An essential role of Nrf2 in American ginseng-mediated anti-oxidative actions in cardiomyocytes

Abstract

Aim of the study: 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.

Materials and 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.

Conclusion: 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.

Fig. 1

Effect of American ginseng on Nrf2 transcriptional activity in H9C2 cells. (A) Cells transfected with Nrf2 transcription reporter gene of ARE-luc and internal control gene of pRL-TK-luc were cultured in serum-free DMEM for 24 hours to induce a quiescent status, and then treated with or without American ginseng extract (Am. g. ) as indicated. Nrf2 transcriptional activity was assessed by dual luciferase assay as described in “Methods”. Left panel was the time-course analysis (Am. g. 50 µg/ml), *p < 0.05 vs non-treated group (Am. g., 0), n=4. Right panel was the dose-response analysis (American ginseng treatment for 12 hours). *p < 0.05 vs non-treated group (Am.g. 0), n=4. (B) Quiescent cells were treated with or without American ginseng extract (Am. g., 50 µg/ml) as indicated. Nrf2 protein expression as well as nuclear translocation was examined by immunochemical staining of Nrf2 and confocal microscopic analysis. Red is Nrf2. Green is F-actin. Blue is nuclei. Results were representatives of three separated experiments. (C) Quiescent cells were treated with or without American ginseng extract (Am. g., 50 µg/ml) as indicated. Expression of Nrf2 downstream genes including HO-1, Txn-1, Txnrd-1, NQO-1, SOD-2, and γGCLc was quantified by Q-PCR. *p < 0.05 vs Vehicle (non-treated), n=4.

Fig. 1

Effect of American ginseng on Nrf2 transcriptional activity in H9C2 cells. (A) Cells transfected with Nrf2 transcription reporter gene of ARE-luc and internal control gene of pRL-TK-luc were cultured in serum-free DMEM for 24 hours to induce a quiescent status, and then treated with or without American ginseng extract (Am. g. ) as indicated. Nrf2 transcriptional activity was assessed by dual luciferase assay as described in “Methods”. Left panel was the time-course analysis (Am. g. 50 µg/ml), *p < 0.05 vs non-treated group (Am. g., 0), n=4. Right panel was the dose-response analysis (American ginseng treatment for 12 hours). *p < 0.05 vs non-treated group (Am.g. 0), n=4. (B) Quiescent cells were treated with or without American ginseng extract (Am. g., 50 µg/ml) as indicated. Nrf2 protein expression as well as nuclear translocation was examined by immunochemical staining of Nrf2 and confocal microscopic analysis. Red is Nrf2. Green is F-actin. Blue is nuclei. Results were representatives of three separated experiments. (C) Quiescent cells were treated with or without American ginseng extract (Am. g., 50 µg/ml) as indicated. Expression of Nrf2 downstream genes including HO-1, Txn-1, Txnrd-1, NQO-1, SOD-2, and γGCLc was quantified by Q-PCR. *p < 0.05 vs Vehicle (non-treated), n=4.

Fig. 2

Effect of American ginseng on Nrf2 binding to ARE on the promoters of its target genes in H9C2 cells. Cells were stimulated with or without American ginseng extract (Am. g., 50 µg/ml) for 12 hours and subjected to Chip assay as described in “Methods”. *p < 0.05 vs vehicle, n=4. TSS, transcriptional start site.

Fig. 3

Effect of Nrf2 knockdown on American ginseng-mediated suppression of ROS and RNS formation in H9C2 cells. Cells infected with adenovirus of control scramble shRNA or Nrf2 shRNA for 48 hours, and then treated with Ang II (0.5 µM) and American ginseng extract (Am. g., 200 µg/ml) for 1 hour as indicated. Free radical formation was determined as described in “Methods”. *p < 0.05 vs Ad-scramble (−); #p < 0.05 vs Ang II (+) of Ad-scramble;§p < 0.05 vs Ang (+) of Ad-Nrf2 shRNA; †p < 0.05 vs Ad-Nrf2 shRNA (−); n=6.

Fig. 4

Effect of Nrf2 knockdown on American ginseng-mediated inhibition of H₂O₂-induced cell death in H9C2 cells. Cells infected with adenovirus of control scramble shRNA or Nrf2 shRNA for 48 hours, and then treated with H₂O₂ (50 µM) for 12 hours. American ginseng extract (Am. g., 50 µg/ml) was added 12 hours before H₂O₂ treatment. Cell viability was assessed by the Cytotoxicity Detection Kit ^PLUS (Roche) as described in “Methods”. *p < 0.05 vs Ad-scramble (−); #p < 0.05 vs H₂O₂ (+) of Ad-scramble; n=6.

Fig. 5

American ginseng-induced activation of Nrf2 in the murine heart. (A) Representatives of immunochemical staining of Nrf2 from C57BL/6J mice (n=3) 6 hours after single gavages with American ginseng extract (Am. g., 50 mg/kg). Vehicle (Veh) is saline. Red is Nrf2. Green is F-actin. Blue is nuclei. (B) Semi-quantification of Nrf2 protein expression and Q-PCR analysis of Nrf2 downstream gene expression. Semi-quantification was performed using Image-Pro Plus software as described in “Methods”. Expression of HO-1, NQO-1 and Txn-1 mRNAs was used as biomarkers to indicate Nrf2 activation in the heart. Results are fold changes relative to the control treated with vehicle. *p < 0.05 vs basal (0), n=4.