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      Isothiocyanate from Moringa oleifera seeds mitigates hydrogen peroxide-induced cytotoxicity and preserved morphological features of human neuronal cells

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          Abstract

          Reactive oxygen species are well known for induction of oxidative stress conditions through oxidation of vital biomarkers leading to cellular death via apoptosis and other process, thereby causing devastative effects on the host organs. This effect is believed to be linked with pathological alterations seen in several neurodegenerative disease conditions. Many phytochemical compounds proved to have robust antioxidant activities that deterred cells against cytotoxic stress environment, thus protect apoptotic cell death. In view of that we studied the potential of glucomoringin-isothiocyanate (GMG-ITC) or moringin to mitigate the process that lead to neurodegeneration in various ways. Neuroprotective effect of GMG-ITC was performed on retinoic acid (RA) induced differentiated neuroblastoma cells (SHSY5Y) via cell viability assay, flow cytometry analysis and fluorescence microscopy by means of acridine orange and propidium iodide double staining, to evaluate the anti-apoptotic activity and morphology conservation ability of the compound. Additionally, neurite surface integrity and ultrastructural analysis were carried out by means of scanning and transmission electron microscopy to assess the orientation of surface and internal features of the treated neuronal cells. GMG-ITC pre-treated neuron cells showed significant resistance to H 2O 2-induced apoptotic cell death, revealing high level of protection by the compound. Increase of intracellular oxidative stress induced by H 2O 2 was mitigated by GMG-ITC. Thus, pre-treatment with the compound conferred significant protection to cytoskeleton and cytoplasmic inclusion coupled with conservation of surface morphological features and general integrity of neuronal cells. Therefore, the collective findings in the presence study indicated the potentials of GMG-ITC to protect the integrity of neuron cells against induced oxidative-stress related cytotoxic processes, the hallmark of neurodegenerative diseases.

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          Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research.

          Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line which has been used as an in vitro model for neurotoxicity experiments. Although the neuroblastoma is usually differentiated by all-trans-retinoic acid (RA), both RA-differentiated and undifferentiated SH-SY5Y cells have been used in neuroscience research. However, the changes in neuronal properties triggered by RA as well as the subsequent responsiveness to neurotoxins have not been comprehensively studied. Therefore, we aim to re-evaluate the differentiation property of RA on this cell line. We hypothesize that modulation of signaling pathways and neuronal properties during RA-mediated differentiation in SH-SY5Y cells can affect their susceptibility to neurotoxins. The differentiation property of RA was confirmed by showing an extensive outgrowth of neurites, increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin and synaptic associated protein-97, and decreased expression of inhibitor of differentiation-1. While undifferentiated SH-SY5Y cells were susceptible to 6-OHDA and MPP+, RA-differentiation conferred SH-SY5Y cells higher tolerance, potentially by up-regulating survival signaling, including Akt pathway as inhibition of Akt removed RA-induced neuroprotection against 6-OHDA. As a result, the real toxicity cannot be revealed in RA-differentiated cells. Therefore, undifferentiated SH-SY5Y is more appropriate for studying neurotoxicity or neuroprotection in experimental Parkinson's disease research.
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            Comparison between proliferative and neuron-like SH-SY5Y cells as an in vitro model for Parkinson disease studies.

            The molecular mechanisms underlying the cellular lost found in the nigrostriatal pathway during the progression of Parkinson's disease (PD) are not completely understood. Human neuroblastoma cell line SH-SY5Y challenged with 6-hydroxydopamine (6-OHDA) has been widely used as an in vitro model for PD. Although this cell line differentiates to dopaminergic neuron-like cells in response to low serum and retinoic acid (RA) treatment, there are few studies investigating the differences between proliferative and RA-differentiated SH-SY5Y cells. Here we evaluate morphological and biochemical changes which occurs during the differentiation of SH-SY5Y cells, and their responsiveness to 6-OHDA toxicity. Exponentially growing SH-SY5Y cells were maintained with DMEM/F12 medium plus 10% of fetal bovine serum (FBS). Differentiation was triggered by the combination of 10 microM RA plus 1% of FBS during 4, 7 and 10 days in culture. We found that SH-SY5Y cells differentiated for 7 days show an increase immunocontent of several relevant neuronal markers with the concomitant decrease in non-differentiated cell marker. Moreover, cells became two-fold more sensitive to 6-OHDA toxicity during the differentiation process. Time course experiments showed loss of mitochondrial membrane potential triggered by 6-OHDA (mitochondrial dysfunction parameter), which firstly occurs in proliferative than neuron-like differentiated cells. This finding could be related to the increase in the immunocontent of the neuroprotective protein DJ-1 during differentiation. Our data suggest that SH-SY5Y cells differentiated by 7 days with the protocol described here represent a more suitable experimental model for studying the molecular and cellular mechanisms underlying the pathophysiology of PD. (c) 2010 Elsevier B.V. All rights reserved.
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              Increased Reactive Oxygen Species Formation and Oxidative Stress in Rheumatoid Arthritis

              Background Rheumatoid arthritis (RA) is an autoimmune inflammatory disorder. Highly reactive oxygen free radicals are believed to be involved in the pathogenesis of the disease. In this study, RA patients were sub-grouped depending upon the presence or absence of rheumatoid factor, disease activity score and disease duration. RA Patients (120) and healthy controls (53) were evaluated for the oxidant—antioxidant status by monitoring ROS production, biomarkers of lipid peroxidation, protein oxidation and DNA damage. The level of various enzymatic and non-enzymatic antioxidants was also monitored. Correlation analysis was also performed for analysing the association between ROS and various other parameters. Methods Intracellular ROS formation, lipid peroxidation (MDA level), protein oxidation (carbonyl level and thiol level) and DNA damage were detected in the blood of RA patients. Antioxidant status was evaluated by FRAP assay, DPPH reduction assay and enzymatic (SOD, catalase, GST, GR) and non-enzymatic (vitamin C and GSH) antioxidants. Results RA patients showed a higher ROS production, increased lipid peroxidation, protein oxidation and DNA damage. A significant decline in the ferric reducing ability, DPPH radical quenching ability and the levels of antioxidants has also been observed. Significant correlation has been found between ROS and various other parameters studied. Conclusion RA patients showed a marked increase in ROS formation, lipid peroxidation, protein oxidation, DNA damage and decrease in the activity of antioxidant defence system leading to oxidative stress which may contribute to tissue damage and hence to the chronicity of the disease.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: MethodologyRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: ResourcesRole: SupervisionRole: Validation
                Role: ConceptualizationRole: Data curationRole: MethodologyRole: ResourcesRole: SupervisionRole: Validation
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                3 May 2018
                2018
                : 13
                : 5
                : e0196403
                Affiliations
                [1 ] UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia
                [2 ] Department of Biochemistry, Kaduna State University, Main Campus, Kaduna, Nigeria
                [3 ] Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia
                [4 ] Laboratory of Natural Product, Institute of Bioscience, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia
                [5 ] Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia
                [6 ] Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia
                [7 ] Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia
                [8 ] Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia
                University of PECS Medical School, HUNGARY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                ‡ These authors also contributed equally to this work.

                Author information
                http://orcid.org/0000-0002-6635-4931
                http://orcid.org/0000-0001-9205-3764
                Article
                PONE-D-18-05240
                10.1371/journal.pone.0196403
                5933767
                29723199
                9ba1abc1-bb0b-4275-a121-4f93f7968d83
                © 2018 Jaafaru et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 16 February 2018
                : 12 April 2018
                Page count
                Figures: 7, Tables: 0, Pages: 17
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100004530, Universiti Putra Malaysia;
                Award ID: GP-IPS (Vot no. 9537300)
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100004530, Universiti Putra Malaysia;
                Award ID: GP (Vot no.9628600)
                Award Recipient :
                The work was funded by Universiti Putra Malaysia through GP-IPS (Vot no. 9537300) and GP (Vot no.9628600) to Ahmad Faizal Abdull Razis (AFAR); http://www.rmc.upm.edu.my/. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Developmental Biology
                Cell Differentiation
                Neuronal Differentiation
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cell Death
                Apoptosis
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Neurons
                Biology and Life Sciences
                Neuroscience
                Cellular Neuroscience
                Neurons
                Biology and Life Sciences
                Developmental Biology
                Cell Differentiation
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Neurons
                Neuronal Dendrites
                Neurites
                Biology and Life Sciences
                Neuroscience
                Cellular Neuroscience
                Neurons
                Neuronal Dendrites
                Neurites
                Research and Analysis Methods
                Specimen Preparation and Treatment
                Staining
                Cell Staining
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cell Death
                Neuronal Death
                Research and Analysis Methods
                Microscopy
                Light Microscopy
                Fluorescence Microscopy
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                All relevant data are within the paper.

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