Dang-Gui Buxue Tang Protects against Oxidant Injury by Enhancing Cellular Glutathione in H9c2 Cells: Role of Glutathione Synthesis and Regeneration

Abstract

In order to investigate the biochemical mechanism of Dang-Gui Buxue Tang (DBT) involved in its cardioprotective action, the effects of DBT and related preparations on the cellular level of reduced glutathione (GSH) and on susceptibility to menadione-induced toxicity were examined in H9c2 cardiomyocytes. Treatment with herbal extract prepared from the fresh root of Astragalus membranaceus (RAM) or Angelica sinensis (RAS) alone and their combinations (D_{1 : 1} - D_{10 : 1}) in varying ratios of RAM to RAS (1 : 1 to 10 : 1, respectively) increased cellular GSH in a concentration-dependent manner, with the effect produced by the D_{5 : 1} extract, an authentic formula of DBT, being the most potent. The enhancement of cellular GSH was found to correlate positively with the degree of cytoprotection against menadione toxicity. Both GSH-enhancing and cytoprotective effects of DBT were largely abolished by GSH depletion as a result of buthionine sulfoximine (BSO)/phorone treatment. The DBT-induced increase in the cellular GSH level and the associated cytoprotection were also suppressed by the treatment with BSO, an inhibitor of GSH synthesis, or 1,3-bis(2-chloroethyl)-1-nitrosourea, an inhibitor of GSH regeneration. The results indicate that DBT treatment protects against oxidant injury in H9c2 cells, and that the cytoprotective action is causally related to the increase in cellular GSH level, which is likely mediated by the enhancement of GSH synthesis and regeneration.

Abbreviations

BCNU:1,3-bis(2-chloroethyl)-1-nitrosourea

BSO:buthionine sulfoximine

DBT:Dang-Gui Buxue Tang

FBS:fetal bovine serum

GCL:γ-glutamate cysteine ligase

GR:glutathione reductase

GSH:reduced glutathione

LDH:lactate dehydrogenase

PBS:phosphate-buffered saline

PHO:phorone

RAM:Radix Astragali membranaceus

RAS:Radix Angelicae sinensis

TCM:traditional Chinese medicine

Key words

Astragalus membranaceus - Angelica sinensis - glutathione - menadione - buthionine sulfoximine - 1,3-bis(2-chloroethyl)-1-nitrosourea - H9c2 cells

References

• 1 Chen S Y, Li F. A clinical guide to Chinese herbs and formulae. London; Churchill Livingstone 1993: 77
• 2 Ding Y X, Quian Y Y, Xu J Z, Long C Y, Gong S Z, Li X N. et al . Changes in peripheral blood status and microcirculation in haemolytic anaemic animals: Effects of treatment with ginseng, deer antler and ‘blood-enriching’ injections.  Chin Tradit Herbal Drug. 1986;  17 44-5
• 3 Nie K. Drug-pair of radix Astragali and radix Angelicae sinensis on cardiovascular system.  Zhongguo Zhong Xi Yi Jie He Za Zhi. 1996;  16 379-81
• 4 Mak D HF, Chiu P Y, Dong T TX, Tsim K WK, Ko K M. Dang-Gui Buxue Tang produces a more potent cardioprotective effect than its component herb extracts and enhances glutathione status in rat heart mitochondria and erythrocytes. Phytother Res, in press
• 5 Wang D, Shen W, Tian Y, Sun Z, Jiang C, Yuan S. Protective effect of active components extracted from Radix Astragali on human erythrocyte membrane damages caused by reactive oxygen species.  Zhongguo Zhong.Yao Za Zhi. 1996;  21 746-8
• 6 Zheng H Z, Dong Z H, She J. Modern study of chinese drugs and clinical applications. Beijing; Xueyuan Press 1997 Vol. 2: 1822
• 7 Tao J Y, Ruan Y P, Mei Q B, Liu S, Tian Q L, Chen Y Z. et al . Studies on the antiasthmatic effect of ligustilide of Dang Gui, Angelica sinensis (Oliv.) Diels.  Acta Pharmacol Sin. 1984;  19 561-5
• 8 Song Z H, Ji Z H, Lo C K, Dong T TX, Zhao K J, Li O TW. et al . Chemical and biological assessment of a traditional Chinese herbal decoction prepared from Radix Astragali and Radix Angelicae Sinensis: Orthogonal array design to optimize the extraction of chemical constituents.  Planta Med. 2004;  70 1222-7
• 9 Dong T TX, Zhao K J, Gao Q T, Zhao N J, Zhu T T, Li J. et al . Chemical and biological assessment of a Chinese herbal decoction containing Radix Astragali and Radix Angelicae Sinensis: Determination of drug ration in having optimized properties.  J Agric Food Chem. 2006;  54 2767-74
• 10 Griffith O W. Biologic and pharmacologic regulation of mammalian glutathione synthesis.  Free Radic Biol Med. 1999;  27 922-35
• 11 Meredith M J, Reed D J. Depletion in vitro of mitochondrial glutathione in rat hepatocytes and enhancement of lipid peroxidation by adriamycin and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU).  Biochem Pharmacol. 1983;  32 1383-8
• 12 Hescheler J, Meyer R, Plant S, Krautwurst D, Rosenthal W, Schultz G. Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart.  Circ Res. 1991;  69 1476-86
• 13 Griffith O W. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine.  Anal Biochem. 1980;  106 207-12
• 14 Seelig G F, Meister A. Glutathione biosynthesis: gamma-glutamylcysteine synthetase from rat kidney.  Methods Enzymol. 1985;  113 379-90
• 15 Chiu P Y, Mak D HF, Poon M KT, Ko K M. In vivo antioxidant action in a lignan-enriched extract of Schisandra fruit and an anthraquinone-containing extract of Polygonum roost in comparison with schisandrin B and emodin.  Planta Med. 2002;  68 951-6
• 16 Li P C, Mak D HF, Poon M KT, Ip S P, Ko K M. Myocardial protective effect of Sheng Mai San (SMS) and a lignan-enriched extract of Fructus Schisandrae, in vivo and ex vivo .  Phytomedicine. 1996;  3 217-21
• 17 Dickinson D A, Forman H J. Cellular glutathione and thiols metabolism.  Biochem Pharmacol. 2002;  64 1019-26
• 18 Wu G, Fang Y Z, Yang S, Lupton J R, Turner N D. Glutathione metabolism and its implications for health.  J Nutr. 2004;  134 489-92
• 19 Monks T J, Hanzlik R P, Cohen G M, Ross D, Graham D G. Quinone chemistry and toxicity.  Toxicol Appl Pharmacol. 1992;  112 2-16
• 20 Morrison H, Jernstrom B, Nordenskjold M, Thor H, Orrenius S. Induction of DNA damage by menadione (2-methyl-1,4-naphthoquinone) in primary cultures of rat hepatocytes.  Biochem Pharmacol. 1984;  33 1763-9
• 21 Nicotera P, McConkey D, Svensson S A, Bellomo G, Orrenius S. Correlation between cytosolic Ca²⁺ concentration and cytotoxicity in hepatocytes exposed to oxidative stress.  Toxicology. 1988;  52 55-63
• 22 Carlberg I, Mannervik B. Glutathione reductase.  Methods Enzymol. 1985;  113 484-90
• 23 Meister A, Anderson M E. Glutathione.  Annu Rev Biochem. 1983;  52 398-401
• 24 Liu M, Sainsbury M, Tabor M W, Shertzer H G. Mechanisms of protection from menadione toxicity by 5,10-dihydroindeno[1,2-b]indole in a sensitive and resistant hepatocyte line.  Biochem Pharmacol. 1993;  46 1491-9