| Summary: | 博士 === 國立中興大學 === 食品暨應用生物科技學系所 === 98 === Hydrogen peroxide (H2O2) induces cellular oxidative stress associated with neurodegenerative diseases. The main citrus flavonoids such as hesperidin (Hdn), hesperetin (Htn), and neohesperidin are known to exhibit antioxidant activities and could traverse the blood-brain barrier. Hence, in this study, we first evaluated the neuroprotection of these flavanones against H2O2-induced cytotoxicity in PC12 cells. The results demonstrated for the first time that at both physiological (0.8-4 μM) and high (20-50 μM) concentrations, the flavanones exhibited multiple mechanisms including inhibition of caspase-3 activation, calcium homeostasis and mitochondrial potential maintenances, alleviating membrane and DNA damages, scavenging ROS, and enhancing activities of antioxidant enzymes to protect the cells from oxidative damage. These dietary antioxidants thus are potential candidates for intervening in neurodegenerative diseases.
Biological activities of dietary flavonoids have been attributed to their antioxidant and signaling properties. However, their physiological benefits might come from their metabolites. Therefore we further studied the effects of Htn (a metabolite of Hdn) and its structural counterparts, isorhamnetin (a metabolite of quercetin) and isosakuranetin (a metabolite of naringin), on kinases related to survival signaling as well as other cytoprotective actions. Based on their structural differences and the concentration (0.8 or 50 μM) used, these flavonoids differentially activated pro-survival signaling molecules, including Akt/protein kinase B, p38 mitogen-activated protein kinase, and inhibited the activation of c-jun N-terminal kinase, which triggers apoptosis. Our results demonstrate that signaling actions of these flavonoids are involved in their neuroprotection against oxidative stress and that they act more as signaling molecules than antioxidants.
Htn is known to activate estrogen receptor (ER). Physiological concentration of estrogen could mediate its neuroprotective actions via both ER and tyrosine kinase receptors (Trks) signaling. We next tested whether Htn (0.1, 1 and 50 μM) protected PC12 cells from H2O2-induced oxidative damage via ER- and/or TrkA-mediated actions. We found that only low concentration (0.1 or 1 μM) of Htn via ER- and TrkA-mediated actions to inhibit increases of ROS, intracellular calcium level, and caspase-3 activity in the cells. The later two were more dependent on TrkA-mediated actions in 1 μM Htn treated cells. Meanwhile Htn (1 μM) stimulated the activation of Akt, extracellular signal-regulated kinase (ERK), and cAMP response element-binding protein (CREB) as well as induced brain-derived neurotrophic factor, PPARγ coactivator 1α(PGC-1α) and seladin-1 (selective Alzheimer’s disease indicator-1) via both ER and TrkA in the cells. These proteins are known to exhibit beneficial activities of anti-apoptosis, anti-oxidative damage and anti-amyloid-induced neurotoxicity. Thus neuroprotective effects of Htn, at low concentrations, are attributed to its stimulation on receptor signaling.
It is known that TrkA triggers MAPK/ERK, PI-3K/Akt, phospholipaseγ(PLCγ)/PKC and cAMP/PKA pathways; membrane ER triggers MAPK/ERK, PKA, Akt/PKB or PKC pathway. We finally investigated whether Htn induced targeted proteins, PGC-1α (regulated by CREB) and seladin-1 (regulated by ER), as well as triggers signaling pathways mentioned above in PC12 cells under normal culturing condition. The results showed that Htn induced rapid (15 min) and sustained (∼24 h) expressions of the targeted proteins. Htn also activated PI-3K, PKA, PKC, MAPK/ERK and CREB as well as induced targeted proteins via both ER and TrkA. Such Htn triggering ER- and TrkA-mediated parallel pathways could converge on different transcriptional factors and promote their protein regulation. This novel mechanism explains why Htn known with relatively low antioxidant and estrogen activities could exhibit multiple actions of neuroprotection in studies of this dissertation. It broadens the approach to using dietary flavonoids for its intervention in neurodegenerative disorders as well.
This study reveals for the first time that citrus flavonoids possess multiple mechanisms of neuroprotection against oxidative stress. Such action might be more attributed to their signaling regulations, especially to those mediated by receptors as physiological concentrations of flavonoids used. It also suggests that using dietary flavonoids with low dose and cytotoxicity as well as no side effects for the intervention in neurodegenerative disorders is promising.
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