Mechanism for hepato-protective action of Liangxue Huayu Recipe (LHR): Blockade of mitochondrial cytochrome c release and caspase activation

Liangxue Huayu Recipe (LHR) as a classical prescription is clinically employed to treat liver diseases in traditional Chinese medicine. In this study, we attempt to show that LHR attenuates hepatocyte apoptosis and hepatic injury induced by lipopolysaccharide (LPS) and d-galactosamine (GalN) in rats. The present study was also designed to examine whether LHR had the protective effects on d-GalN and tumor necrosis factor-α (TNF-α)-treated human L02 hepatocytes and its possible association with the mitochondrial pathway. LHR is composed of three traditional Chinese medicines: Herba Rehmannia, Rhubarb and Radix Paeoniae Rubra. LHR at 541, 1082 and 2164 mg/kg was orally administered to model and normal rats for 7 days. The effects of LHR on serum levels of liver enzymes, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were measured. Hepatocyte apoptosis in vivo was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method. Apoptosis in vitro and related morphological changes of human L02 hepatocytes were determined by high content screening (HCS) assay. The expression levels of Bcl-2, Bax and cytochrome c were detected by Western-blot analysis in L02 cells. In addition, the activities of caspase-3 and caspase-9 were tested by enzyme-linked immunosorbent detector. It revealed that LHR pretreatment effectively ameliorated the GalN/LPS-induced elevation of serum ALT and AST levels, and attenuated hepatocyte apoptosis in the rat model characterized by the addition of GalN/LPS. In subsequent experiments in vitro, LHR also attenuated GalN/TNF-α-induced apoptosis in human L02 hepatocytes. Furthermore, LHR improved the mitochondrial function, inhibited the upregulation of Bax/Bcl-2 protein ratio, decreased the release of cytochrome c from the mitochondria into the cytosol, as well as inhibited caspase-3 and caspase-9 activation in this cell model. These results indicate that LHR is effective in attenuating hepatocyte apoptosis both in vivo and in vitro, and this effect is partly mediated through the activation of the mitochondrial pathway and subsequent regulation of particular pro-apoptotic gene expression.