| Alzheimer's disease(AD)is the most common degenerative neurological disease,of which the pathogenesis is not yet fully understood.Studies have shown that there are a large number of A?,especially A?42 deposition in the brain of AD patients.A? is one of the key factors induce and promote the pathogenesis of AD.However,the mechanism underlying neurotoxicity of A?42 is still undefined,though the studies proved it could cause oxidative stress and inflammatory response.Calycosin is extracted from traditional Chinese medicine Astragalus membranaceus,which has anti-oxidative,anti-osteoporosis,anti-tumor and immunoregulatory activity.However,whether or not Calycosin can reduce the neurotoxicity of A? has not been reported.This study was divided into three parts to explore the protective effects of Calycosin on AD and the underlying mechanism in vitro and in vivo.Part ?: Calycosin attenuate A? induced oxidative stress and inflammation in PC12 cellsIn this part of study,we observed the effects of calycosin treatment on oxidative stress,neuroinflammation and cell viablity in PC12 cells exposed to A?42 in vitro.The results showed that A?42 could induce the production of reactive oxygen species(ROS)in PC12 cells,which was confirmed by DCFH-DA.And calycosin can reduce the ROS content in PC12 cells and down-regulate the mitochondrial membrane potential damage caused by A?42.Moreover,the calycosin can up-regulate SOD activity and down-regulate the level of MDA.The results indicated that the calycosin had the effect of reducing the oxidative stress in PC12 cells induced by A?42.The expression of TNF-? and IL-1? in PC12 cells was significantly increased by the treatment of A?42by q RT-PCR and ELISA.The expression and release of inflammatory factors in PC12 cells induced by A?42 were significantly decreased by calycosin in a dose-dependent manner,indicating that calycosin could alleviate the inflammatory response inducedby A?42 in vitro.At the end of this study,CCK-8 and LDH activity and apoptosis detection were used to detect A?42-mediated cell injury and apoptosis,and the results showed that calycosin could protect PC12 cells from A?42-induced neurotoxicity in a dose-dependent manner.In summary,the results of this study show that calycosin can protect A?42-exposed PC12 cells by reducing A?42-induced oxidative stress and neuroinflammatory response.However,the mechanism by which the calycosin played a neuroprotective effect is not yet fully understood.In the second part of this study,the mechanism underlying antioxidant and anti-inflammatory effects of calycosin was further explored.Part ? Calycosin modulates oxidative stress and inflammation in PC12 cells via PKC/Nrf2 pathwayIn this part of the study,we further explored on the mechanism underlying antioxidant and anti-inflammatory effects of calycosin.It was found that both low-dose and high-dose calycosin had no significant effect on the expression of Nrf2 in PC12 cells.However,the expression of Nrf2 in the nucleus was significantly increased after stimulation with calycosin in a dose-dependent manner.Knockdown of Nrf2 in PC12 cells via RNA interference significantly weakened the antioxidant and anti-inflammatory ability of calycosin,as well as the neuroprotective effect of calycosin.A PKC-specific inhibitor,calphostin C,inhibited the effect of calycosin on Nrf2 translocation.Furthermore,calphostin C also inhibited the effect of calycosin on membrane potential,MDA level and the inflammatory cytokines TNF-? and IL-1?induced by A?,and attenuated the protective effect of calycosin on cell viability and apoptosis.In summary,the results show that calycosin can promote the activation of Nrf2 through PKC signaling pathway,leading to antioxidant and anti-inflammatory ability to protect PC12 cells exposed to A?.However,in vitro studies do not adequately address the therapeutic effect of calycosin on AD.Therefore,in the next part of the study,we will further observe and explore the effect of calycosin on oxidative stress,inflammatory response and memory learning ability in APP/PS1 mice.Part ?: Therapeutic effects of Calycosin on APP/PS1 mice and the underlying mechanismBased on its anti-oxidant and estrogenic properties,we tested the effects of chronic calycosin administration at three different doses on various behavioural and neuropathological markers in transgenic AD mice.Endpoints of the study were motor activity(rotarod),locomotion(actophotometer),learning,memory(Morris water maze and active avoidance),hippocampal amyloid beta,tau protein,IL-1?,TNF-?,malondialdehyde(MDA),and glutathione(GSH)levels.Untreated transgenic AD mice showed impaired learning and memory along with hippocampal amyloid beta and tau protein deposition,as well as elevated pro-inflammatory mediators(increased IL-1? and TNF-?)and signs of oxidative stress(increased MDA and decreased GSH).The administration of calycosin rectified in a dose-dependent manner the learning and memory impairments,as well as the various pathological markers in brain and peripheral tissues of the AD mice.In addition,these beneficial effects of calycosin in transgenic AD animals were significantly abolished by co-administration of calphostin C,a selective protein kinase C(PKC)inhibitor.We conclude that calycosin rescues many aspects of brain function in AD model mice via activation of the PKC pathway,and may have therapeutic potential for the treatment of AD.. |
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