Cardioprotective role of a magnolol and honokiol complex in the prevention of doxorubicin-mediated cardiotoxicity in adult rats.

Doxorubicin (DOXO) induces marked cardiotoxicity, though increased oxidative stress while there are some documents related with cardioprotective effects of some antioxidants against organ-toxicity during cancer treatment. Although magnolia bark has some antioxidant-like effects, its action in DOXO-induced heart dysfunction has not be shown clearly. Therefore, here, we aimed to investigate the cardioprotective action of a magnolia bark extract with active component magnolol and honokiol complex (MAHOC; 100 mg/kg) in DOXO-treated rat hearts. One group of adult male Wistar rats was injected with DOXO (DOXO-group; a cumulative dose of 15 mg/kg in 2-week) or saline (CON-group). One group of DOXO-treated rats was administered with MAHOC before DOXO (Pre-MAHOC group; 2-week) while another group was administered with MAHOC following the 2-week DOXO (Post-MAHOC group). MAHOC administration, before or after DOXO, provided full survival of animals during 12-14 weeks, and significant recoveries in the systemic parameters of animals such as plasma levels of manganese and zinc, total oxidant and antioxidant statuses, and also systolic and diastolic blood pressures. This treatment also significantly improved heart function including recoveries in end-diastolic volume, left ventricular end-systolic volume, heart rate, cardiac output, and prolonged P-wave duration. Furthermore, the MAHOC administrations improved the structure of left ventricles such as recoveries in loss of myofibrils, degenerative nuclear changes, fragmentation of cardiomyocytes, and interstitial edema. Biochemical analysis in the heart tissues provided the important cardioprotective effect of MAHOC on the redox regulation of the heart, such as improvements in activities of glutathione peroxidase and glutathione reductase, and oxygen radical-absorbing capacity of the heart together with recoveries in other systemic parameters of animals, while all of these benefits were observed in the Pre-MAHOC treatment group, more prominently. Overall, one can point out the beneficial antioxidant effects of MAHOC in chronic heart diseases as a supporting and complementing agent to the conventional therapies.

An Overexpression of SLC30A6 Gene Contributes to Cardiomyocyte Dysfunction via Affecting Mitochondria and Inducing Activations in K-Acetylation and Epigenetic Proteins.

Intracellular free Zn2+ ([Zn2+]i) is less than 1-nM in cardiomyocytes and its regulation is performed with Zn2+-transporters. However, the roles of Zn2+-transporters in cardiomyocytes are not defined exactly yet. Here, we aimed to examine the role of an overexpression and subcellular localization of a ZnT6 in insulin-resistance mimic H9c2 cardiomyoblasts (IR-cells; 50-µM palmitic acid for 24-h incubation). We used both IR-cells and ZnT6-overexpressed (ZnT6OE) cells in comparison to those of H9c2 cells (CON-cells). The IR-cells have higher ZnT6-protein levels than CON-cells while this level was similar to those of ZnT6OE-cells. The [Zn2+]i in IR-cells was increased significantly and mitochondrial localization of ZnT6 was demonstrated in these cells by using confocal microscopy visualization. Furthermore, electron microscopy analysis demonstrated abnormal morphological appearance in both IR-cells and ZnT6OE-cells characterized by irregular mitochondrion cristae and condensed and dilated cisterna in the sarcoplasmic reticulum. Mitochondria were similarly depolarized in both IR-cells and ZnT6OE-cells. The protein expression level of a mitofusin protein MFN2 in the IR-cells was decreased, significantly, whereas, it was found significantly upregulated in both ZnT6-OE-cells and IR-incubated ZnT6OE-cells, which demonstrates the role of ZnT6-overexpression but not IR. Additionally, the total protein level of a mitochondrial fission protein, dynamin-related protein 1, DRP1 was found to be increased over 1.5-fold in IR-cells while this increase was found to be higher in the ZnT6OE-cells than those of IR-cells, demonstrating an additional effect on IR-increase. ZnT6-overexpression induced also significant increases in K-acetylation, trimethylation of histone H3 lysine27, and mono-methylation of histone H3 lysine36, in a similar manner to those of IR-cells. Overall, our data point out an important contribution of ZnT6-overexpression to IR-induced cellular changes, such as alteration in mitochondria function and activation of epigenetic modifications.

Morphological and Functional Analysis of Cardiac Ameliorations in Elderly Rats Supplemented with a Magnolol Extract Complex / Análisis Morfológico y Funcional de las Mejoras Cardíacas en Ratas Ancianas Suplementadas con un Complejo de Extracto de Magnolol

SUMMARY: Magnolia bark extract supplementation has an anti-oxidative role in mammalians. However, its role in physiological aged-associated heart insufficiency is not known yet. Therefore, we investigated the effects of a magnolia bark complex, including magnolol and honokiol components (MAHOC), in elderly rat hearts (24-month-old aged group). One group of aged rats was supplemented with MAHOC (400 mg/kg/d, for 12 weeks) besides the standard rat diet while the second group of elderly rats and adult rats (to 6-month- old adult-group) were only fed with the standard rat diet. The morphological analysis using light microscopy has shown marked myofibrillar losses, densely localized fibroblasts, vacuolizations, infiltrated cell accumulations, and collagen fibers in the myocardium of the elderly rats compared to the adults. We also detected a markedly increased amount of degenerated cardiomyocytes including the euchromatic nucleus. The MAHOC supplementation of the elderly rats provided marked ameliorations in these abnormal morphological changes in the heart tissue. Furthermore, electrophysiological analysis of electrocardiograms (ECGs) in the supplemented group showed significant attenuations in the prolonged durations of P-waves, QRS-complexes, QT-intervals, and low heart rates compared to the unsupplemented elderly group. The biochemical analysis also showed significant attenuations in the activity of arylesterase and total antioxidant status in the myocardium of the supplemented group. We further determined significant attenuations in the activity of a mitochondrial enzyme succinate dehydrogenase, known as a source of reactive oxygen species (ROS), and the decreased level of ATP/ADP in the heart homogenates of the supplemented group. Moreover, under in vitro conditions by using an aging-mimicked cardiac cell line induced by D-galactose, we demonstrated that MAHOC treatment could provide prevention of depolarization in mitochondria membrane potential and high-level ROS production. Overall, our data presented significant myocardial ameliorations in physiological aging-associated morphological alterations parallel to the function and biochemical attenuations with MAHOC supplementation, at most, through recoveries in mitochondria.

La suplementación con extracto de corteza de magnolia tiene un papel antioxidante en los mamíferos, sin embargo, su rol en la insuficiencia cardíaca asociada al envejecimiento fisiológico aún no se conoce. Por lo anterior, investigamos los efectos de un complejo de corteza de magnolia, incluidos los componentes magnolol y honokiol (MAHOC), en corazones de ratas seniles (grupo de edad de 24 meses). La alimentación de grupo de ratas seniles se complementó con MAHOC (400 mg/kg/d, durante 12 semanas) además de la dieta estándar, mientras que el segundo grupo de ratas seniles y ratas adultas (hasta el grupo de adultos de 6 meses) solo recibió la dieta estándar para ratas. El análisis morfológico mediante microscopía óptica ha mostrado marcadas pérdidas miofibrilares, fibroblastos densamente localizados, vacuolizaciones, acumulaciones de células infiltradas y fibras de colágeno en el miocardio de las ratas seniles en comparación con las adultas. También detectamos una cantidad notablemente mayor de cardiomiocitos degradados, incluido el núcleo eucromático. La suplementación con MAHOC de las ratas seniles proporcionó mejoras marcadas en estos cambios morfológicos anormales en el tejido cardiaco. Por otra parte, el análisis de los electrocardiogramas (ECG) en el grupo suplementado mostró atenuaciones significativas en las duraciones prolongadas de las ondas P, los complejos QRS, los intervalos QT y las frecuencias cardíacas bajas, en comparación con el grupo de ratas seniles sin suplementación alimenticia. El análisis bioquímico también mostró atenuaciones significativas en la actividad de la arilesterasa y el estado antioxidante total en el miocardio del grupo suplementado. Determinamos además atenuaciones significativas en la actividad de la enzima mitocondrial succinato deshidrogenasa, conocida como fuente de especies reactivas de oxígeno (ROS), y la disminución del nivel de ATP/ADP en los homogeneizados de corazón del grupo suplementado. Además, en condiciones in vitro mediante el uso de una línea de células cardíacas, imitando el envejecimiento inducido por D- galactosa, demostramos que el tratamiento con MAHOC podría prevenir la despolarización en el potencial de membrana de las mitocondrias y la producción de ROS de alto nivel. En general, nuestros datos presentaron mejoras miocárdicas significativas en alteraciones morfológicas asociadas con el envejecimiento fisiológico paralelas a la función y atenuaciones bioquímicas con la suplementación con MAHOC, como máximo, a través de recuperaciones en las mitocondrias.

Overexpression of Slc30a7/ZnT7 increases the mitochondrial matrix levels of labile Zn2+ and modifies histone modification in hyperinsulinemic cardiomyoblasts.

BACKGROUND: Cellular free Zn2+ concentrations ([Zn2+]) are primarily coordinated by Zn2+-transporters, although their roles are not well established in cardiomyocytes. Since we previously showed the important contribution of a Zn2+-transporter ZnT7 to [Zn2+]i regulation in hyperglycemic cardiomyocytes, here, we aimed to examine a possible regulatory role of ZnT7 not only on [Zn2+]i but also both the mitochondrial-free Zn2+ and/or Ca2+ in cardiomyocytes, focusing on the contribution of its overexpression to the mitochondrial function. METHODS: We mimicked either hyperinsulinemia (by 50-µM palmitic acid, PA-cells, for 24-h) or overexpressed ZnT7 (ZnT7OE-cells) in H9c2 cardiomyoblasts. RESULTS: Opposite to PA-cells, the [Zn2+]i in ZnT7OE-cells was not different from untreated H9c2-cells. An investigation of immunofluorescence imaging by confocal microscopy demonstrated a ZnT7 localization on the mitochondrial matrix. We demonstrated the ZnT7 localization on the mitochondrial matrix by using immunofluorescence imaging. Later, we determined the mitochondrial levels of [Zn2+]Mit and [Ca2+]Mit by using the Zn2+ and Ca2+ sensitive FRET probe and a Ca2+-sensitive dye Fluo4, respectively. The [Zn2+]Mit was found to increase significantly in ZnT7OE-cells, similar to the PA-cells while no significant changes in the [Ca2+]Mit in these cells. To examine the contribution of ZnT7 overexpression on the mitochondria function, we determined the level of reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) in these cells in comparison to the PA-cells. There were significantly increased production of ROS and depolarization in MMP and increases in marker proteins of mitochondria-associated apoptosis and autophagy in ZnT7-OE cells, similar to the PA-cells, parallel to increases in K-acetylation. Moreover, we determined significant increases in trimethylation of histone H3 lysine27, H3K27me3, and the mono-methylation of histone H3 lysine36, H3K36 in the ZnT7OE-cells, demonstrating the role of [Zn2+]Mit in epigenetic regulation of cardiomyocytes under hyperinsulinemia through histone modification. CONCLUSIONS: Overall, our data have shown an important contribution of high expression of ZnT7-OE, through its buffering and muffling capacity in cardiomyocytes, on the regulation of not only [Zn2+]i but also both [Zn2+]Mit and [Ca2+]Mit affecting mitochondria function, in part, via histone modification.

Astaxanthin Enhances Gingival Wound Healing following High Glucose-Induced Oxidative Stress.

Fibroblasts of the gingiva play a key role in oral wound healing in diabetes. In this study, effects of astaxanthin (ASTX), a xanthophyll carotenoid, were tested on gingival fibroblasts in a wound healing assay in vitro. The aim of this study was to determine whether ASTX can recover delayed wound healing or not when oxidative stress is elevated by high glucose exposure. For this purpose, human gingival fibroblasts were incubated with or without ASTX following exposure to systemic doses of low glucose (LG) and high glucose (HG) in culture media (5- and 25-, 50 mM D-glucose in DMEM Ham's F12) following 24 hours of incubation. Levels of ROS (Reactive oxygen species) were determined for each experimental group by confocal microscopy. Cell proliferation and viability were assessed by an automated cell counter with trypan blue assay. Wound healing assay was designed in 60 mm petri dishes. Cells were exposed to 5-, 25-, and 50 mM glucose for 24 hours, and a straight line free of cells was created upon full confluency. 100 µM ASTX was added to the recovery group, simultaneously. Cells were monitored with JuLIⓇ-Br Cell History Recorder. ROS levels were significantly increased with increasing glucose levels, while cell proliferation and viability demonstrated a negative correlation with increasing oxidative stress. ROS levels significantly decreased in the 100 µM ASTX-treated group compared to the gingival fibroblasts treated with 50 mM HG medium-only, as well as growth rate and viability. Wound healing was delayed in a dose-dependent manner following high glucose exposure, while ASTX treatment recovered wounded area by 1.16-fold in the 50 mM HG group. Our results demonstrated that ASTX enhances gingival wound healing through its antioxidative properties following high glucose induced oxidative stress. Therefore, ASTX can be suggested as a promising candidate to maintain oral health in chronic wounds of the oral tissues related to diabetes.