Orexin-A Protects Human Neuroblastoma SH-SY5Y Cells Against 6-Hydroxydopamine-Induced Neurotoxicity: Involvement of PKC and PI3K Signaling Pathways.
Parkinson's disease (PD) is a common neurodegenerative disorder that is characterized by progressive and selective death of dopaminergic neurons. Multifunctional neuropeptide orexin-A is involved in many biological events of the body. It has been shown that orexin-A has protective effects in neurodegenerative disease such as PD. However, its cellular mechanisms have not yet been fully clarified. Here, we investigated the intracellular signaling pathway of orexin-A neuroprotection in 6-hydroxydopamine (6-OHDA)-induced SH-SY5H cells damage as an in vitro model of PD. The cells were incubated with 150 μM 6-OHDA, and the viability was examined by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay. Mitochondrial membrane potential and intracellular calcium were measured by fluorescent probes. Western blotting was also used to determine cyclooxygenase type 2 (COX-2), nuclear factor erythroid 2 related factor 2 (Nrf2), and HSP70 protein levels. The data showed that 6-OHDA has decreasing effects on cell viability, Nrf2, and HSP70 protein expression and increases the level of mitochondrial membrane potential, intracellular calcium, and COX-2 protein. Orexin-A (500 pM) significantly attenuated the 6-OHDA-induced cell damage. Furthermore, Orexin-A significantly prevented the mentioned effects of 6-OHDA on SH-SY5Y cells. Orexin 1 receptor antagonist (SB3344867), PKC, and PI3-kinase (PI3K) inhibitors (chelerythrin and LY294002, respectively) could suppress the orexin-A neuroprotective effect. In contrast, blockage of PKA by a selective inhibitor (KT5720) had no effects on the orexin protection. The results suggest that orexin-A protective effects against 6-OHDA-induced neurotoxicity are performed via its receptors, PKC and PI3K signaling pathways.
1 Department of Biology, Faculty of Sciences, ShahidBahonar University of Kerman , Kerman, Iran .
Hamzeh
H
Pasban-Aliabadi
1 Department of Biology, Faculty of Sciences, ShahidBahonar University of Kerman , Kerman, Iran .|2 Laboratory of Molecular Neuroscience, Kerman Neuroscience Research Center (KNRC), Kerman University of Medical Sciences , Kerman, Iran .
Saeed
S
Esmaeili-Mahani
1 Department of Biology, Faculty of Sciences, ShahidBahonar University of Kerman , Kerman, Iran .
Mehdi
M
Abbasnejad
D053119:Benzophenanthridines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002867:Chromones / Q000494:pharmacology; D051546:Cyclooxygenase 2 / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009025:Morpholines / Q000494:pharmacology; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D009447:Neuroblastoma / Q000473:pathology*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009498:Neurotoxins / Q000633:toxicity*; D064446:Orexin Receptors / Q000378:metabolism; D000068797:Orexins / Q000494:pharmacology*; D016627:Oxidopamine; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*
D053119:Benzophenanthridines; D002227:Carbazoles; D002867:Chromones; D018840:HSP70 Heat-Shock Proteins; D009025:Morpholines; D051267:NF-E2-Related Factor 2; D018696:Neuroprotective Agents; D009498:Neurotoxins; D064446:Orexin Receptors; D000068797:Orexins; D047428:Protein Kinase Inhibitors; D011758:Pyrroles; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C057416:KT 5720; D016627:Oxidopamine; C016299:chelerythrine; D051546:Cyclooxygenase 2; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D002118:Calcium
6-OHDA; PI3K; PKC; Parkinson's disease; SH-SY5H cells; orexin-A