1. Yan Wu, Xiangru Lu, Fuli Xiang, and Qingping Feng
Ginseng Protects the Heart from Ischemia and Reperfusion Injury via PI3K/Akt/eNOS Pathway
Yan Wu, Xiangru Lu, Fuli Xiang, and Qingping Feng

2. Background

3. Ischemic Heart Disease (IHD) Main Cause: Atherosclerosis
Other Causes:
Prolonged coronary spasm
Allergic conditions
Severe anemia, e.g., sickle cell crisis
Acquired hypercoagulable states

4. Myocardial infarction (MI)

5. Ischemia-reperfusion (I/R) injury
Thrombolytic and Fibrinolytic therapy
Coronary angioplasty
coronary artery bypass surgery
Ischemia-reperfusion (I/R) injury
Ischemic period < 5 min
5 – 20 min
> 20 min
Cardioprotection
Ischemic preconditioning
Short periods of heat stress (41-42 oC, 15 min)
Ischemic postconditioning

6. Ginseng

7. ? Hypothesis PI3K/Akt/eNOS pathway Ginseng Ischemia-reperfusion injury
We hypothesize that ginseng protects the heart from ischemia-reperfusion injury via PI3K/Akt/eNOS signaling pathway.

8. Methods

9. In vivo experiments Experiment 1: Experiment 2:
Adult male C57BL/6 and eNOS-/- mice were treated with Ginseng root aqueous extract (50 mg/kg/day) or water by oral gavage for 7 days.
Akt phosphorylation and eNOS protein expression were assessed by western blotting.
Experiment 2: .
After pretreatment with ginseng for 7 days, mice were subjected to 45 min of myocardial ischemia followed by 3 hours of reperfusion.
LY294002, a PI3K/Akt pathway inhibitor, was administered by i.p. (7.5 mg/kg) 15 min before coronary reperfusion.
Ischemic area and infarct size were assessed by Evans blue and triphenyltetrazolium chloride (TTC) staining, respectively.

10. In vitro experiments Experiment 1: Experiment 2:
Neonatal cardiomyocytes were treated with Ginseng root aqueous extract at 0, 0.5, 2 and 8 μg/ml for 24 hrs.
The levels of Akt phosphorylation and eNOS protein expression were measured by western blotting analysis.
Experiment 2:
After pretrement with ginseng for 24 hrs, cardiomyocytes were subjected to anoxia for 2 hrs followed by reoxygenation for a further 2 hrs (A/R), normoxia for 4 hrs as a control (N/N).
Caspase-3 activity assay was performed in cardiomyocytes after N/N or A/R to examine the extent of apoptotic cell death.

11. Results

12. Effects of ginseng on myocardial Akt phosphorylation in WT mice
Ginseng treatment (50mg/kg) 1 3 5 7
days
Phospho-Akt
60KDa
Total-Akt
60KDa
*P<0.05 vs. control (day 0). N=4.

13. Effects of ginseng on myocardial eNOS protein expression in WT mice
Ginseng treatment (50mg/kg) 1 3 5 7
days
Total-eNOS
140kDa
α-actinin
100kDa
*P<0.05 vs. control (day 0). N=4.

14. Myocardial infarct size measurement
Ischemic Area
Infarcted Area

15. Effects of ginseng on infarct size following I/R in WT and eNOS-/- mice
I/R+GS
I/R+GS
+LY294002
(**P<0.01 vs. I/R in WT mice; †P<0.01, ‡P<0.01 vs. I/R+GS in WT mice). N=6.

16. Effects of ginseng on Akt phosphorylation in neonatal cardiomyocytes
Ginseng concentration
0.5 2 8
(g/ml)
Phospho-Akt
60 kDa
Total-Akt
60 kDa
*P<0.05 vs. control (0 g/ml), N=3.

17. Effects of ginseng on eNOS protein expression in neonatal cardiomyocytes
Ginseng concentration
0.5 2 8
(μg/ml)
Total-eNOS
140 kDa
α-actinin
100 kDa
. * P<0.05 vs. control (0 g/ml), N=3.

18. Role of PI3-K/Akt signaling in ginseng-induced eNOS protein expression in neonatal cardiomyocytesWT
GS+LY GS LY
Total-eNOS
140 kDa
α-actinin
100 kDa
*P<0.05 vs. WT control; #P<0.05 vs. GS treatment group. N=3.

19. Effects of ginseng on caspase-3 activity in cardiomyocytes during A/R
*P<0.05 vs. WT in N/N; #P<0.05 vs. WT with A/R. N=6.

20. Summary
Pretreatment with ginseng significantly increased Akt phosphorylation and eNOS protein expression under basal conditions in both intact heart and neonatal cardiomyocytes.
Under basal conditions, ginseng increased eNOS protein expression via PI3K/Akt pathway in neonatal cardiomyocytes.
Ginseng pretreatment significantly reduced myocardial infarct size during I/R.
Ginseng significantly decreased caspase-3 activity after A/R injury.

21. Conclusion
Ginseng pretreatment protects the heart against ischemia-reperfusion injury via PI3K/Akt/eNOS signaling pathway.
The protection of ginseng in ischemia-reperfusion injury is mediated, at least in part, by inhibiting cardiomyocyte apoptosis.
Beneficial effects of ginseng on ischemia-reperfusion injury suggest that ginseng products may be used as a cardioprotective agent.

22. Thank you!