated mitochondria with triptolide results in swollen mitochondria in a concentration-dependent manner. Moreover, triptolide significantly activated mitochondrial permeability transition at 5 and 10 mu mol/L (P < 0.05 and P < 0.01). When HL7702 cells were exposed to a various concentration triptolide for 24 h, mitochondrial membrane depolarization and increase of ROS were caused by triptolide in a concentration-dependent manner. Triptolide significantly induced the mitochondrial membrane depolarization at 0.1 and 0.5 mu mol/L (P < 0.05 and P < 0.01) and the increase of ROS at 0.1 and 0.5 mu mol/L (P < 0.05 and P < 0.01). Triptolide could induce mitochondrial impairment, which may be one of the mechanisms by which hepatotoxicity occurs.