Study On The Effect And Mechanism Of Early Intervention With Extracts Of Chinese Herbal Compound On Cognitive Funtion Of APP Transgenic Mice

Background:The mild cognitive impairment of old people, has now been recognized as the "Early clinical dementia status",patients with MCI and early AD have many similarities,both of them are central nervous system lesions characterized by Progressive cognitive decline. Epidemiological survey shows,with the accelerated aging of the global population,mild cognitive impairment and the incidence of dementia showed a continually rising trend,the number of Global prevalence of dementia by 2030 will reach 65.7 million,the year of 2050 will be increased to 100 million and 15.4 million,but so far, cognitive impairment and dementia as a worldwide medical problem, the pathogenesis is not very clear,In particular, the lack of effective treatments and measures. The treatment of Traditional Chinese Medicine (TCM), based on syndrome differentiation, has superiority and prospect at the prevention and treatment of MCI and AD which is beyond the reach of western medicine, especially by the role of Chinese herbal compound, which has many curative pathways and targets to MCI and AD. Huannao Yicong Fang (HNYCF) is a Chinese herbal compound based on"Deficiency-Blood Stasis-Turbidity-Toxin",the pathogenesis of MCI and AD in TCM.It has been confirmed by Early experiments and clinical research that HNYCF has good preventive and therapeutic effects to MCI and early AD,In this experiment, we use modern technology to extract the fractions of HNYCF, In-depth study its mechanism and effective channel to APP695V717I transgenic mouse model,so as to provide basis to the TCM is more widely used in clinical treatment of MCI and early Alzheimer's disease.Objective:Based on cognitive dysfunction in the pathogenesis of the Cholinergic system dysfunction and free radical imbalance theory, APP695V717I transgenic mice 3 month-old were used as models in this study,application of the extract of HNYCF (herbal extract) on the model of early intervention,observe the behavior of the model, generation of beta amyloid protein(Aβ),cholinergic system and oxidative stress-related index changes,discussion the mechanism of HNYCF in the treating of MCI and AD.Methods:APP695V717I transgenic mice 3 month-old were used as models in this study, which were randomly divided into model group, Donepezil group, the extract of HNYCF large dose group and the extract of 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 7 month-old. This research was divided into three parts. PartⅠ: Morris water maze test was performed to measure the spatial learning,memory and other cognitive-related capacity.Step-down test (Passive avoidance conditioning) was performed to observe the learning and memory ability of single passive avoidance response, This part was to study 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. PartⅢ:Determination the content of ACh, AchE, ChAT by ELISA and Bradford method,This part was to study the extract of HNYCF on central cholinergic function of APP transgenic mice. 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 free radical-induced oxidative stress in the brain of APP transgenic mice.Results:PartⅠ:The Early intervention with extract of HNYCF to improve the disorder of cognitive function in APP transgenic mice of the experimental observation of behavior.Morris water maze test results show that:Compared with the control group, model mice through the security platform for the location of the original number, in the fourth quadrant swimming time and distance were significantly shortened and reduced (P<0.01 or P<0.05); after treatment, mice in the Morris water maze in the position of crossing the number of security platform, in the fourth quadrant swimming time and distance compared with model group was significantly increased (P<0.05 or P<0.01);Step down test results show that:Compared with the control group, the number of errors of model group was significantly higher (P<0.05), latency was significantly shorter (P<0.05); Compared with model group, the extract of HNYCF large dose group had significantly fewer errors, there was significant difference (P<0.05), Donepezil hydrochloride group, the extract of HNYCF small dose group group showed a decreasing trend in the number of errors, but no significant difference (P>0.05);Compared with model group,the extract of HNYCF large dose group was significantly longer latency (P<0.01),Donepezil hydrochloride group, low dose group also extended incubation period of trend, but no significant difference (P>0.05).PartⅡ:Effect of early intervention with extract of HNYCF on the pathway of APP transgenic mouse brain AβgenerationComparing to the model group, the levels of expression of APP in hippocampus CA1 region of mice in the HNYCF group decreased obviously (P<0.01), including the number and area proportion of positive cells;the levels of expression of APP in hippocampus CA1 region of mice in the 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).Part III:Effect of early intervention with extract of HNYCF fractions on the cholinergic system and related indicators of Free radical-induced oxidative stress of APP transgenic mice.Comparing to the model group, model mice hippocampus ACh and ChAT levels were significantly reduced, AchE were significantly increased, through statistical analysis, the difference of the data was significant (P<0.05); Compared with model group, group of donepezil hydrochloride, the extract of HNYCF large and small dose group can effectively improve the content of ACh in hippocampus of mice, through statistical treatment, there was significant difference (P<0.05, P<0.01);Compared with model group, donepezil group and the extract of HNYCF large dose group could decrease the AchE consent of the hippocampus in mice, the difference was significant (P<0.05);Compared with model group, donepezil hydrochloride, the extract of HNYCF large and small dose group could significantly increase the content of ChAT in hippocampus of mice, through the statistical treatment, there was significant difference (P<0.05, P<0.01).Compared with the control group, model group mice cortex ACh, ChAT levels were significantly reduced, AchE was significantly increased, the statistical difference was statistically significant (P<0.05); Compared with model group, the extract of HNYCF large and small dose group could significantly improve the model mice in the ACh content of cortex, the statistical difference was statistically significant (P<0.05); Compared with model group, the mice treated by the administration to determine the content of AchE cerebral cortex, the statistical treatment, no significant difference (P<0.05); Compared with model group, the extract of HNYCF small dose group can improve the model mice brain cortex ChAT levels, the statistical difference was statistically significant (P<0.05),the donepezil hydrochloride group and the extract of HNYCF large dose group have no significant change in ChAT levels (P<0.05).Compared with the control group,serum SOD activity in model group were significantly different (P<0.01); Compared to the Serum SOD activity of the model group,the extract of HNYCF large and small dose group were significantly higher (P<0.05 or P<0.01); Compared to the Serum SOD activity of the model group,Donepezil hydrochloride group significantly increased (P<0.05); Compared with the control group, the Serum MDA levels of the model group,were significantly different (P<0.01); Compared to the Serum MDA levels of the model group,each treatment group of serum MDA content showed a decreasing trend, but no significant difference (P>0.05).Conclusions:Apply HNYCF effective fractions to early intervention, to a certain extent, improve the APP transgenic mouse model of spatial learning, memory and other cognitive function as well as the learning and memory ability of single passive, The mechanism may be related to the protective effect of the extract of HNYCF on the Cerebral cortex and hippocampus of the central cholinergic system function, and to improve the free radical induced oxidative stress in brain.