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NEUROPHARMACOLOGY

Chondroitin Sulfate Protects SH-SY5Y Cells from Oxidative Stress by Inducing Heme Oxygenase-1 via Phosphatidylinositol 3-Kinase/Akt

Instituto Teófilo Hernando and Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain (N.C., T.V., M.V., A.G.G., M.G.L.); Servicio de Farmacología Clínica, Hospital Universitario la Princesa, Madrid, Spain (A.G.G.); Instituto Universitario de Investigación Gerontológico y Metabólica, Hospital de la Princesa, Madrid, Spain (M.G.L., A.G.G.); and Bioibérica SA, Barcelona, Spain (E.M., J.V.)

We investigated the mechanism of the neuroprotective properties of chondroitin sulfate (CS), an endogenous perineuronal net glycosaminoglycan, in human neuroblastoma SH-SY5Y cells subjected to oxidative stress. Preincubation with CS for 24 h afforded concentration-dependent protection against H₂O₂-induced toxicity (50 µM for 24 h) measured as lactic dehydrogenase released to the incubation media; cell death was prevented at the concentrations of 600 and 1000 µM. Cell death caused by a combination of 10 µM rotenone plus 1 µM oligomycin-A (Rot/oligo) was also reduced by CS at concentrations ranging from 0.3 to 100 µM; in this toxicity model, maximum protection was achieved at 3 µM (48%). No significant protection was observed in a cell death model of Ca²⁺ overload (70 mM K⁺, for 24 h). H₂O₂ and Rot/oligo generated reactive oxygen species (ROS) measured as an increase in the fluorescence of dichlorofluorescein diacetate-loaded cells. CS drastically reduced ROS generation induced by both H₂O₂ (extracellular ROS) and Rot/oligo (intracellular ROS). CS also increased the expression of phosphorylated Akt and heme oxygenase-1 by 2-fold. The protective effects of CS were prevented by chelerythrine, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), cycloheximide, and Sn(IV)-protoporphyrin IX. Taken together, these results show that CS can protect SH-SY5Y cells under oxidative stress conditions by activating protein kinase C, which phosphorylates Akt that, via the phosphatidylinositol 3-kinase/Akt pathway, induces the synthesis of the antioxidant protein heme oxygenase-1.

Address correspondence to: Dr. Manuela G. López, Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo 4, E-28029 Madrid, Spain, E-mail: manuela.garcia{at}uam.es