Salvianic acid A protects L-02 cells against γ-irradiation-induced apoptosis via the scavenging of reactive oxygen species

Abstract

Salvianic acid A (SAA) is the main hydrophilic active ingredient of Salvia miltiorrhiza bunge, which has long been used to treat liver and heart disease in China. In the present study, we investigated the radioprotective effects of SAA against γ-radiation-induced apoptosis in cultured human embryo liver L-02 cells. The results demonstrated that SAA markedly inhibited γ-radiation induced apoptosis, decreased DNA damage, and increased the intracellular antioxidative ability of the L-02 cells. SAA exhibited radioprotection by decreasing the generation of reactive oxygen species, inhibiting the release of mitochondrial cytochrome C, blocking the activation of caspase-3, and down regulating the expression of Bax and P53 and up regulating the expression of Bcl-2. This indicated that SAA pretreatment inhibited the caspase-dependent mitochondria apoptosis pathway. The radioprotection of the SAA pretreatment was also evidenced by an increased survival ratio, maintaining the antioxidant enzyme levels in the liver, inhibition of oxidative stress, and relative low liver and renal toxicity compared with estriol exposure. In conclusion, SAA may be an effective radioprotector against γ-radiation induced apoptosis in L-02 cells and damage in mice, the antioxidant potency of SAA might be correlated with the beneficial radioprotectant effects observed.

Introduction

Ionizing radiation (IR) has been used in medicine (radiotherapy and radiodiagnosis), industries, and research laboratories for several decades. However, the increasing use of radiation has resulted in accidental and occupational exposure threatening human health. It is known that exposure to ionizing radiation gives rise to genomic instability leading to mutagenesis, carcinogenesis, and cell death (Snyder and Morgan, 2003). Exposure to γ-radiation at sub-lethal doses induces a variety of cellular and sub-cellular damage in many living organisms (Garrison and Uyeki, 1988). The γ-rays are absorbed directly by DNA, leading to single or double-strand breaks, base damage, and DNA–DNA or DNA-protein cross-linkages (Reily, 1994, Weiss, 1997). However, the detrimental effects of IR on biological tissues, for the most part, are mediated via increased production of reactive oxygen species (ROS), and it induces the production of free radicals such as hydrogen radicals, hydroxyl radicals, singlet oxygen and peroxyl radicals in a cascade pathway. Approximately 65% of the DNA damage is caused by the indirect effect of these free radicals (Ward, 1988). Furthermore, these free radicals can trigger the oxidation of lipids, amino acids, and saccharides leading to the formation of various secondary free radicals, which can chemically modify DNA, proteins, and lipids, causing cellular damage (Kalpana et al., 2009).

A radioprotector could be used as an adjunct to radiotherapy safeguarding the normal tissue during the intended radiation exposure. Various radioprotective strategies have been explored, including thiols, growth factors and cytokines (Vijayalaxmi et al., 2004, Weiss and Landauer, 2000). However, these compounds have many shortcomings, including relatively high toxicity and unfavorable routes of administration, which delay their application and efficacy (Maisin, 1998). For these reasons, the search for new less toxic radioprotectors is crucial to develop improved strategies for protecting normal cells from radiation-induced damage.

Salvia miltiorrhiza bunge is a traditional Chinese medicine with many pharmacological effects, including protection of cardiac muscle, dilatation of blood vessels, inhibition of arteriosclerosis and thrombus, improvement of the microcirculation, regulation of restoration and regeneration of tissue, antisepsis and anti-inflammation (Xu and Fu, 2006). The formulation derived from this herb-like compound, termed the Danshen Dripping Pill, the Danshen Pian and the Danshen Injection have been developed and used in clinics in China, Korea and Russia (Zhao et al., 2006).

Salvianic acid A (SAA) is the major water-soluble component of Salvia miltiorrhiza bunge with the molecular structure d(+)-(3,4-dihydroxyphenyl) lactic acid (Wang et al., 2005). Previous studies have shown that SAA inhibits apoptosis of myocardiac cells induced by angiotensin (Ang II), and inhibits hypertrophy of myocardiac myocytes (Guo et al., 2006). It can also protect the liver from injury by decreasing the amount of endotoxin and improving the hepatic microcirculation (Zhou et al., 2006). Moreover, SAA was found to protect neuroblastoma cells (SH-SY5Y) from death induced by 1-methyl-4-phenylpyridiniumion, and it not only protects these cells from apoptosis, but also from increased levels of ROS. SAA inhibited the activation of caspase-3, stimulated the accumulation of Bcl-2 and reduced Bax (Wang and Xu, 2005). Additionally, SAA was reported to scavenge lipid free radicals and inhibit lipid peroxidation effectively (Wang et al., 2005).

However, only a few studies have been undertaken on the in vitro radioprotective effects of SAA. Therefore, the aim of our present study is to investigate the radioprotective effects of SAA on γ-radiation induced apoptosis in cultured human embryo liver L-02 cells.