Study On The Effect And Mechanism Of Effective Fractions Of Chinese Herbal Compound On Learning And Memory Of APP Transgenic Mice

Alzheimer's disease (AD) is a common neurodegenerative disease, characterized by progressive cognitive function deterioration. As the aging of human beings in the world, the incidence of AD is increasingly high. But so far, as a world medical problem, the pathogenesis of AD has not been clear, and its ideal drug treatment has still been deficient. The treatment of Traditional Chinese Medicine (TCM), based on syndrome differentiation, has superiority and prospect at the prevention and treatment of AD, especially by the role of Chinese herbal compound, which has many curative pathways and targets to AD. Huannao Yicong Fang (HNYCF) is a Chinese herbal compound based on "Deficiency-Blood Stasis-Turbidity-Toxin", the pathogenesis of AD in TCM. It has been proved that HNYCF has good preventive and therapeutic effects to AD. Now, we use modern technology to extract the effective fractions of HNYCF, and study further its mechanism and effective channel to AD animal model, so as to provide basis to the clinical treatment of AD.Objective:According to beta-amyloid cascade hypothesis in AD, this study is to observe effects of the effective fractions of HNYCF (herbal extract) on generation of beta amyloid protein (Aβ), inflammatory and oxidative stress in the brain of AD animal model, to explore the mechanism of HNYCF in the treating of AD.Methods:APP695V717I transgenic mice 3 month-old were used as AD models in this study, which were randomly divided into model group, Donepezil group, HNYCF large dose group and HNYCF small dose group. Normal control adopted the same age and background C57BL/6J mice. The animals were administered intragastrically by the drug or water from 3 month-old to 9 month-old. This research was divided into three parts. PartⅠ:Morris water maze test was performed to measure the spatial learning and memory ability. Step-down test was performed to observe the learning and memory ability of single passive avoidance response. This part was to study effects of HNYCF on the behavior of APP transgenic mice. PartⅡ:The expression ofβ-amyloid precursor protein (APP),β-site APP cleaving enzyme (BACE) and Aβin hippocampus CA1 region was detected by immunohistochemistry with image analysis. Amyloid deposition were observed with Congo red staining. This part was to study effects of HNYCF on generation of Aβin the brain of APP transgenic mice. PartⅢ: The expression of nuclear factor kappa B (NF-κB) and peroxisome proliferator-activated receptorγ(PPARγ) in hippocampus CA1 region were detected by immunohistochemistry with image analysis. The content of inflammatory cytokines interleukin-6 (IL-6) and high sensitivity C-reactive protein (hs-CRP) in the brain cortex and hippocampus homogenate were detected with radioimmunoassay (RIA). The content of total protein in the brain cortex and hippocampus homogenate was detected with biuret method. The activity of superoxide dismutase (SOD) in serum was detected with colorimetry. The content of malondialdehyde (MDA) in serum was detected with Thibabituric Acid (TBA). This part was to study effects of HNYCF on inflammatory cytokines and oxidative stress in the brain of APP transgenic mice.Results:PartⅠ:Behavioral experiments suggested the effects of HNYCF on the behavior of APP transgenic mice. Morris water maze test showed that the times of crossing platform of HNYCF group and Donepezil group were much more than that in the model group (P＜0.05), the swimming time and distance of all treated groups in the fourth quadrant where the original platform put were prolonged distinctly (P< 0.05 or P<0.01). The mice step-down test manifested that there was no statistical significant difference about wrong times among all treated groups and the model group (P>0.05), but the escape latency of HNYCF large dose group and Donepezil group was significantly longer than model group (P<0.05 or P<0.01), and the escape latency of HNYCF small dose group had the trend of extension but had no statistical significant difference comparing to the model group (P>0.05).PartⅡ:Comparing to the model group, the levels of expression of APP in hippocampus CA1 region of mice in the HNYCF group and Donepezil group decreased obviously (P<0.01), including its number and area proportion of positive cells. Comparing to the model group, the number of positive cells of BACE in the HNYCF group decreased significantly (P<0.01), and Donepezil group had the trend to reduce, but there was no statistical significant difference (P>0.05). The expression of Aβin hippocampus CA1 region of mice in all the treated groups decreased obviously (P＜0.05 or P<0.01), comparing with model group. Congo red staining showed that there were orange deposition scattered in the cortex and hippocampus of mice in the model group, which had sizes, uneven coloring, and intensively in the hippocampus. Comparing to the model group, HNYCF group and Donepezil group had shallow and limited orange staining, and its number decreased significantly.Part III:After treated with HNYCF and Donepezil, the number of NF-κB positive cells in hippocampus CA1 region in the APP transgenic mice decreased obviously (P<0.05 or P<0.01), comparing to the model group. The number of PPARy positive cells increased significantly in the brain of mice in all treated groups (P<0.05). The content of IL-6 in the brain cortex in HNYCF group was lower than that of model group (P<0.05 or P<0.01), and that in Donepezil group was also decreased, but not significantly different (P>0.05). Comparing with the model group, the content of IL-6 in hippocampus in HNYCF small dose group and Donepezil group decreased obviously (P<0.05), and which was in HNYCF large dose group also reduced, but there was no significant difference (P>0.05). Comparing the content of hs-CRP in the brain tissue, there was no significant difference among all treated groups and the model group (P>0.05), but all the treated groups had the trend of reduction. The activity of SOD in serum of HNYCF group was significantly higher than model group (P<0.05 or P<0.01), but there was no siginificant difference between Donepezil group and model group (P>0.05). Comparing the content of MDA in serum, there was no significant difference among all treated groups and the model group (P>0.05).Conclusions:The effective fractions of HNYCF can ameliorate evidently the spatial learning and memory ability and that of single passive avoidance response of APP transgenic mice. The effective fractions of HNYCF can decrease the expression of APP and itsβ-secretase, reduce the production and accumulation of Aβ, and reduce the amyloid deposition in the brain of APP transgenic mice. The effective fractions of HNYCF can increase the expression of PPARy, inhibit the expression of NF-κB, decrease the content of inflammatory cytokines, and alleviate inflammatory response in the brain of APP transgenic mice. The effective fractions of HNYCF can also improve the ability of scavenging oxygen free radicals, alleviate oxidative stress in APP transgenic mice. This study suggests that HNYCF can reduce the neuron toxicity of Aβin the brain of APP transgenic mice, alleviate the induced inflammatory response and oxidative stress, and improve their learning and memory ability. Briefly, this study suggests HNYCF has many effective targets in treating AD, can prevent and treat AD by anti-Aβneurotoxicity, anti-inflammatory and anti-oxidation.