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      Drug Design, Development and Therapy (submit here)

      This international, peer-reviewed Open Access journal by Dove Medical Press focuses on the design and development of drugs, as well as the clinical outcomes, patient safety, and programs targeted at the effective and safe use of medicines. Sign up for email alerts here.

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      Protective effects of five compounds from Livistona chinensis R. Brown leaves against hypoxia/reoxygenation, H 2O 2, or adriamycin-induced injury in H9c2 cells

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          Abstract

          Purpose: Discovering new antimyocardial ischemia drug candidates that are highly efficient, have low toxicity, and originate from natural products is a popular trend for new cardiovascular drug development at present. The ethanol extract of Livistona chinensis leaves showed a favorable antioxidant activity in our preliminary screening test. This study aims to screen out antioxidants from the herb leaves further and evaluate their efficacy in acute myocardial ischemia treatment at the cellular level.

          Materials and methods: Guided with online 1, 1-diphenyl-2-picrylhydrazyl (DPPH)–high-performance liquid chromatography (HPLC) screening, antioxidants were first separated and isolated from the ethanol extract of L. chinensis leaves by preparative-HPLC. Subsequently, offline DPPH approach was used to validate the free radical scavenging activity of the components. Ultimately, the resulting antioxidants were evaluated against the hypoxia/reoxygenation (H/R)-, H 2O 2-, or adriamycin (ADM)-induced injury in H9c2 cells to verify their cardioprotective effects in vitro.

          Results: Five antioxidant ingredients, namely, orientin, isoorientin, vitexin, isovitexin, and tricin, were quickly distinguished and isolated from L. chinensis leaves. The IC 50 values of these ingredients were further examined by offline DPPH assay, as follows: 15.51±0.22, 6.64±0.38, 11.86±0.24, 8.89±0.66, and 31.86±0.24 μg/mL, respectively. Out of these ingredients, isoorientin showed the strongest antioxidation, which was equivalent to that of the positive control drug (vitamin C, IC 50: 6.99±0.62 μg/mL). Using H/R-, H 2O 2-, and ADM-induced H9c2 cell injury models, the five ingredients had different extents of cardioprotective effects in vitro. In particular, isoorientin showed the strongest protection. All the five ingredients also showed insignificant cytotoxic effect to normal H9c2 cells.

          Conclusion: The ethanol extract of L. chinensis leaves contained five antioxidants with low cardiac cytotoxicity. Isoorientin possessed the strongest antioxidation, which can predominantly account for the myocardial protection effects within the extract.

          Most cited references36

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          Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart.

          Peter Schultz, W Rosenthal, D. Krautwurst … (1991)
          Morphological, electrophysiological, and biochemical properties of H9c2 cells, a permanent cell line derived from rat cardiac tissue, were studied. Although the lectin binding pattern revealed similar sugar residues in the surface coat of H9c2 cells and isolated rat cardiocytes, heart-specific morphological structures could not be detected in H9c2 cells. Under physiological ionic conditions, H9c2 cells exhibited an outwardly rectifying, transient K+ current. When this current component was blocked by Ba2+ and Cs+, we observed an inward current through Ca2+ channels (15.8 +/- 2.2 pA/pF, n = 18, measured as Ba2+ current) that showed all characteristics of cardiac L-type currents. The activation kinetics were fast, and the current was stimulated by isoproterenol. The effect of isoproterenol was mimicked by forskolin or intracellularly applied cAMP. In radioligand binding experiments, we identified a specific, saturable, stereoselective and reversible high-affinity [3H]-(+)PN 200-110 binding with a dissociation constant Kd = 0.53 +/- 0.28 nM and a maximal specific binding of Bmax = 129.3 +/- 16.1 fmol/mg protein. There was an additional low-affinity/high-capacity binding site, which is unlikely to be related to a Ca2+ channel protein. Signal-transducing G proteins in membranes were characterized by [32P]ADP-ribosylation catalyzed by bacterial toxins and by the use of various antibodies. Cholera toxin substrates of 42 and 45 kd were identified that apparently correlated to Gs alpha-subunits. Pertussis toxin substrates of 40-41 kd were tentatively identified as Gi alpha-subunits. The G protein Go was absent or at least extremely low in concentration.
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            What does the commonly used DCF test for oxidative stress really show?

            Ulf Brunk, Tino Kurz, C Frennesson … (2010)
            H(2)DCF-DA (dihydrodichlorofluorescein diacetate) is widely used to evaluate 'cellular oxidative stress'. After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol [H(2)DCF (dihydrodichlorofluorescein)] that may be oxidized to fluorescent DCF (2',7'-dichlorofluorescein) by a process usually considered to involve ROS (reactive oxygen species). It is, however, not always recognized that, being a hydrophilic molecule, H(2)DCF does not cross membranes, except for the outer fenestrated mitochondrial ones. It is also not generally realized that oxidation of H(2)DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor H(2)O(2), directly oxidizes H(2)DCF. Consequently, oxidation of H(2)DCF requires the presence of either cytochrome c or of both redox-active transition metals and H(2)O(2). Redox-active metals exist mainly within lysosomes, whereas cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H(2)DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to H(2)O(2), showing that by itself it is unable to oxidize H(2)DCF. Cells that were either exposed to the lysosomotropic detergent MSDH (O-methylserine dodecylamide hydrochloride), exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.
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              Sirtuin-3 (SIRT3) Protein Attenuates Doxorubicin-induced Oxidative Stress and Improves Mitochondrial Respiration in H9c2 Cardiomyocytes.

              Kyle G Cheung, Laura Cole, Bo Xiang … (2015)
              Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electron transfer resulting in the production of reactive oxygen species (ROS). Sirtuin-3 (SIRT3) is a class III lysine deacetylase that is localized to the mitochondria and regulates mitochondrial respiration and oxidative stress resistance enzymes such as superoxide dismutase-2 (SOD2). The purpose of this study was to determine whether SIRT3 prevents DOX-induced mitochondrial ROS production. Administration of DOX to mice suppressed cardiac SIRT3 expression, and DOX induced a dose-dependent decrease in SIRT3 and SOD2 expression in H9c2 cardiomyocytes. SIRT3-null mouse embryonic fibroblasts produced significantly more ROS in the presence of DOX compared with wild-type cells. Overexpression of wild-type SIRT3 increased cardiolipin levels and rescued mitochondrial respiration and SOD2 expression in DOX-treated H9c2 cardiomyocytes and attenuated the amount of ROS produced following DOX treatment. These effects were absent when a deacetylase-deficient SIRT3 was expressed in H9c2 cells. Our results suggest that overexpression of SIRT3 attenuates DOX-induced ROS production, and this may involve increased SOD2 expression and improved mitochondrial bioenergetics. SIRT3 activation could be a potential therapy for DOX-induced cardiac dysfunction.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Drug Des Devel Ther
                Journal ID (iso-abbrev): Drug Des Devel Ther
                Journal ID (publisher-id): DDDT
                Journal ID (pmc): dddt
                Title: Drug Design, Development and Therapy
                Publisher: Dove
                ISSN (Electronic): 1177-8881
                Publication date (Electronic): 08 May 2019
                Publication date Collection: 2019
                Volume: 13
                Pages: 1555-1566
                Affiliations
                [1 ]Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University , Fuzhou 350122, People’s Republic of China
                Author notes
                Correspondence: Hong Yao; Xinhua LinDepartment of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University , Fuzhou350122, People’s Republic of ChinaTel +865 912 286 2016Fax +865 912 286 2016Email yauhung@ 123456126.com ; xhlin1963@ 123456sina.com
                Article
                Publisher ID: 201816
                DOI: 10.2147/DDDT.S201816
                PMC ID: 6514125
                PubMed ID: 31190736
                SO-VID: 1f6730e4-ef16-45b0-bcfe-30e6506d485a
                Copyright © © 2019 Li et al.
                License:

                This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License ( http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms ( https://www.dovepress.com/terms.php).

                History
                Date received : 16 January 2019
                Date accepted : 25 March 2019
                Page count
                Figures: 7, Tables: 2, References: 41, Pages: 12
                Categories
                Subject: Original Research

                ScienceOpen disciplines: Pharmacology & Pharmaceutical medicine
                Keywords: effect-directed analysis,antioxidant,protective effects,flavone,h9c2 cell
                Data availability:
                ScienceOpen disciplines: Pharmacology & Pharmaceutical medicine
                Keywords: effect-directed analysis, antioxidant, protective effects, flavone, h9c2 cell

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