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

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