Mechanism of Rhein-Induced Apoptosis in Rat Primary Hepatocytes: Beneficial Effect of Cyclosporine A

Past observational and toxicity studies have established an association between the deaths of children and consumption of Cassia occidentalis (CO) seeds. We recently reported chemical evidence of this association following the identification of toxic anthraquinones (AQs), viz. aloe-emodin, chrysophanol, emodin, physcion, and rhein, in CO seeds (Panigrahi, G. K. et al. (2015), Chem. Res. Toxicol. DOI: 10.1021/acs.chemrestox.5b00056 ). Of these five AQs, earlier studies have shown rhein to be the most cytotoxic AQ in hepatocytes. Therefore, the present study was designed to investigate the effect of rhein on rat primary hepatocytes. Results indicated that rhein (50 mu M) causes apoptosis in rat primary hepatocytes by generating reactive oxygen species (ROS), increasing intracellular Ca2+, decreasing the mitochondrial membrane potential, and depleting intracellular glutathione content. At the molecular level, rhein-induced DNA damage results in overexpression of gamma-H(2)AX protein (2.5-fold), thereby causing enhancement of p53 (4.5-fold) and p21 (3.6-fold), leading to intrinsic pathway-mediated apoptosis involving Bax, bcl2, cytochrome c, caspases 3 and 9, and poly-ADP ribose polymerase. Further, it was observed that rhein-induced ROS generation is also involved in the modulation of signaling molecules like MAPK kinases, including ERK1/2, p38, and JNK, and mitochondrial energetics proteins, including complexes II-V, p-AMPK, and Sirt-1. It was shown that 100 nM cyclosporine A was the most effective among the different protective agents at preventing apoptosis in hepatocytes by interfering in various metabolic pathways which were found to be altered by rhein.