Panax ginseng Polysaccharide Protected H9c2 Cardiomyocyte From Hypoxia/Reoxygenation Injury Through Regulating Mitochondrial Metabolism and RISK Pathway

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.

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“…The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XFe24 (Seahorse Agilent) extracellular flux analyser, as previously described [22]. Three injections were performed in order to change cells metabolism, namely: oligomycin (1 µg/mL)—a complex V inhibitor; FCCP (3 µM)—a classic uncoupling agent, and antimycin A+Rotenone (2.5 µM + 2 µM)—inhibitors of complex I and III, respectively [23]. The OCR parameters recorded in the analysis were as follows: (i) the basal respiration (before oligomycin addition); (ii) the leak state (after oligomycin addition)—the amount of O 2 consumption needed to sustain the proton gradient; (iii) the maximal respiration (after FCCP addition)—respiration in the presence of a classic uncoupler of oxidative phosphorylation; (iv) the ATP turnover (the difference between the basal respiration and the leak state)—the amount of O 2 consumption used for ATP production; (v) the reserve capacity (the difference between the maximal and the basal respiration).…”

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“…Levels of intracellular and mitochondrial ROS were measured using a DCFH-DA or mitoSOX probe as previously method (Zuo et al, 2018). In brief, at 0, 4, 8, 16, 24, and 48 h after corresponding treatment, cells were harvested, collected, and washed with serum-free DMEM media.…”

“…Mitochondrial respiration is an indicator of both the functional bioenergetics capacity of mitochondria and overall cellular health (Zuo et al, 2018;Pan et al, 2018). In the study, we used an XFp Extracellular Flux Analyzer (Seahorse Biosciences, North Billerica, MA, USA) to evaluate the OCR.…”

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“…However, the injury caused by hypoxia may be averted. A recent study performed by Zuo et al showed that an acid polysaccharide fraction of ginseng (AP1) was the most effective fraction at protecting cardiomyocytes from hypoxia [135]. AP1 exerted a protective effect by increasing the expression of both GR and the estrogen receptor, which in turn mediated the activation of the RISK pathway and eNOS-dependent mechanisms to resist reperfusion injury [136].…”