scholarly journals Hepatocyte Growth Factor Protects Endothelial Barrier Against Oxidative Stress and Mitochondria-dependent Apoptosis

2021 ◽  
Author(s):  
Shan-Shan Meng ◽  
Fei-Ping Xia ◽  
Jing-Yuan Xu ◽  
Xi-Wen Zhang ◽  
Ming Xue ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Complex I ◽  
Mitochondrial Membrane ◽  
Mtor Inhibitor ◽  
Endothelial Barrier ◽  
Ros Production ◽  
Endothelial Apoptosis ◽  
Junction Protein ◽  
Endothelial Junction ◽  
Hepatocyte Growth

Abstract Background: Pulmonary microvascular endothelial cells(PMVECs) were incomplex and endothelial barrier was destroyed in the pathogenesis progress of acute lung injury(ALI)/acute respiratory distress syndrome(ARDS). Previous studies have demonstrated that hepatocyte growth factor (HGF) could decrease endothelial apoptosis. Nevertheless, the mechanism by which HGF-suppressed oxidative stress contributes to endothelial mitochondria-dependent apoptosis is poorly understood. Methods: In our current study, we introduced lipopolysaccharide(LPS)-induced PMEVCs with HGF treatment. To investigate the effects of mTOR/STAT-3 pathway in endothelial oxidative stress and mitochondria-dependent apoptosis, mammalian TOR (mTOR) inhibitor rapamycin and signal transducer and activator of transcription 3 (STAT-3) inhibitor S3I-201 were respectively used to inhibit mTOR/STAT-3 signaling. Moreover, lentivirus vector-mediated HGF, mTORC1(raptor) and mTORC2(rictor) gene knockdown modification were introduced to evaluate mTORC1 and mTORC2 pathway. Calcium measurement, ROS production, mitochondrial membrane potential and protein complex I expression, cell proliferation, apoptosis and endothelial junction protein were detected to evaluate HGF effects.Results: Our study demonstrated that HGF protected endothelium via the suppression of ROS production and intracellular calcium uptake, which leading to increased mitochondrial membrane potential (JC-1 and mitochondria tracker green detection)and specific proteins(complex I), decreased endothelial apoptosis specific protein(Caspase-3), raised anti-apoptosis mRNA level(Bcl-2 and Bcl-xL), and increased endothelial junction proteins (VE-cadherin and occludin). Reversely, mTOR inhibitor rapamycin and STAT-3 inhibitor S3I-201 could raise oxidative stress and mitochondria-dependent apoptosis even with HGF treatment in LPS-induced endothelial cells. Similarly, mTORC1 as well as mTORC2, have the same protective effects in mitochondria damage and apoptosis. Conclusion: In all, these reveal that mTOR/STAT-3 signaling mediate the HGF suppression effects to oxidative level, mitochondria-dependent apoptosis and endothelial junction protein in LPS-stimulated PMVECs, which contributing to the endothelial survival and barrier integrity.

scholarly journals Altered expressions of endothelial junction protein of placental capillaries in premature infants with intraventricular hemorrhage

2016 ◽  
Vol 25 (3) ◽  
pp. 143-50 ◽  
Author(s):  
Maria Ekawati ◽  
Ninik Mujihartini ◽  
Ahmad A. Jusuf ◽  
Nani Dharmasetiawani ◽  
Sri W.A. Jusman ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Premature Infants ◽  
Intraventricular Hemorrhage ◽  
Tissue Response ◽  
Cranial Ultrasound ◽  
Cross Sectional ◽  
Altered Expression ◽  
Hypoxia Group ◽  
Junction Protein ◽  
Endothelial Junction

Background: Placental hypoxia may lead to oxidative stress, which inflicts damage to capillary protein junction. The aim of this study was to evaluate altered expression of endothelial junction protein of capillaries in hypoxia condition and to observe its correlation with the incidence of  intraventricular hemorrhage in premature infants.Methods: A cross-sectional study was conducted by using placental tissues of premature infants as amodel of capillary integrity (29 hypoxic and 29 non-hypoxic). Hypoxia inducible factor (HIF)-1α was measured to define placental tissue response to hypoxia; malondialdehyde (MDA) and glutathione (GSH) served as markers of oxidative stress. The expressions of junctional proteins, N-cadherin and occludin were analyzed by immunohistochemistry. Intraventricular hemorrhage (IVH) was detected by cranial ultrasound at the third day. Unpaired t test, Mann-Whitney, and Chi-square tests were used to analyze the data.Results: The HIF-1α and MDA levels were slightly, but not significantly, higher in hypoxia group {13.64±8.70 pg/mg protein and 10.31 pmol/mg tissue (ranged 1.92–93.61), respectively}  compared to non- hypoxia group {10.65±5.35 pg/mg protein and 9.77 pmol/mg tissue (ranged 2.42–93.31)}. GSH levels were not different in both groups (38.14 (ranged 9.44–118.91) and  38.47(ranged 16.49–126.76) ng/mg protein, respectively. mRNA expression of N-cadherin (0.13) and occludin (0.096) were significantly lower in hypoxia comparedto non-hypoxia group (p=0,001), while protein expression of  N-cadherin (3.4; 75.9; 6.9; 13.8%) and occludin  (20.7; 3.4; 69.0; 3.4; 6.9%)  in hypoxia group was not associated with IVH (p=0.783 and p=0.743).Conclusion: Hypoxia altered expression of endothelial junction protein in placental capillaries, but no association with intraventricular hemorrhage was observed.

scholarly journals Mitochondrial Alterations and Enhanced Human Leukocyte/Endothelial Cell Interactions in Type 1 Diabetes

2020 ◽  
Vol 9 (7) ◽  
pp. 2155
Author(s):  
Francesca Iannantuoni ◽  
Aranzazu M. de Marañon ◽  
Zaida Abad-Jiménez ◽  
Francisco Canet ◽  
Pedro Díaz-Pozo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Type 1 Diabetes ◽  
Adhesion Molecules ◽  
Mitochondrial Function ◽  
Mitochondrial Membrane ◽  
Diabetic Patients ◽  
Ros Production ◽  
Mitochondrial Ros ◽  
Type 1 Diabetic Patients

Type 1 diabetes has been associated with oxidative stress. This study evaluates the rates of oxidative stress, mitochondrial function, leukocyte–endothelium interactions and adhesion molecules in type 1 diabetic patients. The study population consisted of 52 diabetic patients and 46 body-composition and age-matched controls. We assessed anthropometric and metabolic parameters, oxidative stress and mitochondrial function by evaluating reactive oxygen species (ROS) production, mitochondrial ROS production, mitochondrial membrane potential and superoxide dismutase (SOD) and catalase (CAT) expression in polymorphonuclear leukocytes from type 1 diabetic patients. In addition, we evaluated interactions between leukocytes and human umbilical vein endothelial cells (HUVEC), and serum expression of adhesion molecules (P-selectin, VCAM-1 and ICAM-1), proinflammatory cytokines (IL-6 and TNFα) and myeloperoxidase (MPO). HbA1C and glucose levels were higher in diabetic patients than in control subjects, as expected. Mitochondrial function was altered and leukocyte–endothelium interactions were enhanced in diabetic patients, which was evident in the increase in total and mitochondrial ROS production, higher mitochondrial membrane potential, enhanced leukocyte rolling and adhesion, and decreased rolling velocity. Furthermore, we observed an increase in levels of adhesion molecules P-selectin, VCAM-1, and ICAM-1 in these subjects. In addition, type 1 diabetic patients exhibited an increase in proinflammatory mediators TNFα and MPO, and a decreased expression of SOD. The enhancement of leukocyte–endothelium interactions and proinflammatory markers correlated with glucose and HbA1Clevels. Mitochondrial alteration, oxidative stress, and enhanced leukocyte–endothelium interactions are features of type 1 diabetes and may be related to cardiovascular implications.

Olfactomedin 4 Is Essential for Superoxide Production and Sensitizes Oxidative Stress-Induced Apoptosis in Neutrophils.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1356-1356
Author(s):  
Wenli Liu ◽  
Yueqin Liu ◽  
Ruihong Wang ◽  
Cuiling Li ◽  
Chuxia Deng ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Respiratory Chain ◽  
Complex I ◽  
Mitochondrial Membrane ◽  
Mitochondrial Respiratory Chain ◽  
H2o2 Production ◽  
Induced Apoptosis ◽  
Mitochondrial Respiratory

Abstract Abstract 1356 Poster Board I-378 Introduction Olfactomedin 4 (OLFM4), also called hGC-1, GW112 and pDP4, was first identified and specifically expressed in hematopoietic myeloid cells. OLFM4 expression in myeloid cells is regulated by transcription factors, PU1 and NF-κB. It has significant homology in its C-terminal domain with other olfactomedin-related proteins. OLFM4 encodes a 510 amino acid N-linked glycoprotein. The exact biological function of OLFM4, especially in neutrophils, is currently undefined. To characterize the in vivo function of OLFM4, we generated OLFM4 deficient mice (OLFM4-/-) and investigated its potential role in neutrophil functioins. Results 1) In this study, we showed that OLFM4 is a secreted glycoprotein and is also localized in the mitochondria, cytoplasm and cell membrane fractions of neutrophils. We demonstrated that OLFM4 interacts with GRIM-19 (Genes associated with Retinoid-IFN-induced Mortality-19), an apoptosis related protein, in the neutrophil mitochondria using co-immuoprecipitation assay. GRIM-19 is a subunit of complex I of mitochondrial respiratory chain and is essential for maintenance of mitochondrial membrane potential. Our result suggests that OLFM4 appears to be a novel component of complex I of mitochondrial respiratory chain and may be involved in regulation of mitochondrial membrane potential. 2) Mice heterozygous (OLFM4+/-) and homozygous (OLFM4-/-) for the null mutation in OLFM4 appeared to have normal development, fertility, and viability relative to wild-type (WT) mice. Whole blood analysis, differential leukocyte counts, blood chemistry and bone marrow smears were normal in OLFM4-/- mice, suggesting that OLFM4 is not essential for normal development and hematopoiesis in mice. 3) In response to LPS, fMLP and E.coli bacteria challenge, neutrophils from OLFM4-/- mice showed significantly reduced superoxide (O2−) and hydrogen peroxide (H2O2) production compared with WT mice. These results suggest that OLFM4 is an essential component to mediate O2− and H2O2 production in the neutrophil mitochondria under inflammation stimuli. 4) Exogenous H2O2 induced neutrophil apoptosis in a time and dose dependent manner in WT mice, but this induction of apoptosis was significantly reduced in OLFM4-/- mice. This result suggests that OLFM4 sensitizes and mediates H2O2-induced apoptosis in neutrophils. 5) Furthermore, we demonstrated that H2O2-stimulated mitochondrial membrane permeability reduction and caspase-3 and caspase-9 activation were inhibited in the neutrophils of OLFM4-/- mice. This result confirmed our hypothesis that OLFM4 may be involved in maintenance of mitochondrial membrane potential and suggests that OLFM4 may have opposite role as GRIM-19. 6) Moreover, Bax association with mitochondria and the cytoplasmic translocation of Omi/HtrA2 and Smac/DIABLO in response to H2O2 were inhibited in the neutrophils of OLFM4-/- mice. Conclusion Our results suggest: 1) OLFM4 has multiple subcellular localizations including mitochondria, cytoplasm, and cell membrane in neutrophils. The interaction of OLFM4 with GRIM-19 in the mitochondria suggests that OLFM4 is novel component of complex I of mitochondrial respiratory chain in the mitochondria of neutrophils, 2) OLFM4 is a novel mitochondrial molecule that is essential for O2− and H2O2 production in the neutrophils in the presence of inflammation stimuli, 3) Loss of OLFM4 in neutrophils does not trigger spontaneous apoptosis. However, OLFM4 sensitizes oxidative stress-induced apoptosis in mouse neutrophils. OLFM4 is involved in the regulation of mitochondria membrane potential and sensitizes cytoplasmic translocation of Omi/HtrA2 and Smac/DIABLO and caspases-3 and caspase-9 mediated apoptosis in the presence of oxidative stress. Disclosures No relevant conflicts of interest to declare.

Effect of thyroid hormone on mitochondrial properties and oxidative stress in cells from patients with mtDNA defects

2009 ◽  
Vol 296 (2) ◽  
pp. C355-C362 ◽  
Author(s):  
Keir J. Menzies ◽  
Brian H. Robinson ◽  
David A. Hood
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Thyroid Hormone ◽  
Protein Expression ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Membrane ◽  
Ros Production ◽  
Mitochondrial Mass ◽  
Mtdna Mutations

Mitochondrial (mt)DNA mutations contribute to various disease states characterized by low ATP production. In contrast, thyroid hormone [3,3′,5-triiodothyronine (T3)] induces mitochondrial biogenesis and enhances ATP generation within cells. To evaluate the role of T3-mediated mitochondrial biogenesis in patients with mtDNA mutations, three fibroblast cell lines with mtDNA mutations were evaluated, including two patients with Leigh's syndrome and one with hypertrophic cardiomyopathy. Compared with control cells, patient fibroblasts displayed similar levels of mitochondrial mass, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), mitochondrial transcription factor A (Tfam), and uncoupling protein 2 (UCP2) protein expression. However, patient cells exhibited a 1.6-fold elevation in ROS production, a 1.7-fold elevation in cytoplasmic Ca2+ levels, a 1.2-fold elevation in mitochondrial membrane potential, and 30% less complex V activity compared with control cells. Patient cells also displayed 20–25% reductions in both cytochrome c oxidase (COX) activity and MnSOD protein levels compared with control cells. After T3 treatment of patient cells, ROS production was decreased by 40%, cytoplasmic Ca2+ was reduced by 20%, COX activity was increased by 1.3-fold, and ATP levels were elevated by 1.6-fold, despite the absence of a change in mitochondrial mass. There were no significant alterations in the protein expression of PGC-1α, Tfam, or UCP2 in either T3-treated patient or control cells. However, T3 restored the mitochondrial membrane potential, complex V activity, and levels of MnSOD to normal values in patient cells and elevated MnSOD levels by 21% in control cells. These results suggest that T3 acts to reduce cellular oxidative stress, which may help attenuate ROS-mediated damage, along with improving mitochondrial function and energy status in cells with mtDNA defects.

scholarly journals Mir-141-3p Regulates Apoptosis and Mitochondrial Membrane Potential via Targeting Sirtuin1 in a 1-Methyl-4-Phenylpyridinium in vitro Model of Parkinson’s Disease

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Yumin Zheng ◽  
Li Dong ◽  
Na Liu ◽  
Xiaoguang Luo ◽  
Zhiyi He
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Western Blot ◽  
Pc12 Cells ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Luciferase Reporter ◽  
Ros Production ◽  
Reporter Assay ◽  
Induced Apoptosis

Objectives. Parkinson’s disease (PD) is a common neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons in the substantia nigra. The present study investigated miR-141-3p/sirtuin1 (SIRT1) activity in a 1-methyl-4-phenylpyridinium- (MPP+-) induced PC12-cell model of PD. Methods. PC12 cells were exposed to MMP+ following induction of differentiation by nerve growth factor (NGF). miR-141-3p and SIRT1 expressions were examined using RT-qPCR and western blot. Cell viability was evaluated using the MTT assay. Apoptosis percentage, reactive oxygen species (ROS) production, and mitochondrial membrane potential (Δψm) were evaluated using flow cytometry. Expression of Nuclear factor-kappa B- (NF-κB-) related proteins was determined by western blot. Bioinformatic analysis, RT-qPCR, and luciferase reporter assay were used to confirm the interaction between miR-141-3p and SIRT1. Results. miR-141-3p was upregulated, and SIRT1 was downregulated in MPP+-treated PC12 cells. MPP+ treatment also upregulated nitric oxide synthase 1 (Nos1) and α-synuclein. miR-141-3p induced apoptosis, oxidative stress, mitochondrial dysfunction, and downregulated the SIRT1 mRNA expression. The luciferase reporter assay showed that SIRT1 was the target of miR-141-3p. SIRT1 transfection attenuated apoptosis, ROS production and maintained Δψm. SIRT1 also downregulated Nos1, tumor necrosis factor-α (TNF-α), interleukin 1 beta (IL-1β), interleukin 6(IL-6) and upregulated B cell lymphoma 2 (Bcl-2) protein. In addition, SIRT1 activator resveratrol blocked the effects of miR-141-3p mimic on Nos1, α-synuclein, and mitochondrial membrane potential. SIRT1 inhibitor sirtinol reversed the biological effects of miR-141-3p. Conclusion. Increased miR-141-3p induced apoptosis, oxidative stress, and mitochondrial dysfunction in MPP+-treated PC12 cells by directly targeting the SIRT1 expression. Our study provided a potential therapeutic strategy for PD.

scholarly journals Thrombopoietin Protects Cardiomyocytes from Iron-Overload Induced Oxidative Stress and Mitochondrial Injury

2015 ◽  
Vol 36 (5) ◽  
pp. 2063-2071 ◽  
Author(s):  
Shing Chan ◽  
Godfrey Chifung Chan ◽  
Jieyu Ye ◽  
Qizhou Lian ◽  
Jianliang Chen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Iron Overload ◽  
Protective Effect ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Lipid Hydroperoxides ◽  
Ros Production ◽  
H9c2 Cells ◽  
Induced Apoptosis

Background/Aims: Thalassaemia accompanied with iron-overload is common in Hong Kong. Iron-overload induced cardiomyopathy is the commonest cause of morbidity and mortality in patients with β-thalassaemia. Chronic iron-overload due to blood transfusion can cause cardiac failure. Decreased antioxidant defence and increased ROS production may lead to oxidative stress and cell injury. Iron-overload may lead to heart tissue damage through lipid peroxidation in response to oxidative stress, and a great diversity of toxic aldehydes are formed when lipid hydroperoxides break down in heart and plasma. Methods: Iron entry into embryonic heart H9C2 cells was determined by calcein assay using a fluorometer. Reactive oxygen species (ROS) production in cells treated with FeCl3 or thrombopoietin (TPO) was monitored by using the fluorescent probe H2DCFDA. Changes in mitochondrial membrane potential of H9C2 cells were quantified by using flow cytometry. Results: We demonstrated that iron induced oxidative stress and apoptosis in cardiomyocytes, and that iron increased ROS production and reduced cell viability in a dose-dependent manner. Iron treatment increased the proportion of cells with JC-1 monomers, indicating a trend of drop in the mitochondrial membrane potential. TPO exerted a cardio-protective effect on iron-induced apoptosis. Conclusions: These findings suggest that iron-overload leads to the generation of ROS and further induces apoptosis in cardiomyocytes via mitochondrial pathways. TPO might exert a protective effect on iron-overload induced apoptosis via inhibiting oxidative stress and suppressing the mitochondrial pathways in cardiomyocytes.

TPO exerts a protective effect on iron-overload induces apoptosis in cardiomyocytes via mitochondrial pathways

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4668-4668 ◽  
Author(s):  
Mo Yang ◽  
Shing Chan ◽  
Jie yu Ye ◽  
Godfrey ChiFung Chan
Keyword(s):  
Oxidative Stress ◽  
Iron Overload ◽  
Protective Effect ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Ros Production ◽  
H9c2 Cells ◽  
Plot Analysis ◽  
Iron Treatment ◽  
Induced Apoptosis

Thalassaemia companied with iron-overload is common in Hong Kong. Iron overload induced cardiomyopathy is the commonest cause of morbidity and mortality in b-thalassaemia patients. One of the causes of cardiac failure is chronic iron overload of blood transfusion. Some studies showed that iron overload can cause toxic effect in heart cells. Iron-overload induced cardiomyopathy damages are the major complications in patients with beta-thalassaemia major. Iron-overload may induce apoptosis in cardiomyocytes. Our previous study showed TPO has cardiac protective effect (Li et al, Circulation, 2007). In this study, we demonstrated firstly that iron induced oxidative stress can cause apoptosis in cardiomyocytes. By using H9C2 cells, we showed that iron increased reactive oxygen species (ROS) production (n=3) and reduced cell viability in a dose-dependent manner (0-0.6 mM) (n=6). Apoptotic cells were found to be significantly increased under iron treatment (0.3 mM, 72 hrs) in the AnnexinV/PI assay (n=6). The expression of active caspase-3 significantly increased in iron-treated cells. Furthermore, iron treatment increased the proportion of cells containing JC-1 monomers, indicating a trend in the drop of mitochondrial membrane potential (n=6). Secondly, we found that TPO exerted cardio-protective effect on iron-induced apoptosis. H9C2 cells were cultured in the presence of iron (0.3 mM) with or without TPO (50 ng/mL). The ROS production was significantly decreased with the addition of TPO at 50 ng/mL (n=3). Dot-plot analysis of AnnexinV/PI staining demonstrated that TPO significantly reduced the population of apoptotic cells (n=6). Incubation with TPO also decreased the iron-induced caspase-3 expression (n=6). Flow cytometric dot-plot analysis also showed trends of amelioration of the increase in JC-1 monomers in the iron plus TPO group (n=6), indicating a trend in attenuation of the drop of mitochondrial membrane potential. Our findings suggest that iron-overload lead to generation of ROS which further induces apoptosis in cardiomyocytes via mitochondrial pathways and TPO might exert a protective effect on iron-overload induced apoptosis via inhibiting oxidative stress and mitochondrial pathway in cardiomyocytes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Significance of Mitochondrial Reactive Oxygen Species in the Generation of Oxidative Stress in Spermatozoa

2008 ◽  
Vol 93 (8) ◽  
pp. 3199-3207 ◽  
Author(s):  
Adam J. Koppers ◽  
Geoffry N. De Iuliis ◽  
Jane M. Finnie ◽  
Eileen A. McLaughlin ◽  
R. John Aitken
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Complex I ◽  
Mitochondrial Membrane ◽  
Reactive Oxygen ◽  
Human Spermatozoa ◽  
Ros Generation ◽  
Oxygen Species ◽  
Mitochondrial Ros ◽  
Sperm Movement

Abstract Context: Male infertility has been linked with the excessive generation of reactive oxygen species (ROS) by defective spermatozoa. However, the subcellular origins of this activity are unclear. Objective: The objective of this study was to determine the importance of sperm mitochondria in creating the oxidative stress associated with defective sperm function. Method: Intracellular measurement of mitochondrial ROS generation and lipid peroxidation was performed using the fluorescent probes MitoSOX red and BODIPY C11 in conjunction with flow cytometry. Effects on sperm movement were measured by computer-assisted sperm analysis. Results: Disruption of mitochondrial electron transport flow in human spermatozoa resulted in generation of ROS from complex I (rotenone sensitive) or III (myxothiazol, antimycin A sensitive) via mechanisms that were independent of mitochondrial membrane potential. Activation of ROS generation at complex III led to the rapid release of hydrogen peroxide into the extracellular space, but no detectable peroxidative damage. Conversely, the induction of ROS on the matrix side of the inner mitochondrial membrane at complex I resulted in peroxidative damage to the midpiece and a loss of sperm movement that could be prevented by the concomitant presence of α-tocopherol. Defective human spermatozoa spontaneously generated mitochondrial ROS in a manner that was negatively correlated with motility. Simultaneous measurement of general cellular ROS generation with dihydroethidium indicated that 68% of the variability in such measurements could be explained by differences in mitochondrial ROS production. Conclusion: We conclude that the sperm mitochondria make a significant contribution to the oxidative stress experienced by defective human spermatozoa.

scholarly journals The protective effect of palm tocotrienol-rich fraction against H2O2- induced oxidative stress in neonatal rat cardiomyocytes

2017 ◽  
Author(s):  
Noor Shareena Aisha Abdul Khalid ◽  
Zakiah Jubri
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Mitochondrial Membrane ◽  
Neonatal Rat ◽  
Ros Production ◽  
Rat Cardiomyocytes ◽  
Apoptotic Gene ◽  
Neonatal Rat Cardiomyocytes ◽  
Ldh Activity ◽  
Tocotrienol Rich Fraction

Background: Oxidative stress plays an important role in the pathogenesis of heart diseases. Antioxidants such as palm tocotrienol-rich fraction (TRF) can reduce oxidative stress, hence preventing and reducing the risk of heart disease. This study was aimed to determine the protective effects of TRF against hydrogen peroxide (H2O2 ) - induced oxidative stress in neonatal rat cardiomyocytes (NRCM). Methods: The NRCM were divided into five groups: (1) control, (2) cells treated with TRF (10 µg/ml) for 24 hours, (3) cells subjected to H2O2 (0.5 mM ) for 30 minutes, (4) cells pre-treated with TRF, and (5) cells post-treated with TRF. The IC50 of H2O2 (0 – 5 mM ) and the effective dose of TRF (0 – 25 µg/ml) were determined using the MTS cell viability assay. Meanwhile, ELISA was used to measure the level of reactive oxygen species (ROS). The presence of superoxides and H2O2 were detected by dihydroethidium and 5-(and-6 ) - carboxy -2′,7′-dichlorodihydrofluorescein diacetate respectively. Flowcytometry analysis was conducted to determine the presence of apoptosis and measure the mitochondrial membrane potential, whereby the former involved the use of Annexin V-FITC stain while the latter JC-1 stain. The gene expressions of antioxidant (SOD, CAT, GPx) and apoptosis (Bax, Bcl-2, Caspase-3) enzymes were studied using qRT -PCR. Results: The IC50 of H2O2 was 0.5 mM while the effective dose of TRF 10 µg/ml. The cells which were subjected to H2O2 showed a decrease in NRCM viability and significant increase (p < 0.05) in ROS production. LDH activity and green fluorescence intensity (which indicated mitochondrial depolarisation ) were increased following H2O2 induction . With reference to the control, the H2O2- induced group had a higher percentage of late apoptotic cells, which was associated with the upregulation of the pro-apoptotic gene, Bax, and downregulation of the anti-apoptotic gene, Bcl-2 (p < 0.05). H2O2 also upregulated GPx expression , apart from downregulating CAT and Cu/Zn SOD expression (p < 0.05). The pre- and post-treatment groups had increased cell viability and reduced ROS production. Pre-treatment with TRF protected the cell membranes and mitochondria from H2O2- induced injury, as reflected by the reduction in extracellular LDH activity and apoptosis (the latter of which was associated with the downregulation of Bax). Meanwhile, the expression of GPx, Cat, and Cu/Zn SOD was reduced in the post-treatment group. Conclusion: By scavenging for ROS, palm TRF directly protects the cell membrane from H2O2- induced injury, leading to a decrease in oxidative stress. Thus, palm TRF maintains the mitochondrial membrane potential and prevents apoptosis secondary to decreased Bax expression.

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