Citrus polymethoxyflavone,5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone, promotes neuronal differentiation through cAMP/PKA/CREB signaling pathway in PC12 cells

• Main Authors: Hui-Chi Lai, 賴慧綺
• Other Authors: Jui-Hung Yen
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/21724320751442339880

碩士 === 慈濟大學 === 分子生物暨人類遺傳學系碩士班 === 99 === Neurodegenerative diseases such as Alzheimer’s disease (AD) are characterized by the progressive degeneration, dysfunction and loss of neuronal cells in the nerve system. Patients with neuronal dysfunction in the brain accompanied losses of cognition and learning memory. Recently, several evidences suggest that the neurotrophic action of dietary flavonoids, such as citrus flavonoids, may transverse the blood-brain-barrier (BBB) and exert beneficial effects on neurogenesis, neuronal differentiation, neuronal survival, and may prevent cognitive losses associated with neurodegeneration. The aim of this study is to investigate the neurotrophic effects and mechanism of 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-OH-HxMF), a citrus hydroxyl polymethoxyflavone (PMF), on promoting neuronal cell neurite outgrowth and differentiation. In the present study, we found that 5-OH-HxMF significantly induces neurite outgrowth along with increased mRNA and protein expression of the neuronal cell differentiation marker, growth-associated protein-43 (GAP-43), in PC12 cells. These results indicated that 5-OH-HxMF could promote neuronal cell differentiation. It is known that activation of cAMP response element binding protein (CREB) may play an essential role in neuronal cell differentiation, we investigated whether 5-OH-HxMF activate CREB in PC12 cells. We found that 5-OH-HxMF stimulated phosphorylation of CREB protein and activated the transcription of cAMP response element (CRE)-mediated luciferase reporter gene, which was inhibited by KG-501, a specific antagonist for the CREB-CBP complex. To identify the molecular signaling pathways involved in the CREB activation, the pathway selectively specific inhibitors including U0126, Bisindolylmaleimide I, LY294002, KN62, SQ22536 and H-89 were used. Our results show that 5-OH-HxMF induced CREB activation significantly attenuated by adenylate cyclase inhibitor (SQ22536) and protein kinase A (PKA) inhibitor (H-89), respectively. Moreover, we also found that CREB activation is independent on the ERK / MAPK, PI3-K, PKC and CaMK II signaling pathways. Our results indicated that 5-OH-HxMF stimulated CREB phosphorylation through the cAMP/PKA pathway. Consistently, our results show that 5-OH-HxMF increased the level of intracellular cAMP rapidly and downstream component, PKA activity in PC12 cells. Finally, we found that the adenylate cyclase inhibitor SQ22536 and the PKA inhibitor H-89 significantly blocked the potentiation of 5-OH-HxMF-induced neurite outgrowth. In conclusion, the above findings reveal that 5-OH-HxMF possessed the neurotrophic action for promoting neurite outgrowth and neuronal cell differentiation. 5-OH-HxMF induced neurite outgrowth via increases of intracellular cAMP levels and PKA activity, further enhanced CREB phosphorylation and CRE dependent transcription in PC12 cells. This study thus enhances our understanding on 5-OH-HxMF may be beneficial for prevention and therapeutic use for neurodegenerative disorders.