| Alzheimer’s disease is the most common neurodegenerative disease, presented progressive change. Major clinical manifestations of AD are cognitive impairment, memory impairment and abnormal behavior. AD is characterized by the formation of β-amyloid plaques, neurofibrillary tangles and the loss of neurons and synapses. Many studies have shown that Aβ, as the core of the common pathways of AD, is a key factor in the occurrence and development of AD. Therefore, the study on various influencing factors around Aβ in the development process of AD and its regulation mechanism has become a hot spot in life sciences. Anthocyanins is a category indican derivatives widespread in plant foods. Many studies have shown that anthocyanins have strong antioxidant activity and neuroprotective effects, but its mechanism is obscure. Epigenetics and autophagy mechanisms have been proved to be associated with synaptic loss, learning and memory impairment during the process of AD. Based on that, we put forward a hypothesis that “anthocyanins may play a neuroprotective role in AD by regulating the expression of the epigenetic and autophagy related proteinsâ€.ObjectivesTo observe the effects of the blueberry extracts in the perinatal period on cognitive dysfunction in the APP/PS1 transgenic mice; and the protective effects of Cyanidin-3-O-β-glucoside against injury induced by Aβ25-35 in SH-SY5 Y cells. To explore the modulating mechanism of epigenetics and autophagy of anthocyanins in AD.Methods1. In vivo experiment: Twenty-eight adult APP/PS1 mice and six wild mice with agematched were cohabitated to get pregnant mice. APP/PS1 pregnant mice were divided into positive control group(AD group) and blueberry intervention group(AD+BB group); the wild pregnant mice were negative control group(CT group).Since the pregnant on the first day, the mice in CT and AD group were fed AIN-93 diet until grown up to the offspring weaning(21 d); The mice in AD+BB group mice were given AIN-93 diet containing 2% blueberry extracts until grown up to the offspring weaning(21d). The positive offspring of AD group and AD+BB group, the negative control group all offspring are chosen to be the experimental objects. After the intervention, the offspring growing until 9 months are tested. The ability of learning and memory of mice was evaluated by Morris water maze test including the directional navigation experiment and the space exploration experiment. The pathological changes of hippocampus and cortex in mice were observed by HE and immunohistochemical staining method.The brain tissue content of Aβ1-42 was analyzed by ELISA Kit. And the cell apoptosis of hippocampus and cortex in mice were examined by TUNEL staining method. The protein expression of Caspase-3,LC3 B,Beclin-1,Mecp2 and AcH3(Lys9)in mice brain tissues were analyzed by Western blot.2. In vitro experiment: SH-SY5 Y cells were cultured in vitro. First, SH-SY5 Y cells were divided into control group and Aβ25-35-treated groups(the Aβ25-35 concentrations are 5, 10, 15, 20, 25μmol/L, respectively). Second, SH-SY5 Y cells were divided into control group, Aβ25-35-treated group and Cy-3G pre-treatment groups(the Cy-3G concentrations are 10, 25, 50, 100μmol/L, respectively). Third, SH-SY5 Y cells were divided into control group, Aβ25-35-treated group and Cy-3G pre-treatment groups(the time of intervention are 3, 6, 12, 24 h, respectively). Finally, SH-SY5 Y cells were divided into control group, Aβ25-35 group, Aβ25-35+Cy-3G group and Cy-3G group.Cell viability was measured by MTT assay; Apoptosis rate of SH-SY5 Y cells was detected by Hoechst staining method; The intracellular ROS level was determined by fluorescence spectrophotometry. The protein expression of Mecp2, DNMT3 a, Ac-H3(Lys9), Ac-H4(Lys12), LC3 B and Beclin-1 in SH-SY5 Y cells were analyzed by Western blot.Results1. In vivo experiment(1) In the directional navigation experiment, compared with CT group, the latent time of mice in AD group was extended significantly(P<0.05); While compared with AD group, the latent time of mice in AD+BB group was obviuously shortened(P<0.05).In the space exploration experiment, compared with CT group, the times crossing platform of mice in AD group was dropped significantly(P<0.01); While compared with AD group, there was increased significantly in AD + BB group(P<0.05).(2) By HE staining, neuronal atrophy in AD mice were observed. Compared with AD group, the tissue structure and cell morphology in hippocampus and cortex were improved significantly in the AD + BB group, the cells arranged neatly and distributed evenly. By immunohistochemical staining method, there was an obvious decline in the number of senile plaques from the hippocampus and cortex in AD + BB group than that in AD group(P<0.05).Compared with CT group, there was an increase in the brain Aβ1-42 levels of AD group(P<0.01); Compared with AD group, the brain Aβ1-42 levels were significantly decreased in AD + BB group(P<0.05).(3) Compared with mice in CT group, the AD mice displayed a further significant increase in the content of MDA in serum and brain tissue(P<0.05), and the serum activities of SOD and GSH-Px were dropped significantly(P<0.05, P<0.01), there was a decline in the activity of SOD in the brain tissue(P<0.05); Compared with AD group, the AD+BB mice displayed a further significant decrease in the content of MDA in serum and brain tissue(P<0.05, P<0.01), while there was an increase in the activity of GSH-Px in serum(P<0.05), the activity of SOD in the brain tissue was also increased significantly(P<0.01).Compared with CT group, there was an obvious increase in the apoptosis rate of neuron of hippocampus and cortex in AD group(P<0.05); Compared with AD group, there was an obviously decline in AD+BB group(P<0.05).Compared with CT group, the protein expression of Caspase-3 in mice brain tissue was expressed at greater level in AD group(P<0.05); Compared with AD group, there was an obviously decline in AD+ BB group(P<0.01).(4) Compared with CT group, LC3 B and Beclin-1 protein expression in AD group were up-regulated significantly(P<0.05; P<0.01); Compared with AD group, LC3 B and Beclin-1 protein expression in AD+BB group were down-regulated significantly(P<0.05; P<0.01).Compared with CT and AD+BB group, the protein expression of Mecp2 and AcH3K9 in the brain tissue of AD group had a down-regualted and up-regulated tendency respectively, but there was no significant difference.2. In vitro experiment(1) For Aβ25-35-treated SH-SY5 Y cells, the optimal concentration and treatment time of protective Cy-3G were 100μmol/L and 12 h respectively. Compared with control group, after Aβ treatment, the cell viability was dropped sharply(P<0.05), the cell apoptosis ratio increased obviously(P<0.01), and the generation of ROS increased obviously(P<0.01), while Cy-3G pre-treatment can reverse the above changes.(2) Compared with control group, the protein expression of Mecp2 in Aβ25-35 group cells had an up-regulated tendency; Compared with Aβ25-35 group, the protein expression of Mecp2 in Aβ25-35+Cy-3G group had a down-regualted tendency, but there was no significant difference.Compared with control group, the protein expression of DNMT3 a in SH-SY5 Y cells increased obviously in Aβ25-35 group(P<0.05); Compared with Aβ25-35 group, the protein expression of DNMT3 a decreased significantly in Cy-3G+Aβ25-35 group(P<0.05).Compared with control group, the protein expression of Ac-H3(Lys9) and Ac-H4(Lys12) in SH-SY5 Y cells increased obviously in Aβ25-35 group(P<0.01); Compared with Aβ25-35 group, the protein expression of Ac-H3(Lys9) and Ac-H4(Lys12) decreased significantly in Cy-3G+Aβ25-35 group(P<0.01).Compared with control group, the viability of HDAC in SH-SY5 Y cell nucleus increased significantly in Aβ25-35 group(P<0.01); Compared with Aβ25-35 group, the viability of HDAC decreased significantly in Cy-3G+Aβ25-35 group(P<0.05).(3) Compared with control group, the protein expression of LC3 B and Beclin-1 in SHSY5 Y cells increased obviously in Aβ25-35 group(P<0.05); Compared with Aβ25-35 group, the protein expression of LC3 B and Beclin-1 decreased significantly in Cy-3G+Aβ25-35 group(P<0.05).Conclusion1. Blueberry extract intervention in the perinatal period can improve cognitive dysfunction in the APP/PS1 transgenic mice significantly.2. Cy-3G pre-treatment can obviously protect SH-SY5 Y cells against the injury induced by Aβ25-35.3. Anthocyanins play a neuroprotective role in AD animal and cell models, it may associate with antiapoptosis, antioxidation, epigenetic modulation and autophagy regulation. |
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