| A large body of evidence indicates that oxidative stress is a salient pathological feature in many neurodegenerative diseases, including Alzheimer's disease. In addition to signs of systemic oxidative stress, at the biochemical and neuropathological level, neuronal degeneration in these disorders has been shown to coincide with several markers of oxidative damage to lipids, nucleic acids, and proteins in affected brain regions. Given the pathogenic impact of oxidative stress therapeutic strategies aimed to blunt this processes is considered an effective way to confer neuroprotection.Alzheimer's disease (AD) is neuropathologically characterized by deposition of?-amyloid (A?) plaques and intracellular neurofibrillary tangles and loss of neurons in the brain. Although the cause of AD remains unclear, several lines of evidence suggest that A?-induced oxidative stress plays an important role in the pathogenesis or progression of AD. A?induces oxidative stress (OS), and OS promotes the production of A?. Recent studies showed that OS contributes to A?accumulation. A?, in turn, induces OS resulting in increased levels of?-and?-secretases, which further enhances A?production, thereby providing a vicious circle among A?production/accumulation, OS and?-/?-secretase for the cleavage of APP. Thus, one promising preventive or therapeutic intervention in AD may attenuate or suppress the oxidative stress-dependent, A?-mediated cytotoxicity. Therefore, numerous antioxidants, such as Huperzine A, Curcumin, Ginkgo biloba and Ginsenoside Rgl have been demonstrated to inhibit A?-induced neurotoxicity.Astaxanthin (ATX) is a red carotenoid pigment, which is mainly found in certainmarine animals and plants such as fish, shrimps and algae. It is a potent anti-oxidant without provitamin-A activity. ATX possesses many important biological functions. Of note, ATX has antioxidative activity. It is thought that ATX is an excellent candidate for testing with neurological diseases, including PD and AD. Indeed, it has been reported recently that ATX has neuroprotective effects. ATX inhibits 6-hydroxydopamine (6-OHDA)-induced neuronal apoptosis in vitro. ATX can reduce ischemia-related injury in brain tissue through the inhibition of oxidative stress, reduction of glutamate release, and anti-apoptosis. However, themechanisms by which ATX mediates its therapeutic effects in vitro are unclear, and remains an interesting speculation that awaits further investigation. Acteoside,2-(3,4-Dihydroxyphenyl)ethyl-0-R-L-rhamnopyranosyl-(1?3)-4-O-caffe oyl-?-D-glucopyranoside. was a phenylethanoid glycoside first extracted fromVerbascumsinuatumand named "verbascoside". The chemical structure of this corn-pound was elucidated by Birkofer, who also introduced the new name "acteoside". Previous studies have shown that acteoside has various pharmacological activities, such as anti-inflammatory, hepatoprotective). anti-apoptotic and antioxidative activities. Recently, it has been suggested that acteoside has neuroprotective activities. Acteoside inhibits neuronal death induced by 1-methyl-4-phenylpyridinium ion (MPP+) and glutamate. Although acteoside exhibited neuroprotective activity, the protective effect of acteoside against A?-induced cytotoxicity has not been reported.Heme oxygenase-1 (HO-1) activity protects against oxidative stress. Particularly interesting is the role played by HO-1 in AD, a neurodegenerative disorder that involves a chronic inflammatory response associated with both oxidative brain injury and A?-associated pathology. HO-1 protein levels are increased in AD patients and colocalize with?-amyloid in senile plaques, suggesting activetion of the antioxidant defence. Also, HO-1 is found in neurofibrillary tangles associated with Tau. Amyloid precursor protein (APP) overexpressing transgenic mice also reproduce the accumulation of HO-1 close to senile plaques, further demonstrating a relevant role in clinical and experimental AD. Astroglia associated with senile plaques in hippocampus appear to be particularly predisposed to HO-1 over-expression.These data and the observation that HO-1 is induced in response to?-amyloid in a dose-dependent manner suggest that HO-1 induction is an attempt to mitigate the oxidant injury associated with?-amyloid aggregates. Snyder and colleagues'studies implicate that HO1 overexpression does not influence the processing of APP into the AP peptides, familial alzheimer's disease (FAD) mutant forms of APP elicit substantially greater inhibition of HO proteins.HO-1 activity protects against oxidative stress. Particularly interesting is the role played by HO-1 in AD, a neurodegenerative disorder that involves a chronic inflammatory response associated with both oxidative brain injury and A?-associated pathology. HO-1 protein levels are increased in AD patients and colocalize with?-amyloid in senile plaques, suggesting activetion of the antioxidant defence. Also. HO-1 is found in neurofibrillary tangles associated with Tau. Astroglia associated with senile plaques in hippocampus appear to be particularly predisposed to HO-1 over-expression. Amyloid precursor protein (APP) overexpressing transgenic mice also reproduce the accumulation of HO-1 close to senile plaques, further demonstrating a relevant role in clinical and experimental AD. These data and the observation that HO-1 is induced in response to?-amyloid in a dose-dependent manner suggest that HO-1 induction is an attempt to mitigate the oxidant injury associated with?-amyloid aggregates. Snyder and colleagues'studies implicate that HO-1 overexpression does not influence the processing of APP into the A?peptides, FAD mutant forms of APP elicit substantially greater inhibition of HO proteins, thereby render the neuron more vulnerable to oxidative stress-induced toxicity. Therefore, we speculate that HO-1 may involve in the mechanism by which inhibit the A?-induced neurotoxicity, and the upregulation of HO-1 may pharmacological mechanism of natural compoundsThe protective role played by HO-1 and its products in AD raised new possibilities regarding the possible use of natural substances, which are able to increase HO-1 levels, as potential drugs for the prevention and treatment of AD.These and other results have stimulated the search for pharmacological compounds capable of up-regulating HO-1 expression, as such compounds would theoretically have potential as treatments for these and other neurologic disorders. In this light very promising, in this study, we investigated whether astaxanthin, a red carotenoid pigment, and Acteoside, a phenylethanoid glycoside upregulate HO-1 expression, thereby protecting the SH-SY5Y cells from A?25-35-induced oxidative cell death, and explored the underlying protective mechanisms. The results were summarized as follows:1 Astaxanthin (ATX) upregulates HO-1 expression through ERK1/2 pathway and its protective effect against beta-amyloid-induced cytotoxicity in SH-SY5Y cells(1) ATX protects against?-amyloid-induced neurotoxicity1) ATX ameliorated A?25-35-induced cell injury, ATX concentration-dependently inhibit A?25-35-induced SH-SY5Y cell viability loss.2) ATX prevented A?25-35-triggered apoptosis, ATX protected SH-SY5Y cells against A?25-35-induced cell apoptosis and death by Hoechst staining assay, and ATX dose-dependently inhibit the AP25-35-triggered activation of caspase-3.3) ATX ameliorated A?25-35-induced oxidative stress, the intracellular ROS was analyzed by H2DCF-DA assay, and DCF fluorescence results evaluated by luorescent spectrometer demonstrated that AP25-35 induced the production of ROS, whereas ATX ameliorated the A?25-35-induced ROS production.4) ATX inhibited AP25-35-induced dissipation of the mitochondrial membrane potential (MMP) monitored using the Rh123 in SH-SY5Y cells.5) A?25-35 induced the downregulation of Bcl-2/Bax ratio, while ATX pretreatment increase the Bcl-2/Bax ratio in a dose-dependent manner. 6) AP25.35 induced the activation of JNK and p38MAPK. while ATX concentration-dependently suppressed the A?25-35-induced activation of JNK and p38MAPK in SH-SY5Y cells.(2) Astaxanthin inhibited beta-amyloid-induced cytotoxicity by upregulating HO-1 expression.1) ATX induced HO-1 protein expression in a dose-dependent and time-dependent manner in vitro.2) A?25-35 concentration-dependently induced HO-1 protein expression and astaxanthin potentiate A?25-35-induced HO-1 protein expression.3) The inhibitor of HO-1 activity, ZnPP dose-dependently reversed the protective effect of astaxanthin against A?25-35-induced cytotoxicity in SH-SY5Y cells, suggesting that astaxanthin-induced HO-1 protects against A?25-35-mediated cytotoxicity in SH-SY5Y cells.4) ATX activated ERK1/2 in a dose-dependent and time-dependent manner SH-SY5Y cells, and the specific ERK inhibitor, PD98059, blocked the ATX-induced HO-1 expression in concentration-dependent manner. These results indicated that ERK kinase pathways involve in ATX-induced HO-1 expression in SH-SY5Y cells.5) The specific ERK inhibitor, PD98059. dose-dependently reversed ATX-mediated protection against the cytotoxic effect of AP25-35 challenge.2 Acteoside protects against?-amyloid-induced neurotoxicity by upregulatingNrf2-mediated HO-1 expression through ERK1/2 and PI3K/Akt pathway(1) Acteoside prevented A?25-35-induced neurotoxicity1)Acteoside ameliorates A?25-35-induced cell injury, acteoside concentration-dependently inhibit A?25-35-induced SH-SY5Y cell viability loss evaluated by the MTT reduction assay. Meanwhile, the protective effect of acteoside on A?25-35-induced neurotoxicity was also analyzed by calcein-AM assay, showing that acteoside pretreatment significantly attenuated the effects of A?25-35, i.e.the number of cells exhibiting bright green fluorescence diminished after AP25-35 treatment, and acteoside prevented the loss of fluorescence staining caused by A?25-35.2) Acteoside prevents A?25-35-induced apoptosis, pretreatment with acteoside decreased the number of apoptotic cells compared to cells treated with A?25-35 alone, and DNA fragmentation assay mirror the anti-apoptotic effect of acteoside.3) Acteoside inhibited the intracellular ROS was analyzed by H2DCF-DA assay, and DCF fluorescence results evaluated by luorescent spectrometer demonstrated that A?25-35 induced the production of ROS, whereas acteoside ameliorated the A?25-35-induced ROS production in SH-SY5Y cells, suggesting that acteoside prevented A?25-35-induced oxidative injury.4) Acteoside inhibited A?25-35-induced dissipation of the mitochondrial membrane potential (MMP) monitored using the Rh123 in SH-SY5Y cells5) A?25-35 induced the upregulation of Bax/Bcl-2 ratio, while acteoside pretreatment dose-dependently decrease the Bax/Bcl-2 ratio in SH-SY5Y cells.6) A?25-35-induced cytochrome c release. AP25-35 significantly increased the release of cytochrome c from mitochondria to the cytosol, and acteoside pretreatment inhibited the release of cytochrome c in a dose-dependent manner.7) A?25-35 induced the induction and activation of caspase-3 and acteoside inhibit the A?25-35-triggered activation of caspase-3 in a dose-dependent manner in SH-SY5Y cells.(2) Acteoside inhibited beta-amyloid-induced cytotoxicity by upregulating HO-1 expression.1) Western blot analysis indicated that acteoside induces HO-1 protein expression in a dose-dependent and time-dependent manner in PC 12 cells, and immunofluorescence labeling indicated that acteoside induced HO-1 protein expression, which was predominantly localized to the cytoplasm. Acteoside induces HO-1 protein expression in a time-dependent manner in vivo, and immunohistochemistry analysis indicated that HO-1 protein expression locate in the neuron.2) Western blot analysis indicated that acteoside increases the levels of Nrf2 and phosphorylated Nrf2; Western blot analysis indicated that acteoside dose-dependently and time-dependently stimulate a translocalization of Nrf2 protein from the cytosol to the nucleus, which was mirrored by immunofluorescence labeling in PC 12 cells.3) The acteoside-induced expression of HO-1 was markedly supressed by siRNA knock down of Nrf2 gene, indicating that the requirement of Nrf2 for acteoside-induced HO-1 expression.4) Acteoside activates Nrf2 via phosphorylation of ERK1/2 and Akt. Western blot analysis indicated that acteoside activate ERK1/2 and Akt in a time-dependent manner in PC 12 cells, and the specific ERK inhibitor, PD98059, the PI3K/Akt inhibitor, LY294002, inhibited the expression of Nrf2. PD98059 and LY294002 decreased nuclear Nrf2 accumulation occurred following treatment with acteoside by immunofluorescence labeling in PC 12 cells.5) PD98059 and LY294002 blocked the acteoside-induced HO-1 expression in concentration-dependent manner. This results indicated that ERK1/2 and PI3K/Akt pathways involve in ATX-induced HO-1 expression in PC12 cells.6) ZnPP, the inhibitor of HO-1 activity, dose-dependently reversed the protective effect of acteoside against A?25-35-induced cytotoxicity in PC 12 cells, suggesting that astaxanthin-induced HO-1 protects against A?25-35-mediated cytotoxicity in PC 12 cells.7) The PD98059 and LY294002 reversed acteoside-mediated protection against the cytotoxic effect of A?25-35 challenge.Taken together, our present study revealed that the specific activation of HO-1 gene expression by pharmacological modulation can inhibit A?25-35-induced neurotoxicity, providing a direct evidence that the involvement of upregulation of HO-1 against A?25-35-induced neurotoxicity. We for the first time identified two natural compounds, i.e. astaxanthin and acteoside can upregulate HO-1 expression, and these findings suggest that astaxanthin and acteoside protect against P-amyloid-induced neurotoxicity by upregulating HO-1 expression. Astaxanthin (ATX) upregulates heme oxygenase-1 expression through ERK1/2 pathway and its protective effect against beta-amyloid-induced cytotoxicity in SH-SY5Y cells, and Acteoside protects against?-amyloid-induced neurotoxicity by upregulating Nrf2-mediated HO-1 expression through ERK1/2 and PI3K/Akt pathway in PC 12 cells. The results presented here may define HO-1 as a potential therapeutic target in Alzheimer's Disease, and astaxanthin and acteoside, which are able to increase HO-1 levels, as potential drugs for the prevention and treatment of AD. |
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