An essential role of Nrf2 in American ginseng-mediated anti-oxidative actions in cardiomyocytes.
OBJECTIVE
Ginseng has been used as a folk medicine for thousands of years in Asia, and has become a popular herbal medicine world-wide. Recent studies have revealed that ginseng, including American ginseng, exerts antioxidant effects in the cardiovascular system; however, the underlying mechanisms are not fully understood. Thus, we investigated role of Nrf2, a master transcription factor of endogenous anti-oxidative defense systems, in the regulation of American ginseng-mediated anti-oxidative actions in cardiomyocytes.


METHODS
A standardized crude extract of American ginseng was supplied by the National Research Council of Canada, Institute for National Measurement Standards. H9C2 cells, a rat cardiomyocyte cell line, were exposed to angiotensin II (Ang II) or tumor necrosis factor alpha (TNFalpha) to induce oxidative stress that was examined by measuring formation of reactive oxygen and nitrogen species. Oxidative stress-induced cell death was induced by exogenous addition of hydrogen peroxide (H(2)O(2)). Proteins were measured by Western blot and mRNA expression was determined by quantitative real time PCR. Nrf2-driven transcriptional activity was assessed by antioxidant response element (ARE)-luciferase reporter assay. Direct Nrf2 binding to its target gene promoters was determined by chromatin immunoprecipitation assay. Adenoviral over-expression of Nrf2 shRNA was utilized to knock down Nrf2 in H9C2 cells. Immunochemical staining was applied for Nrf2 expression in the heart.


RESULTS
American ginseng induced dramatic increases in Nrf2 protein expression, Nrf2 nuclear translocation, Nrf2 transcriptional activity, direct Nrf2 binding to its target gene promoters, and expression of a group of anti-oxidative genes driven by Nrf2 in H9C2 cells. In addition, American ginseng inhibited Ang II- or TNFalpha-induced free radical formation and H(2)O(2)-induced cell death in H9C2 cells over-expressed with control shRNA but not in the cells over-expressed with Nrf2 shRNA. Finally, oral administration of American ginseng markedly increased Nrf2 activity in murine hearts.


CONCLUSIONS
These results demonstrate that American ginseng suppresses oxidative stress and oxidative stress-induced cell death in cardiomyocytes through activating the Nrf2 pathway, thereby providing cardioprotection against pathological cardiac remodeling.
Li
Jinqing
J
Department of Cell Biology and Anatomy, University of South Carolina, Columbia, SC 29208, USA.
Ichikawa
Tomonaga
T
Jin
Yu
Y
Hofseth
Lorne J
LJ
Nagarkatti
Prakash
P
Nagarkatti
Mitzi
M
Windust
Anthony
A
Cui
Taixing
T
D000284:Administration, Oral; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000008:administration & dosage / Q000494:pharmacology*; D016923:Cell Death; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D005894:Panax; D010936:Plant Extracts / Q000008:administration & dosage / Q000494:pharmacology*; D034622:RNA Interference; D051381:Rats; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation
D000975:Antioxidants; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; C495637:Nfe2l2 protein, rat; D016877:Oxidants; D010936:Plant Extracts; D014409:Tumor Necrosis Factor-alpha; D000804:Angiotensin II; D006861:Hydrogen Peroxide
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