The Cardioprotective Effects And Related Mechanisms Of Action Of American Ginseng In Mouse Myocardial Ischemia-reperfusion And Endotoxemia Respectively

Chapter 1Aqueous extract of American ginseng protects the heart from ischemia and reperfusion injury via upregulation of endothelial nitric oxide synthaseBACKGROUNDEndothelial nitric oxide synthase (eNOS) is constitutively expressed in the heart and produces low levels of nitric oxide (NO) that mediate cellular signaling. NO production from eNOS protects the heart from ischemia and reperfusion (I/R) injury via inhibition of myocardial apoptosis. The anti-apoptotic effects of NO produced from eNOS are mediated by reduction of oxidative stress and inhibition of caspase activation through S-nitrosylation. It is well known that activation of PI3-kinase/Akt signaling upregulates eNOS expression leading to increased eNOS activity in cardiomyocytes.Ginseng has been used as an herbal medicine for more than 2000 years in China, Korea and Japan, and in the last two decades has gained popularity in the United States, Canada and Europe. Two most commonly used species of ginseng include Asian ginseng(Panax ginseng C.A. Meyer) and American ginseng(Panax quinquefolius Linn). Its major active components are ginsenosides and about 40 ginsenosides have been identified. Ginseng has a wide range of pharmacological effects including immune modulation, anti-diabetic and anti-cancer effects. In the cardiovascular system, ginseng has been shown to stimulate nitric oxide release, vasorelaxation, and to improve lipid profiles, and has been used to treat hypertension and heart failure. Recent studies have shown that ginsenosides protects the heart from I/R injury in rats. In addition, it was shown that ginsenosides activate PI3-kinase/Akt/eNOS pathway in an isolated rat heart prepartion.In the present study, we hypothesized that aqueous extract of American ginseng protects the heart from I/R injury via upregulation of PI3-kinase/Akt-mediated eNOS expression. To test this hypothesis, eNOS-/- and wild-type (WT) mice were pretreatment with ginseng aqueous extract from the American ginseng for 7 days before induction of myocardial I/R. Furthermore, cultured neonatal cardiomyocytes from eNOS-/- and WT mice were pretreated with ginseng and subjected to anoxia and reoxygenation (A/R) to simulate I/R.METHODSWT mice were pretreated with ginseng for 7 days by oral gavage after which myocardial Akt activity and eNOS protein expression levels were determined at day 0、1、3、5 and 7 after ginseng treatment by Western blot analysis. In addition, WT and eNOS-/- mice were subjected to I/R injury by the occlusion of the left anterior descending coronary artery for 45 min to induce myocardial ischemia followed by 3 hours of reperfusion through loosing the occluded coronary artery. Evans blue perfusion and TTC staining were conducted to determine myocardial infact size. Caspase-3 activity assay and cell death ELISA were carried out to examine apoptosis in the peri-infarcted myocardium.Neonatal mouse cardiomyocytes from eNOS-/- and WT mice were isolated, purified and cultured. The levels of Akt, phosphorylated Akt, eNOS and a-actinin protein expression in cardiomyocytes treated with ginseng for 24 hours were determined by Western blot analysis. In addition, cardiomtocytes isolated from WT and eNOS-/- mice were treated with ginseng for 24 hours followed by A/R protocol to simulate I/R in vivo to induce cardiomyocyte apoptosis. Caspase-3 activity assay and cell death ELISA were performed to evaluate apoptosis.RESULTSThe myocardial levels of phosphorylated Akt and eNOS protein expression were significantly upregulated at day 5 after ginseng oral administration in WT mice. Furthermore, ginseng-induced myocardial eNOS protein expression was blocked by LY294002, a specific inhibitor for PI3K/Akt pathway. Compared to control group, ginseng pretreatment for 7 days markedly decreased I/R-induced myocardial infarct size and apoptosis, while either deficiency of eNOS or PI3-kinase/Akt pathway blockage by LY294002 completely abrogated the cardioprotection of ginseng during myocardial I/R.Compared to control group, ginseng pretreatment significantly increased Akt phosphorylation and eNOS protein levels in neonatal mouse cardiomyocytes. Similar to in vivo study, this ginseng-induced eNOS upregulation was completely abrogated in cardiomyocytes co-treated with LY294002, suggesting a PI3K/Akt-dependent mechanism. Pretreatment with ginseng dramatically inhibited cardiomyocyte apoptosis induced by A/R, while this anti-apoptotic effect of ginseng was eliminated in eNOS-/- cardiomyocytes or in cardiomyocytes treated with LY294002.CONCLUSIONAqueous extract of American ginseng treatment protects the heart from I/R injury via upregulatoin of PI3-kinase/Akt-dependent eNOS expression and this eNOS-mediated ginseng’s cardioprotection is at least in part by inhibition of apoptosis. American ginseng may serve as a potential therapeutic agent to limit myocardial I/R injury. Chapter 2Aqueous extract of American ginseng inhibits tumor necrosis factor-alpha expression and improves cardiac function in endotoxemia in miceBACKGROUNDSepsis, the systemic inflammatory response syndrome to infection, is the leading cause of death in the critically ill. The major pathological manifestation of this syndrome is myocardial depression, leading to cardiac dysfunction, as a main determinant of death or survival. LPS or endotoxin of Gram negative bacteria is recognized as a major mitogen casuing myocardial depression in septic patients. LPS-induced cardiac dysfunction is mediated by the production of pro-inflammatory cytokines. It is known that tumor necrosis factor-alpha (TNF-α) is a key cyotokine responsible for sepsis-induced cardiac dysfunction. Cardiomyocyte is the major source of myocardial TNF-αduring sepsis. Recent stuies reported that sepsis induces ERK1/2 activation leading to TNF-αproduction, suggesting that ERK1/2 is an upstream signal of TNF-α. On the other hand, some studies demonstrated that ginseng inhibits LPS-induced TNF-αsynthasis and ERK1/2 activation in many cell types. Given ERK1/2 and TNF-αare important mediators of cardiac dysfunction during sepsis, and the inhibitory effects of ginseng on these proteins, we hypothesized that aqueous extract of American ginseng inhibits ERK1/2 activation and TNF-αproduction in cardiomyocytes during LPS-induced endotoxemia in mice. METHODSIn in vivo study, WT mice were treated with ginseng for 5 days by oral gavage, after which LPS injection was conducted to induce endotoxemia. In vivo cardiac function was measured after LPS injection in WT mice with or without ginseng pretreatment. In addition, The myocardium and plasma were harvested to evaluate the levels of TNF-αmRNA (real-time PCR) and protein (ELISA) expression in the myocardium, TNF-αplasma concentration (ELISA), as well as the level of ERK1/2 phosphorylation in the myocardium (Western Blot). Neonatal mouse cardiomyocytes from wild-type (WT) mice born within 24 hours were isolated, purified and cultured. Ginseng pretreated cardiomyocytes for 48 hours, after which LPS was added to induce TNF-a production and ERK1/2 activation. Supernatant and cardiomyocytes were collected to determine the levels of TNF-αmRNA expression (real-time PCR) and release (ELISA), and phosphorylated ERK1/2 protein concentration (Western Blot), respectively.RESULTSThe results of cardiac function measurement showed that indices of cardiac function measured were improved in the ginseng treatment group. Compared to control group, ginseng oral administration to mice for 5 days significantly inhibited LPS-induced myocardial TNF-αmRNA and protein expression, as well as ERK1/2 activation. In addition, TNF-αplasma concentration was decreased as well. Similar to in vivo study, ginseng pretreatment significantly inhibited LPS-induced TNF-αrelease in cultured cardiomyocytes in a dose-dependent manner. Furthermore, TNF-αmRNA and phosphorylated ERK1/2 protein expression levels were markedly decreased in ginseng-treated cardiomyocytes.CONCLUSIONAqueous extract of American ginseng protects from cardiac dysfunction during LPS-induced endotoxemia in mice, possibly via inhibition of ERK1/2-TNF-αpathway in cardiomyocytes.