| Objective: On the basis of A? toxic theory,Alzheimer's disease(AD)animal model was established by injecting A?25-35 into the hippocampus,wheather VTF and VTP could improve the learning and memory dysfunction or hippocampal pathology changes in AD rats.In order to further investigate the molecular mechanism of VTF and VTP in the treatment of AD,with the classic AD pathogenesis theory "inflammatory reaction theory","damaged synaptic theory","APP metabolism","cholinergic injury" and "apoptosis hypothesis" as the starting point,the neuroprotective effect of VTF and VTP clearly defined on AD rats and explored the possible mechanism;In addition,A?25-35 treated PC12 cells as a cell model of AD,to study the effects of VTF on PI3K/Akt signaling pathway.Aim to provide some experimental basis on the pathogenesis of AD,looking for the prevention and treatment of AD and drug targets of VTF,VTP.Methods:(1)The morphology and behavior experiment: bilateral hippocampus CA1 was injected A?25-35 established AD animal model,Morris water maze tested rats' learning and memory,Nissl staining and transmission electron microscopy observed neuron morphological changes in hippocampus.(2)Study on the metabolic mechanism of APP: immunohistochemistry was used to detect the expression of APP,BACE-1 and A?1-42.RT-q RCR and Western Blotting was used to detect the gene and protein expression of APP and BACE-1.(3)Research on the mechanism of apoptosis: TUNEL was used to detect the rats apoptosis of different experimental groups;immunohistochemical and Western Blotting was used to detect the protein expression of Caspase-3 in the rats.A?25-35 induced injury in PC12 cells,WST-1 analyzed cell activity,flow cytometry analyzed effect of VTF on PC12 cell apoptosis;Western Blotting was used to detect the effect of VTF on PI3K/Akt signaling pathway in protein level.(4)Inflammation mechanism:ELISA was used to detect the inflammatory factor TNF-?,IL-6? and IL-1;immunohistochemistry was used to detect the expression of NF-?Bp65;Western Blotting was used to detect the expression of p-I?B-?,I?B-? and p-NF-?Bp65 protein;RT-q RCR was used to detect the expression of NF-?Bp65 m RNA and I?B-? m RNA.(5)Research on the mechanism of cholinergic injury and synaptic plasticity: biochemical methods was used to detect the activity of Ach E and Ch AT.The expression of Ca M,CREB,BDNF and Syt-1 was detected by immunohistochemistry.The expression of BDNF,Ca M,Syt-1,p-CREB and CREB protein was detected by Western Blotting.The expression of Ca M m RNA and CREB m RNA was detected by RT-q RCR.Results:(1)The behavioral and morphological experiments:(1)the place navigation experiment could be obviously observed that the model group rats need to spend more time to find the platform compared with other groups.After the treatment of VTF and VTP,the escape latency was shortened;without platform,the AD model group rats passed through the effective area significantly reduced than the control group;The high-dose group of VTF and VTP passed through the effective area increased than the model group.(2)Nissl staining showed that AD neurons in the hippocampus of the model group were obviously loose,disordered,VTF and VTP groups significantly reduced the injury of hippocampal neurons,deep staining,normal morphology.Transmission electron microscope,the morphology of hippocampus neurons in CA1 area was irregular in AD group,the nuclear membrane was unclear,vacuolar degeneration.After the treatment of VTF and VTP,hippocampus neurons in CA1 area was more regular,nuclear pyknosis and apoptosis neurons was also significantly reduced compared with the model group.(2)Study on the metabolic mechanism of APP:(1)Compared with the control group,expression of APP was Up-regulated in model group;the expression level of APP protein was significantly reduced after treatment with VTF and VTP.(2)Compared with the model group,VTF and VTP inhibited the relative protein expression and transcription levels of BACE1.The expression of A?1-42 also significantly decreased in VTF and VTP group.(3)Study on the mechanism of apoptosis:(1)The activity of Caspase-3 in model group was obviously higher than in the control group.Compared with the model group,the number of apoptotic cell was reduced and expression of Caspase-3 protein down-regulation in VTF and VTP group.(2)Cell viability in model group was significantly lower than the control group.Compared with the model group,the cell activity in VTF group significantly increased and VTF could significantly reduce thePC12 cell apoptosis which induced by A?25-35.(3)Compared with control group,p-Akt/Akt was significantly reduced in model group.Compared with the model group,p-Akt/Akt value was significantly increased in VTF group.Compared with LY294002 group,p-Akt/Akt value was increased significantly in VTF+LY group.(4)The research on the mechanism of inflammation:(1)Compared with model group,the transcription level and the protein expression of nucleus p-NF-?B p65 were significantly decreased in VTF and VTP high-dose group.(2)The VTF and VTP groups,p-I?B/I?B and I?B-? m RNA were decreased compared with the model group.(3)VTF and VTP inhibited the production of inflammatory factors TNF-?,IL-6 and IL-1.(5)Study on the mechanism of cholinergic injury and synaptic plasticity:(1)VTF and VTP could inhibit the expression of Ca M in each group compared with model group.CREB m RNA gene expression,p-CREB/CREB value correlated with VTF and VTP dose.(2)Compared with model group,the expression of BDNF and Syt-1 in VTF group and VTP group were increased.(3)In hippocampus and serum,the activity of Ach E was increased and the activity of Ch AT was decreased in the model group.After the treatment of VTF and VTP,the activity of Ach E was decreased and the activity of Ch AT was increased significantly in hippocampus and serum.Conclusions:(1)The AD animal model was successfully established by A?25-35 which was the core toxic fragment of A?1-42.VTF and VTP could effectively improve the learning and memory abilities of AD rats and repair pathological changes of hippocampal neurons in a certain dose.(2)VTF and VTP adjusted the metabolism of APP by down-regulating the expression level of key protein APP of A? pathway and key enzyme of BACE-1 and its downstream products of A?1-42,thereby reduced the generation of A?,repaired pathological changes of hippocampal neurons in AD rats.(3)VTF and VTP decreased the number of apoptotic cells in the hippocampus of AD rats and down-regulated the expression of Caspase-3 protein,thereby inhibited apoptosis.VTF could alleviate the neurotoxic effects of A?25-35,decrease A?25-35 induced cell apoptosis.VTF could promote the phosphorylation of Akt and inhibit the depressed of phosphorylation level in Akt which induced by the signal pathway blocker LY294002.These results suggested that VTF had neuroprotective effects and reduced the injury of nerve cells by anti apoptotic pathway.(4)VTF and VTP inhibited the protein expression of NF-?Bp65 from gene to protein level thus inhibited the release of inflammatory factor TNF-?,IL-6,IL-1?.VTF and VTP inhibited the phosphorylation of I?B-?,thus inhibited the nuclear translocation of NF-?Bp65 and then inhibited the NF-?B/I?B-? signaling pathway.(5)A?25-35 oligomers could up-regulate the protein expression and gene transcription level of Ca M in rathippocampus,which resulted in synaptic injury in rat hippocampal neurons.VTF and VTP could reduce the protein expression and gene transcription level of Ca M in hippocampus of rats.VTF and VTP could improve the expression of p-CREB,BDNF and Syt-1 in hippocampus of rats,VTF and VTP had protective effects on synaptic injury which induced by A?25-35.VTF and VTP could significantly decrease the activity of Ach E and increase the activity of Ch AT in hippocampus tissue and serum of AD model rats,so as to improve the central cholinergic damage. |
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