Abstract
Background and Objective: Ischemic heart disease (IHD) has been the major issue of public health. Panax ginseng (ginseng) has been verified as an effective traditional Chinese medicines and exerted cardioprotective effect. This study aimed to investigate the polysaccharide fraction of ginseng on hypoxia/reoxygenation (H/R) injury in cardiomyocytes and the underlying mechanisms.Methods: Ginseng was extracted by ethanol and fractionated by high-speed counter current chromatography (HSCCC) and column separation. The cardioprotective effect was evaluated in H9c2 cardiomyocytes underwent H/R treatment. The cell viability, apoptosis and mitochondrial respiration were examined.Results: An acid polysaccharides fraction of ginseng (AP1) was identified the most effective fraction in protecting cardiomyocytes from H/R injury. AP1 restored the mitochondrial function by maintaining mitochondrial membrane potential (MMP), blocking the release of cytochrome C, and increasing the ATP generation and oxygen consumption rate (OCR) of cardiomyocytes. Meanwhile, AP1 induced the expression of glucocorticoid receptor (GR) and estrogen receptor (ER) which further activated reperfusion injury salvage kinase (RISK) pathway. Finally, AP1 increased nitric oxide (NO) production and regulated endothelial function by increasing endothelial NO synthase (eNOS) expression and decreasing inducible NOS (iNOS) expression in H/R injury.Conclusion: The results suggested that AP1 exerted a protective effect in myocardial H/R injury mainly through maintaining myocardial mitochondrial function, thereby inhibiting myocardial H/R caused apoptosis and increasing the expressions of GR and ER, which in turn mediated the activation of RISK pathway and eNOS-dependent mechanism to resist the reperfusion injury.
Highlights
According to the reports of World Health Organization [WHO] in 2014, about 7.4 million people died of ischemic heart diseases (IHD), making IHD the No 1 human killer (World Health Organization, 2014)
The precipitate generated from precipitation in 70% ethanol exhibited the strongest activity (Figure 1D) and was further subjected onto ion-exchange column chromatography on Toyopearl DEAE 650 M
L-NAME (NOS inhibitor) completely suppressed the effect of acidic polysaccharides fraction of ginseng (AP1) on nitric oxide (NO) production, which is accompanied with the completely loss of protection to H9c2 cells (Figure 6A), implying that endothelial nitric oxide synthase (eNOS) dependent NO production is dispensable to the cardioprotective effect of AP1. To further confirm this result, we investigated whether AP1-induced eNOS activation and NO production were inhibited by pretreatment with glucocorticoid receptor (GR), estrogen receptor (ER), PI3K/Akt and ERK inhibitors
Summary
According to the reports of World Health Organization [WHO] in 2014, about 7.4 million people died of IHD, making IHD the No 1 human killer (World Health Organization, 2014). Polysaccharides, obtained from the water-soluble fraction of ginseng, have been demonstrated with immuneregulatory, anti-oxidative, and anti-cancer effects, Abbreviations: AP1, acidic polysaccharides fraction of ginseng; Bax, Bcl-2associated X protein; Bcl-2, B-cell lymphoma 2; CK, creatine kinase; DMEM, dulbecco’s modified Eagle’s medium; DZ, diazoxide; eNOS, endothelial nitric oxide synthase; ER, estrogen receptor; ERK1/2, extracellular signal-regulated kinases; FBS, fetal bovine serum; Gal A, D-galacturonic acid monohydrate; ginseng, Panax ginseng; Glc, D-glucose; GR, glucocorticoid receptor; H/R, hypoxia/reoxygenation; HPGPC, high performance gel filtration chromatography; HPLC, high performance liquid chromatography; HSCCC, high speed counter current chromatography; IHD, ischemic heart diseases; iNOS, inducible nitric oxide synthase; JNK, c-Jun N-terminal kinases; KRB, Krebs-Ringer Bicarbonate; LDH, lactate dehydrogenase; MDA, malondialdehyde; MMP, mitochondrial membrane potential; MVS, mitochondrial Viability Stain; NO, nitric oxide; OCR, oxygen consumption rate; p-eNOS, phosphorylated endothelial nitric oxide synthase; p-ERK1/2, phosphorylated extracellular signal-regulated kinases; p-JNK, phosphorylated c-Jun N-terminal kinases; PMP, 1-pheny-3-methy-5-pyrazolne; RISK, reperfusion injury salvage kinase; ROS, reactive oxygen species; SOD, superoxide dismutase; TCM, traditional Chinese medicine; TFA, trifluoroacetic acid; TLC, thin layer chromatography. This study aimed to investigate the polysaccharide fraction of ginseng on hypoxia/reoxygenation (H/R) injury in cardiomyocytes and the underlying mechanisms
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