| Alzheimer’s disease(AD) is the common type of dementia, also a common neurodegeneration diseases. Prevention and treatment of AD should be studied and solved urgently. It is of great significance and prospects for further research and development of effective Chinese Medicine.Acorus gramineus is one of the most commonly used Chinese Medicine in the treatment of dementia. Related research already verified the therapeutic effect and discussed preliminarily the action mechanism of acorus gramineus and its active components(β-asarone and eugenol) by several vivo and vitro models. Nevertheless, these disease model applied in most research only simulated the chief symptoms of AD such as learning and memory impairment. APPswe/PS1dE9 double-transgenic mice is an AD mouse model expressing mutant amyloid precursor protein (APPswe) to mutant presenilin 1 (PS1dE9). Amyloid plaques are present in this mouse at 4 months and increase with age. The consistent and early onset of beta-amyloid (Aβ) accumulation confirms its utility for studies of biochemical and pathological mechanisms underlying beta-amyloid deposition, as well as exploring new therapeutic treatments. Researching the effect of the active components(β-asarone and eugenol) of acorus gramineuson on learning and memory and Aβin APPswe/PS1dE9 mice is important for further confirming efficacy.Neurotoxicity of Aβaggregates and their deposition in senile plaques are hallmarks of AD. Molecular chaperones protect cells from the deleterious effects of protein misfolding and aggregation. Clusterin, also known as apolipoprotein J, is a versatile chaperone molecule. Clusterin can bind to amyloid-βpeptides, stabilize them and prevent the oligomerization, then enhance the endocytosis of peptides or short fibrils into phagocytic glial cells and mediate its clearance across across the blood-brain barrier and blood-cerebrospinal fluid barrier the BBB via low-density lipoprotein receptor-related protein-1(LRP-1) and LRP-2/megalin. Researching the regulatory effect ofβ-asarone and eugenol on the neuroprotection pathway of clusterin is beneficial to further reveal their action mechanism.To research the effect of acorus gramineus active components(β-asarone and eugenol)on learning, memory, Aβand the neuroprotective pathway of clusterin. Methods42 male APPswe/PS1dE9 double-transgenic mice aged at 4 mouths were divided randomly into model group,β-asarone treated(21.2mg/kg per day) group, eugenol treated (21.2mg/kg per day) group and donepezi treated (2mg/kg per day) group,13 mice per group. In addition,13 male with the same age and background APPswe/PS1dE9 negatively transgenic mice were used as negative control. Distilled water was administered to the negative control group and model group. All mice were oral administered by the drugs from 3 to 7 months of age. After 4 months of treatment, spatial learning and memory was measured by Morris water maze. The amyloid senile plaques in the cortex and hippocampus from mice were detected by congo red staining. The level of soluble Aβoligomers in mice brain tissues was measured by dot blot assay. The relationship between the level of Aβoligomers and performance in morris water maze was analysed by bivariate correlation analysis. The expression of clusterin in mice brain tissue was measured by western blot. The co-expression of clusterin and Aβoligomers in cortex and hippocampus was detected by double labeled immunofluorescence and confocallaser scanning microscopy. The expression of low-density lipoprotein receptor-related protein-1 (LRP-1) and LRP-2/megalin was detected by immunohistochemistry.Experiment 1:The effect of acorus gramineus active components on learning, memory of APP/PS double-transgenic miceCompared with negatively transgenic mice(normal group), the escape latency of APPswe/PS1dE9 double-transgenic mice(model group) was significantly longer in place navigation test(day3～day5, all P<0.01), the total distance was significantly longer in place navigation test (day4:P<0.05; day5:P<0.01), the times of crossing the platform reducedsignificantly (P<0.01). Compared with model group, the escape latency and total distance ofβ-asarone treated group shortened significantly in placenavigation test(day 5, both P<0.05); the escape latency and total distance of eugenol treated group did not show significant change in place navigation test(P>0.05), but showed the trend of shorter; the total distance of donepezil treated group shortened significantly in place navigation test (day 5, P<0.05). Experiment 2:The effect of acorus gramineus active components onβ-amyloid protein in brain from APP/PS double-transgenic mice(1) Amyloid senile plaques could not be found in the cortex and hippocampus from normal group. There were amyloid senile plaques scattered in each area of cortex and hippocampus from model group. Compared with model group, the amyloid senile plaques in hippocampal CA1 area ofβ-asarone treated group reduced significantly (P<0.05); the amyloid senile plaques in hippocampus CA1 area of eugenol treated group showed a decreasing trend; the amyloid senile plaques in hippocampal CA1 area and cortex of donepezil treated group did not show significant change.(2) There was a certain positive correlation between the expression of Aβoligomers and performance(escape latency) in morris water maze(Pearson correlation=0.343, P<0.05). The stronger expression of Aβoligomers, the longer mice escape latency in morris water maze. The Aβoligomers expression of in each group did not show significant difference. The Aβoligomers expression of model group showed a increasing trend compared with nomal group. the Aβoligomers expression ofβ-asarone treated group, eugenol treated group and donepezil treated all showed a decreasing trend compared with model group.Experiment 3:The effect of acorus gramineus active components on clusterin neuroprotective pathway of APP/PS double-transgenic mice(1) Co-localization of clusterin with Aβoligomers was widespread discovered in cortex and hippocampus from APPswe/PS1dE9 double-transgenic mice. The result suggests that there is interaction between clusterin and Aβoligomers. The expression of clusterin in each group did not show significant difference(P>0.05). (2) A certain amount expression of LRP-1 could be discovered in the cortex and hippocampus CA1 area from normal group, mainly in microvessel. Compared with normal group, the LRP-1 expression in cortex and hippocampus CA1 area of model group decreased significantly (both P<0.05). Compared with model group, the LRP-1 expression in the cortex and hippocampus CA1 area ofβ-asarone treated group both increased significantly (both P<0.01); the LRP-1 expression in the cortex of eugenol treated group increased significantly(P<0.01), The expression in hippocampus CA1 also showed a increasing trend; the LRP-1 expression in the cortex and hippocampus CA1 area of donepezil treated group both showed a increasing trend.(3) A certain amount expression of LRP-2/megalin could be discovered in the cortex and hippocampus CA1 area from normal group. Compared with normal group, the LRP-2/megalin expression in cortex of model group decreased significantly(P<0.01), the expression in hippocampus CA1 area also showed a increasing trend. Compared with model group, the LRP-2/megalin expression in the cortex and hippocampus CA1 area ofβ-asarone treated group did not show significant difference; the LRP-2/megalin expression in the cortex of eugenol treated group increased significantly(P<0.01), the expression in hippocampus CA1 also showed a increasing trend; the LRP-2/megalin expression in the cortex and hippocampus CA1 area of donepezil treated group did not show significant difference, but the expression in hippocampus CA1 area showed a increasing trend.(1)β-asarone can effectively improve learning and memory of APPswe/PS1dE9 double-transgenic mice, reduce amyloid senile plaques and up-regulate the expression of LRP-1 in brain from APPswe/PS1dE9 double-transgenic mice.(2) Eugenol can up-regulate the expression of LRP-1 and LRP-2/megalin in brain from APPswe/PS1dE9 double-transgenic mice.(3) There is a certain positive correlation between the expression of Aβoligomers and the ability of learning and memory. There is interaction between clusterin and Aβoligomers in brain from APPswe/PS1dE9 double-transgenic mice.(4) The effect ofβ-asarone on Aβoligomers and clusterin expression need to be verified in further study. The effects of eugenol on learning and memory, amyloid senile plaques, Aβoligomers and clusterin in brain from APPswe/PS1dE9 double-transgenic mice also need to be further comfirmed. |
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