659 JPP 2006, 58: 659–665 ß 2006 The Authors Received October 16, 2005 Accepted January 10, 2006 DOI 10.1211/jpp.58.5.0011 ISSN 0022-3573 Graduate Institute of Traditional Chinese Medicine, Chang Gung University, Taoyuan, Taiwan T.-Y. Lee, H.-H. Chang Center for Traditional Chinese Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan H.-H. Chang National Research Institute of Chinese Medicine, Taipei, Taiwan G.-J. Wang Institute of Traditional Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan J.-H. Chiu Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital and National Yang-Ming University School of Medicine, Taipei, Taiwan Y.-Y. Yang, H.-C. Lin Correspondence: H.-C. Lin, Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei 11217, Taiwan. E-mail: hclin@vghtpe.gov.tw Funding: This work was supported by grant No. NSC91- 2314-B-075-129 from the National Science Council and grant No.VGH92-228 from the Taipei Veterans General Hospital, Taipei, Taiwan. Water-soluble extract of Salvia miltiorrhiza ameliorates carbon tetrachloride-mediated hepatic apoptosis in rats Tzung-Yan Lee, Hen-Hong Chang, Guei-Jane Wang, Jen-Hwey Chiu, Ying-Ying Yang and Han-Chieh Lin Abstract Apoptosis is one of the events that are involved in liver fibrogenesis. Thus, factors that affect apoptosis may be used to modulate liver fibrosis. We have recently reported that Salvia miltiorrhiza plays a protective role in carbon tetrachloride (CCl 4 )-induced hepatic fibrosis. In this study, we aimed to evaluate whether S. miltiorrhiza modulated CCl 4 -induced hepatic apoptosis in rats. Male Wistar rats were given orally either vehicle or water-extract of S. miltiorrhiza (50 mg kg 1 twice daily) for nine weeks beginning from the start of CCl 4 administration. A group of normal rats was included for comparison. Hepatocyte DNA fragmentation and cytosolic caspase-3 and caspase-8 activity were determined in the experimental animals. Hepatic cytosolic Bax, Bcl-2, cytochrome c, and calpain- expressions were measured by Western blot analysis. Hepatic mitochondrial glutathione levels were assessed by colorimetric assay. Compared with normal rats, rats receiving CCl 4 alone showed pro- found DNA fragmentation associated with an increased cytosolic fraction of cytochrome c and calpain- protein expressions and a decreased mitochondrial glutathione level. In contrast, a decreased laddering of DNA fragmentation was noted in rats receiving CCl 4 plus S. miltiorrhiza extract. The mitochondrial glutathione level was significantly increased in rats receiving CCl 4 plus S. miltiorrhiza extract compared with those receiving CCl 4 alone. Additionally, cytosolic caspase-3 activity and cytosolic fractions of Bax, Bcl-2, cytochrome c, and calpain- protein expressions were decreased in rats receiving CCl 4 plus S. miltiorrhiza extract compared with those receiving CCl 4 alone. The cytosolic caspase-8 activity in rats receiving CCl 4 alone was no different from those receiving CCl 4 plus S. miltiorrhiza extract. These results indicated that chronic administration of S. miltiorrhiza ameliorated CCl 4 -mediatd hepatic apoptosis in rats. This effect may be related to the antioxidant properties of S. miltiorrhiza. Introduction Carbon tetrachloride (CCl 4 ) is widely used in animal models to produce chronic liver injury (Gasso et al 1996; Lee et al 2003). CCl 4 is metabolized by cytochrome P450 to reactive trichloromethyl radical, which is quickly conjugated by hepatic glutathione, reacting with membrane lipids to propagate a chain reaction leading to glutathione depletion and lipid peroxidation that induces cell injury (Basu 2003; Beddowes et al 2003). Based on these results, CCl 4 has been assumed to be a typical substance that produces severe oxidative stress. In addition, formation of apoptotic bodies has been observed in animals receiving CCl 4 (Galle 1997). It is well established that hepatocyte apoptosis plays an important mechanism in hepatobiliary diseases (Guicciardi & Gores 2005). In fact, a number of toxic substances including CCl 4 are known to induce apoptosis (Cain & Freathy 2001). Recent evidence has indicated that the generation of lipid peroxidation (Basu 2003; Beddowes et al 2003), mitochondrial dysfunction (Krahenbuhl et al 2000; Hernandez-Munoz et al 2003), disruption of calcium home- ostasis (Hemmings et al 2002), and induction of apoptosis (Cabre et al 1999; Sun et al 2001) are all involved in CCl 4 -induced hepatotoxicity. It is conceivable that both hepatocellular necrosis and apoptosis are involved in CCl 4 -induced hepatic injury. Regardless of the mechanisms, hepatic apoptosis seems to play a pivotal role in CCl 4 - induced acute and chronic liver injury (Shi et al 1998; Cabre et al 1999). Recent studies Downloaded from https://academic.oup.com/jpp/article/58/5/659/6147775 by guest on 21 September 2022