| Objective:1.To confirm the effect of blueberry extract(BB)and Cyanidin-3-O-glucoside(Cy3G)on learning and memory impairment in APP/PS1 transgenic mice.2.To explore the regulation mechanism of BB and Cy3G in lactate metabolism based on transcriptome and metabolome of brain tissue.Methods:1.Effects of BB treatment on learning and memory ability of APP/PS1 mice:The components of anthocyanins in BB were detected by LC-MS.Four-month-old APP/PS1 mice were gavaged with two doses of BB(150 mg/kg·bw and 100 mg/kg·bw)for 4 months,and the cognitive function of mice was evaluated by behavioral experiments.Pathological staining(HE,Congo red,and silver glycine)and transmission electron microscopy were used to analyze the pathological and subcellular changes in hippocampus.ELISA kits were used to detect the contents of Aβ1-42,BDNF and cytokines in brain.2.The mechanism of blueberry extract alleviate cognitive disorders in APP/PS1 mice based on omics technology:4-month-old APP/PS1 mice were gavaged with BB(150 mg/kg·bw)for 4 months.The mRNA levels in brain tissue were detected by next-generation sequencing,and metabolites were detected by LC-MS.RT-PCR,Western blot,and Immunofluorescence staining were used to verify the expression levels of genes and proteins related to lactate metabolism.3.Effects of Cy3G treatment on learning and memory ability of APP/PS1 mice and its regulation mechanism of lactic acid metabolism:6-month-old APP/PS1 mice were gavaged with Cy3G(50 mg/kg·bw)for 2 months,and the cognitive function of mice was evaluated by behavioral experiments.The consumption of 18F-FDG in mice brain tissue was measured by Micro PET/CT.The expression levels of lactate-related proteins were detected by Western blot.Blood glucose and lipid levels of mice were detected by automatic biochemical analyzer.In addition,Cy3G and HIF1α inhibitor PX-478 were used to confirm whether Cy3G promotes lactate metabolism through HIF1α pathway.4.Effects of Cy3G treatment on Aβ-induced astrocyte activation and lactate metabolism:The co-culture model of primary rat astrocytes and neurons was established.The protection of Cy3G on Aβ25-35-induced neuron activity damage and the inhibition of Cy3G on Aβ25-35-induced astrocyte activation were detected by CCK-8.The expression levels of lactate-related proteins were detected by Western blot,and glycolysis rate was detected by Seahorse.In addition,whether Cy3G could promote lactate metabolism through mTOR-HIF1α pathway was evaluated by detecting lactate-related proteins and glycolysis rate after added HIF1α inhibitor PX-478 or mTOR inhibitor Rapa.Results:1.Effects of BB treatment on learning and memory ability of APP/PS1 mice:①Cy3G is the main anthocyanin of BB,accounting for about 27%of the total mass.② In Morris water maze,the latency of APP/PS1 mice was prolonged after training to day 3,and the latency of APP/PS1 mice in high-dose BB gavaged group was significantly shorter than that in AD group on day 4.After the platform was removed,the cross times in the two dose BB gavaged groups was significantly higher than that in the AD group.③ In the open field test,the stand frequencies and total distances of APP/PS1 mice were significantly decreased,and the time spent not moving was prolonged.All of these were reversed after BB treatment.④ In the new object test,the proportion of time spent on novel object and proportion of novel object visits were decreased in APP/PS1 mice and increased in BB treatment groups.⑤Pathological staining showed that the apoptosis of neurons,amyloid deposition and neurofibrillary tangles were increased in APP/PS1 mice and were decreased in BB treatment group.⑥Under transmission electron microscopy,the mitochondria in hippocampal neurons of APP/PS1 mice became swollen and round,while the morphology of mitochondria returned to paramecium after high dose BB treatment.⑦The contents of Aβ1-42,IL-1β and TNF-αin APP/PS1 mice brain tissue were significantly increased compared with the control group;Low-dose BB decreased the contents of Aβ1-42,IL-1β,IL-6 and TNF-α,and high-dose BB increased BDNF content and decreased IL-6 content.2.The mechanism of blueberry extract alleviate cognitive disorders in APP/PS1 mice based on omics technology:① There were 15 metabolites significantly increased and 18 metabolites significantly decreased compared with the control group in APP/PS1 mice brain tissue,involving the TCA cycle,glycolysis,gluconeogenesis and pyruvate metabolic pathways;After BB treatment,6 metabolites were significantly increased and 6 metabolites were significantly decreased,involving glycolysis,gluconeogenesis,and pyruvate metabolic pathways.Colorimetric test showed that the levels of lactate and pyruvate in the brain of APP/PS1 mice were significantly increased compared with the control group,whereas BB had no influence on lactate and pyruvate levels.②Transcriptomic results showed that compared with the control group,289 genes were up-regulated,and 107 genes were down-regulated in APP/PS1 mice.After BB treatment,355 genes were up-regulated,and 649 genes were down-regulated,which involved cholinergic synapse,MAPK signaling pathway,glutamate synapse and LTP.③Compared with the control group,mRNA levels of Hif1α,Ldha,and Glut1 and protein levels of LDHA,LDHB,PKM2,PFKFB3,MCT4,and MCT2 in APP/PS1 mice brain were significantly decreased.After BB intervention,mRNA levels of Hif1α,Hk2,Ldha,Glut1,Mct2,and Mct4 and protein levels of LDHA,LDHB,PFKFB3,HK2,GLUT1,MCT4,and MCT2 were significantly increased.The number and fluorescence intensity of GFAP-labeled astrocytes in the hippocampus of APP/PS1 mice were significantly increased compared with the control group,and the fluorescence intensity of GLUT1,LDHA,and MCT4 in astrocytes and LDHB in neurons were significantly decreased.These were reversed after BB treatment.3.Effects of Cy3G treatment on learning and memory ability of APP/PS1 mice and its regulation mechanism of lactic acid metabolism:① In Morris water maze,the latency of APP/PS1 mice in Cy3G gavaged group was reduced after training to day 4;After the platform was removed,the cross times in the Cy3G gavaged groups was significantly higher than that in the AD group.Cy3G treatment increased the stand frequencies in the open field test,and increased proportion of time spent on novel object and proportion of novel object visits in new objects test.② Cy3G treatment reduced the AUC in oral glucose tolerance test and the serum triglyceride level in APP/PS1 mice.Image in PET/CT showed that the glucose metabolism level in the cerebral cortex of APP/PS1 mice was decreased,and it was increased after Cy3G treatment.Cy3G also promoted the protein levels of PFKFB3,HK2,GLUT1 and HIF1α in APP/PS1 mice.③ The HIF1α inhibitor PX-478 inhibited the improvement of behavior of APP/PS1 mice by Cy3G,increased AUC in oral glucose tolerance test,decreased the level of glucose metabolism in cerebral cortex,and inhibited the expression of HIF1α,PFKFB3,HK2 and GLUT1.4.Effects of Cy3G treatment on Aβ-induced astrocyte activation and lactate metabolism:①CCK-8 assay showed that Aβ25-35 significantly increased the activity of astrocytes;Cy3G significantly inhibited the activity of astrocytes and neurons,and also inhibited the activity of astrocytes induced by Aβ25-35.In the co-culture model,Aβ25-35 inhibited neuronal activity,while the neuronal activity increased significantly after Cy3G treatment.② Cy3G reversed the inhibition of Aβ25-35 on the lactate content in astrocytes.Moreover,Basal glycolysis,maximal glycolysis,and basal oxygen consumption of astrocytes were inhibited after treated with Aβ25-35 for 24 h.The maximum glycolysis rate of astrocytes was increased after treated with Cy3G for 24 h.In addition,Cy3G promoted the compensatory glycolysis rate of reactive astrocytes induced by Aβ25-35 but had no significant effect on basal glycolysis rate and basal oxygen consumption.③The protein levels of GLUT1,MCT4 and HIF1α in reactive astrocytes induced by Aβ25-35 were significantly decreased,and Cy3G treatment significantly increased the protein levels of LDHA,PKM2,PFKFB3,HK2,MCT4,GLUT1,HIF1α,mTOR and p-mTOR.The fluorescence intensities of GLUT1 and LDHA in reactive astrocytes induced by Aβ25-35 were decreased,especially,the content of HIF1α in nuclei were also decreased.Cy3G increased the expression levels of GLUT1 and LDHA,and nuclear HIF1α in reactive astrocytes induced by Aβ25-35.④PX-478 inhibited the decrease of HIF1α,LDHA,HK2,PKM2,PFKFB3,GLUT1 and MCT4 protein levels in Cy3G-induced astrocytes.Rapa also significantly inhibited Cy3G-induced protein levels of p-mTOR,HIF1α,LDHA,PFKFB3,HK2 and GLUT1.Both Rapa and PX-478 significantly inhibited the increase of compensatory glycolysis rate and basal glycolysis rate of astrocytes by Cy3G.But it has no significant effect on basal oxygen consumption.Conclusions:1.Eight-month-old APP/PS1 mice showed cognitive decline,increased inflammatory levels,and decreased number of neurons in brain.These symptoms may be caused by increased reactivity astrocytes and decreased lactate-related proteins,resulting in inadequate energy supply to neurons.2.BB treatment effectively alleviate cognitive impairment in APP/PS1 mice,which may be related to reducing the reactivity astrocytes,increasing the expression level of lactate-related proteins.3.Cy3G is the main anthocyanin of BB.Cy3G treatment increased the ability of glucose uptake in brain tissue,up-regulate the expression of lactate-related proteins,improve the level of glucose and lipid metabolism,and effectively alleviate cognitive decline of AD.4.Cy3G inhibited the activity of reactive astrocytes induced by Aβ,up-regulated the expression of lactate-related proteins and promoted glycolysis through mTOR-HIF1α pathway. |
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