Endoplasmic reticulum stress regulates autophagic response that is involved in Saikosaponin a-induced liver cell damage

Abstract

Saikosaponin a (Ssa) is an active ingredient of the Chinese herbal plant Radix Bupleuri (RB) and has severe hepatotoxicity. However, biomolecular mechanisms involved in Ssa-induced hepatotoxicity are not yet entirely clear. Previous studies reported that Ssd (an isomer of Ssa) as a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitor can induce autophagy in apoptotic defective cells, leading to autophagy-dependent cell death. Therefore, we speculate that endoplasmic reticulum (ER) stress and autophagy may also play an important role in Ssa-induced hepatocyte death. This study aimed to explore the connection between ER stress and autophagy and Ssa-induced hepatotoxicity. Experiments in vitro showed that the cell viability of L-02 cells in the Ssa treatment group decreased, the level of autophagy marker LC3-II/LC3-I and Beclin1 increased, the level of p62 decreased, the colocalization of autophagosome and lysosome increased, and the cell viability was significantly increased after the application of autophagy inhibitors 3-MA. In addition, SSa can induce ER stress in L-02 cells in vitro. Further studies demonstrated that SSa activated the PERK/eIF2α/ATF4/CHOP pathway, IRE1-TRAF2 pathway, ATF6 pathway, and AMPK/mTOR pathway associated with ER stress. Application of ER stress inhibitors 4-PBA can significantly down-regulate the level of autophagy and improve cell viability. Results of in vivo experiments showed that treatment with 150 and 300 mg/kg Ssa significantly elevated the liver/body weight ratio and caused histological injury in mice liver. Furthermore, Ssa treatment induced significantly downregulated p62 expression but upregulated LC3-II, CHOP, and GRP78 expression in mice livers. Taken together, our results showed that SSa can activate endoplasmic reticulum stress, promote toxic autophagy, and then induce cell death. We revealed an alternative mechanism involving autophagy and ERs, by which Ssa induced hepatotoxicity.