Hepatoprotective mechanism of schisandrin B: role of mitochondrial glutathione antioxidant status and heat shock proteins

Abstract

In this study, the time course of schisandrin B- (Sch B-) induced changes in hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (HSP) 25/70 induction was examined to study their differential roles in the hepatoprotection afforded by Sch B pretreatment against carbon tetrachloride (CCl₄) toxicity in mice. Dimethyl diphenyl bicarboxylate (DDB), a nonhepatoprotective analog of Sch B, was also included for comparison. The results indicate that Sch B treatment (2 mmol/kg) produced maximum enhancement in hepatic mtGAS and increases in both hepatic HSP 25 and HSP 70 levels at 24 h after dosing. While the extent of hepatoprotection afforded by Sch B pretreatment against CCl₄ was found to correlate inversely with the elapsed time postdosing, the protective effect was associated with the ability to sustain mtGAS and/or HSP 70 levels in a CCl₄-intoxicated condition. On the other hand, DDB (2 mmol/kg) treatment, which did not sustain mtGAS and HSP 70 level, could not protect against CCl₄ toxicity. Abolition of the Sch B-mediated enhancement of mtGAS by buthionine sulfoximine/phorone did not completely abrogate the hepatoprotective action of Sch B. The results indicate that Sch B pretreatment independently enhances mtGAS and induces HSP 25/70 production, particularly under conditions of oxidative stress, thereby protecting against CCl₄ hepatotoxicity.

Introduction

Schisandrin B (Sch B, Fig. 1) is the most abundant, active dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis (FS), a traditional Chinese herb commonly used as astringent and clinically used for the treatment of viral and chemical hepatitis [1]. Previous studies in our laboratory have shown that Sch B pretreatment protects against carbon tetrachloride- (CCl₄-) induced hepatotoxicity [2], myocardical ischemia/reperfusion injury [3], and brain oxidative damage [4] in rodents. The tissue nonspecific protection is likely afforded by the enhancement of cellular glutathione antioxidant status 2, 3, 4, particularly in the mitochondrion 5, 6. A structure-activity relationship study indicated that dimethyl diphenyl bicarboxylate (DDB, Fig. 1), a synthetic intermediate of schisandrin C (another dibenzocyclooctadiene derivative) lacking the cyclooctadiene ring, did not enhance mitochondrial glutathione status nor did it protect against CCl₄ hepatotoxicity in mice [7]. A recent study in our laboratory has demonstrated that Sch B pretreatment produced a dose-dependent increase in hepatic heat shock protein (HSP) 70 level in mice and afforded protection against tumor necrosis factor-α-induced hepatic apoptosis in D-galactosamine-sensitized mice [8].

Reduced glutathione (GSH) plays an important role in numerous cellular functions, including DNA synthesis, regulation of cytosolic Ca²⁺ homeostasis, and detoxification of reactive oxygen species (ROS) 9, 10, 11. It works together with antioxidant enzymes, such as Se-glutathione peroxidase (GPX), glutathione S-transferases (GST), and glutathione reductase (GRD), in combating ROS and maintaining cellular glutathione status. In this regard, the maintenance of mitochondrial glutathione status was found to be critical for cell survival 12, 13.

HSPs are a family of constitutive and inducible expressed gene products that collectively function to maintain cellular protein conformation during stressful conditions [14]. The synthesis of HSPs, which allows cells to adapt to gradual changes in their environment and to survive in otherwise lethal conditions, can be induced by a variety of mild stresses including exposure to oxidants, heat, hypoxia, and low pH, all of which can affect protein conformation [14]. HSP 70, known to be the major molecular chaperone of eukaryotes, is the cytoprotective stress protein present in all cellular compartments and organelles 15, 16. Expression of small HSPs such as HSP 25/27 has been shown to enhance the survival of mammalian cells exposed to heat or oxidative injury 17, 18. Moreover, the expression of different small HSPs was found to correlate with a decrease in the level of cellular ROS. In these studies, a positive correlation between the level of expressed small HSPs and that of GSH was also observed [19]. However, the interrelationship between the Sch B-induced enhancement of hepatic mitochondrial glutathione antioxidant status (mtGAS) and induction of hepatic HSP remains unclear. Also, whether a Sch B-induced change in hepatic mtGAS can affect mitochondrial function has not been investigated.

In the present study, the time course of Sch B-induced changes in hepatic mtGAS and HSP 25/70 induction was investigated with relation to the changes in susceptibility to CCl₄ hepatotoxicity of mice. The effect of Sch B treatment on hepatic ATP generation capacity in control and CCl₄-intoxicated mice was examined. The effect of DDB was also studied for comparison. Results from the preliminary time course study have been reported in abstract form [20].