The Neuroprotective Effect Of Curcumin On The App/ps1 Double Transgenic Mice

Alzheimer's disease is a kind of age-related neurodegenerative disease that is characterized with progressive worsening of memory and cognition. The typical neuropathological lesions include dysfunction of cholinergic neurons, neuron and synapse loss, accumulation of extracellular Aβresulting in the formation of senile plaques and intracellular hyperphosphorylated tau resulting in the formation of neurofibrillary tangles. The major molecular pathological lesion that occurs in AD is consistently associated with a number of abnormal expressing and processing of proteins. Currently, the growing progression of molecular biology provides useful tools to reveal the mechanisms of protein synthesis, protein transportation and protein accumulation in brain.The present study was designed to observe and quantify the effects of Curcumin on Aβ42/Aβ40 ratio and ADDLs, to study the relationship between ADDLs and insulin signal transduction, to investigate the protective mechanism of Curcumin on neuronal survival, to objectively evaluate the therapeutic effect of Curcumin and to provide theoretical evidence for TCM prevention and treatment of AD.Material and Methods1. APP/PS1 double transgenic mice (APP/PS1 dtg mice) of 3 months and genetic background and age-matched control littermates were intragastric administration once daily for 3 months. TCM treatment groups (high, medium and low dose) were treated with Curcumin. The positive control group was treated with Rosiglitazone (RSG).2. The spatial learning and memory were tested by Morris water maze to evaluate the therapeutic effects of Curcumin on cognitive function.3. To observe the changes of Shank 1 and PSD95 in hippocampus CA1 area of APP/PS1 dtg mice before and after Curcumin treatment using immunohistochemistry method. To observe the changes of neurons and synapses in hippocampus CA1 area of APP/PS1 dtg mice using electronic microscope and to evaluate the therapeutic effect of Curcumin on synapse structures and function.4. To observe the changes of IDE, PS2, Aβ42, Aβ40 and ADDLs in hippocampus of APP/PS1 dtg mice using immunohistochemistry and Western-blot methods and evaluate the therapeutic effects of Curcumin on Aβ* cascade reactions.5. To observe the changes of related proteins such as InR, IRS-1 et al. in insulin signal transduction pathway in hippocampus of APP/PS1 dtg mice using immunohistochemistry, Western-blot and RT-PCR methods and evaluate the therapeutic effects of Curcumin on insulin signal transduction.Results1. The effects of Curcumin on ethology, learning and memory of APP/PS1 dtg mice In Morris water maze test, escape latency and swimming distance of APP/PS1 dtg mice were significantly increased compared with the normal control mice (P<0.01 and PO.05). Compared with APP/PS1 dtg mice, the escape latency of APP/PS1-RSG group, APP/PS1-Curcumin-M group and APP/PS1-Curcumin-H group were significantly shortened (P<0.01 or P<0.05). Nonetheless, the escape latency of APP/PS1 dtg mice treated with Curcumin-H and Curcumin-L were significantly increased compared with APP/PS1 dtg mice treated with RSG (P<0.05). The swimming distance of all treated APP/PS1 dtg mice except APP/PS1-Curcumin-L were significantly decreased compared with APP/PS1 dtg mice (P<0.01 or P<0.05).2. Effects of Curcumin on neurons and synapse associated proteins of APP/PS1 dtg miceUltrastructure:The structures of hippocampal neurons and neuropils were normal in normal control group. Neurons of hippocampal CA1 area in APP/PS1 dtg mice showed varying degrees of degenerative changes, including neuronal shrinkage, markedly increased cytoplasmic lipofuscin and lipid droplets, irregular nuclei shape, some nuclear chromatin condensation; cytoplasmic lipofuscin and lipid droplets increase, dilation of Golgi vesicles, discretion of rough endoplasmic reticulum, depolymerization of polyribosomes, some mitochondria degeneration. In neuropil it is seen that myelin degeneration, reduced synapses and structural abnormalities of synapses and increased aggregation of synaptic vesicles. Curcumin can increase synapses and make these changes improved to varying degrees.The results of immunohistochemistry:Compared with normal group mice, PSD-95and Shankl positive cells of hippocampus CA1 area of APP/PS1 dtg mice were significantly increased (P<0.01).Compared with APP/PS1 dtg mice, PSD-95and Shankl positive cells of hippocampus CA1 area of APP/PS1-RSG and all Curcumin treated groups especially APP/PS1-Curcumin-M group were significantly increased (P<0.05).The results of Western blot:Compared with normal group mice, PSD-95 expression of hippocampus of APP/PS1 dtg mice were significantly increased (P<0.01). Compared with APP/PS1 dtg mice, PSD-95 expression of hippocampus of APP/PS1-RSG and all Curcumin treated groups were significantly increased (P<0.05).3. Effects of Curcumin on Aβof APP/PS1 dtg miceThe results of immunohistochemistry:Compared with normal group mice, PS2 positive cells of hippocampus CA1 area of APP/PS 1 dtg mice were significantly increased (P<0.01) while IDE and NEP positive cells of hippocampus CA1 of APP/PS 1 dtg mice area were significantly reduced. All treated groups showed markedly decreased number of PS2 positive cells of hippocampus CA1 area (P<0.01).Compared with APP/PS 1 dtg mice, IDE positive cells of hippocampus CA1 area of APP/PS 1-Curcumin-H group were significantly increased (P<0.05) and NEP positive cells of hippocampus CA1 area of all Curcumin-treated groups were significantly increased (P<0.05). Compared with normal group mice, Aβ40 and Aβ42 positive cells of hippocampus CA1 area of APP/PS1 dtg mice were significantly increased, Aβ42 positive cells showed more increasing. Compared with APP/PS1 dtg mice, all treated groups showed markedly decreased number of Aβ40 and Aβ42 positive cells of hippocampus CA1 area.Compared with normal group mice, the average gray value of Aβ40, Aβ42 and ADDLs positive cells were significantly decreased (P<0.01). The average gray value of APP/PS1-Curcumin-M and APP/PS1-Curcumin-L groups was significantly increased in comparison with APP/PS1 dtg mice.The results of Western blot:The result of the expression of each protein matched the result of immunohistochemisty.4. Effects of Curcumin on insulin signal transduction of APP/PS1 dtg miceThe results of immunohistochemistry:Compared with normal group mice, InR and IRS-1 positive cells of hippocampus CA1 area of APP/PS1 dtg mice were significantly increased (P<0.05 and P<0.01).Compared with APP/PS1 dtg mice, the increase of InR and IRS-1 positive cells of hippocampus CA1 area of APP/PS1-RSG and APP/PS1-Curcumin-M group were significantly ameliorated (P<0.05 and P0.01).Compared with normal group mice, the expression of p-IRS-1, PI3K, p-PI3K, AKT and p-AKT protein of APP/PS1 dtg mice were significantly decreased (P<0.05 or P<0.01).In comparison with APP/PS1 dtg mice, the expression of p-IRS-1,PI3K, p-PI3K, AKT and p-AKT protein of APP/PS1-RSG and APP/PSl-Curcumin-M group exhibited significant increase (P<0.05 or P<0.01).The results of Western blot:The result of the expression of each protein matched the result of immunohistochemisty.RT-PCR:InR mRNA and IRS-1 mRNA of APP/PS1 dtg mice were significantly increased compared with the normal control mice. All treated groups showed reduced InR mRNA and IRS-1 mRNA expression.ConclusionThe neuroprotecting mechanism of Curcumin is to improve insulin signal transduction, down-regulate the production A042 and ADDLs and enhance the degradation of Aβ42 and ADDLs, hence affecting Aβcascade reactions...