| Alzheimer’s disease (AD) is the most common neurodegenerative disease in the elderly. The pathological features of AD include extracellular P-amyloid protein (Aβ) plaques and intracellular neurofibrillary tangles, accompanied by the loss of neurons and synapses. With the increasing of the world population aging, the incidence of AD is markedly growing, which has become one of the most serious problem of the geriatric medicine. However, the effective treatment method of AD still has not been found.Aβ abnormally deposits in the brain, which is the typical hallmark of AD. As a toxic factor, AP aggregation plays a critical role in the initiation phase of AD pathogenesis. Therefore, a large amount of therapeutic efforts have been focused on reducing the toxicity of Aβ and preventing the formation of Aβ oligomer. In recent years, more and more research on Chinese medicine treatment of AD have been reported. Resveratrol, a natural polyphenolic compound, is widely present in grapes, giant knotweed and other plants. It has long been reported that resveratrol possesses a wide range of biological activities, such as anti-oxidant, anti-inflammatory, anti-cancer and anti-aging effects in numerous organisms. Recently, resveratrol has also attracted the attention from neuroscientists because of its neuroprotective properties.Resveratrol triggers the overexpression of silence signal regulating fator1(SIRT1), a member of the sirtuin family, which is the homologue of silent information regulator factor sir-2and plays an essential role in regulating cellular functions, such as transcriptional silencing of telomeres and life-span extension. SIRT1is also involved in calorie restriction and aging. Recent study found that high expression of SIRT1can enhance the non-amyloidogenic amyloid precursor protein (APP) processing, and reduce the deposition of β-amyloid protein.A previous study showed that SIRT1overexpression in primary neurons enhances cell viability and reduces Aβ secretion and ROCK1expression, suggesting that SIRT1enhances a-secretase-mediated non-amyloidogenic APP processing partly via ROCK1signaling. However, the feedback loop between SIRT1and ROCK1remains unclear.To explore the AD treatment effect of resveratrol and the underlying mechanism, we designed the following experiments:(1) PC12cells were treated with Aβ25-35to establish cell model, which is a commonly used cell line models.(2) we examined the protective effect of resveratrol on the neurotoxic cell model. MTT and LDH assays were employed to determine the cell viability and filtered out the best concentration of protection.(3) Hoechst33342and pI double staining, flow cytometry with Annexin V-FITC/PI double staining and Intercellular calcium ([Ca+]i) were performed to detect the protective effect of resveratrol against Aβ25-35-(4) In order to investigate the protective mechanism of resveratrol, real time quantitative PCR and Western blot were performed to detect the expressions of SIRT1and ROCK1at both the mRNA and protein levels, respectively.(5) SIRT1inhibitor nicotinamide and ROCK.1inhibitor Y-27632were used to further whether SIRT1regulated the ROCK1expression in the neuroprotection against Aβ25-35neurotoxicity by resveratrol.Result shows:(1) At24h in vitro culture, PC12cells grew well and exerted some neurite; when exposed to Aβ25-35, the damage to cells was evident under microscope. The neurites slowly disappeared, and the network was collapsed. Aβ25-35was added into the medium at three doses (10,20and40μmol/L), and its cytotoxic effect became visible after the exposure to20μmol/L Aβ25-35for24h and48h, as evidenced by retracted neurites and some cell debris. Therefore, Aβ25-35of20μM was selected to further assess the protective mechanism of resveratrol. Resveratrol at12.5,25,50and100 μmol/L was added to PC12cells2h prior to the addition of20umol/L Aβ25-35, and the protective effect was compared among groups after24h of treatment. The results of morphologic observation, MTT and LDH showed that the protective effect of50μmol/L resveratrol was more obvious than the other groups (p<0.01), and which was selected for the research of resveratrol protection.(2) The result of Hoechst33342and PI showed that resveratrol significantly reduced the cell apoptosis induced by Aβ25-35(p<0.01). The results of flow cytometry with Annexin V-FITC/PI double staining showed that both early and late apoptosis were obviously inhibited by50μmol/L resveratrol (p<0.01). The lowest [Ca2+]i level was detected from the normal control group; The [Ca2+]i level was increased when exposed to Aβ25-35for24h, with many cells showing high level of green fluorescence intensity. In the presence of resveratrol, the [Ca+]i level was significantly decreased after24h (p<0.01). These data suggested that resveratrol prevents cell apoptosis and decreased the [Ca2+]i level induced by cytotoxic AP25-35.(3)The results of RT-PCR and Western blot were consistant. The SIRT1expression was significantly decreased and ROCK1expression was significantly increased by Aβ25-35(p<0.01). However, the expression of SIRT1and ROCK1were markedly reversed when PC12cells were incubated with resveratrol (p<0.01). These data implied that SIRT1and ROCK1partially participated in the neuroprotection of resveratrol against AP25-35injury. In order to further clear the relationship of SIRT1and ROCK1in the neuroprotective effect of resveratrol, SIRT1inhibitor nicotinamide and ROCK1inhibitors Y-27632were used in the study. The results showed that nicotinamide inhibited the SIRT1expression and activated the ROCK1expression (p<0.01), whereas Y-27632only inhibited the ROCK1expression. These data indicated that ROCK1was a downstream signal molecule and could be suppressed by SIRT1.Our results clearly revealed that resveratrol significantly protected PC12cells and inhibited the β-amyloid-induced cell apoptosis through the upregulation of SIRT1. Moreover, as a downstream signal molecule, ROCK1was negatively regulated by SIRT1. Taken together, our study demonstrated that SIRTl-ROCK1pathway played a critical role in the pathomechanism of AD. |
PDF Full Text Request