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

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.

Curcumin ameliorates experimental autoimmune encephalomyelitis in a C57BL/6 mouse model

“…NADPH oxidation was determined based on a method, described by St. Clair and Chow (St Clair & Chow, 1996), which has been reported in our previous study (Esmaeilzadeh, Gardaneh, Gharib, & Sabouni, 2013). NADPH oxidation was determined based on a method, described by St. Clair and Chow (St Clair & Chow, 1996), which has been reported in our previous study (Esmaeilzadeh, Gardaneh, Gharib, & Sabouni, 2013).…”

“…The level of NADPH oxidation is reflected as an activity indicator for a group of peroxidase enzymes, including GPX-1. NADPH oxidation was determined based on a method, described by St. Clair and Chow (St Clair & Chow, 1996), which has been reported in our previous study (Esmaeilzadeh, Gardaneh, Gharib, & Sabouni, 2013). Briefly, the tissue samples first incubated in a solution that contained glutathione, sodium phosphate buffer (pH 7.2), EDTA, and glutathione reductase for 4 min.…”

Complementation of dopaminergic signaling by Pitx3–GDNF synergy induces dopamine secretion by multipotent Ntera2 cells

“…22 3 | RESULTS Figure 1A shows a panel of our viral constructs, which we used to generate recombinant lentiviruses. 22 3 | RESULTS Figure 1A shows a panel of our viral constructs, which we used to generate recombinant lentiviruses.…”

“…22 Polymerase chain reaction (PCR) products were size-fractionated by 2% agarose gel electrophoresis. 22 Polymerase chain reaction (PCR) products were size-fractionated by 2% agarose gel electrophoresis.…”

“…RNA extraction, reverse transcription, and complementary DNA amplification were performed as standard method. 22 Polymerase chain reaction (PCR) products were size-fractionated by 2% agarose gel electrophoresis. We analyzed the images for the density of DNA bands using UN-Scan-It Gel Analysis Software (Skill Scientific, Inc, Orem, Utah) as described before.…”