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Shikonin Protects Dopaminergic Cell Line PC12 Against 6-Hydroxydopamine-Mediated Neurotoxicity Via Both Glutathione-Dependent and Independent Pathways and by Inhibiting Apoptosis

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Abstract

We have investigated the mechanism of shikonin function on protection of dopaminergic neurons against 6-OHDA-induced neurotoxicity. Treatment of rat pheochromocytoma cell line PC12 by serial dilutions of shikonin determined 10 μM of the compound as its optimum concentration for protection saving nearly 70 % of the cells against toxicity. Reverse transcription-PCR analysis of shikonin-treated cells showed threefold increase in mRNA levels of glutathione peroxidase-1 (GPX-1) as a representative component of the intracellular anti-oxidant defense system. To elucidate shikonin-GPX1 relationships and maximize protection, we transduced PC12 cells using recombinant lentivirus vectors that harbored GPX-1 coding sequence. This change upregulated GPX-1 expression, increased peroxidase activity and made neuronal cells resistant to 6-OHDA–mediated toxicity. More importantly, addition of shikonin to GPX1-overexpressing PC12 cells augmented GPX-1 protein content by eightfold leading to fivefold increase of enzymatic activity, 91 % cell survival against neurotoxicity and concomitant increases in intracellular glutathione (GSH) levels. Depletion of intracellular GSH rendered all cell groups highly susceptible to toxicity; however, shikonin was capable of partially saving them. Subsequently, GSH-independent superoxide dismutase mRNA was found upregulated by shikonin. As signs of apoptosis inhibition, the compound upregulated Bcl-2, downregulated Bax, and prevented cell nuclei from undergoing morphological changes typical of apoptosis. Also, a co-staining method demonstrated GPX-1 overexpression significantly increases the percent of live cells that is maximized by shikonin treatment. Our data indicate that shikonin as an antioxidant compound protects dopaminergic neurons against 6-OHDA toxicity and enhances their survival via both glutathione-dependent and direct anti-apoptotic pathways.

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Acknowledgments

This study was supported by a Grant (420) from NIGEB.

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The authors declare no conflict of interest

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Authors and Affiliations

  1. National Institute of Genetic Engineering and Biotechnology (NIGEB), Pazhoohesh Blvd, Tehran-Karaj HWY Kilometer 15, PO Box 14965/161, Tehran, I. R. Iran

    Emran Esmaeilzadeh, Mossa Gardaneh, Ehsan Gharib & Farzaneh Sabouni

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  1. Emran Esmaeilzadeh
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  2. Mossa Gardaneh
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  3. Ehsan Gharib
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  4. Farzaneh Sabouni
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Correspondence to Mossa Gardaneh.

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Esmaeilzadeh, E., Gardaneh, M., Gharib, E. et al. Shikonin Protects Dopaminergic Cell Line PC12 Against 6-Hydroxydopamine-Mediated Neurotoxicity Via Both Glutathione-Dependent and Independent Pathways and by Inhibiting Apoptosis. Neurochem Res 38, 1590–1604 (2013). https://doi.org/10.1007/s11064-013-1061-9

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  • DOI: https://doi.org/10.1007/s11064-013-1061-9

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