Study On The Pharmacological Effects And Mechanisms Of Anti-Alzheimer’s Agent DZH Based On The Multi-target-directed Drug Design Stretegy

Objective:To evaluate the multiple target action of DZH for the treatment of Alzheimer’s disease.Methods:1. To evaluate the AChE inhibitiory effedects, the estrogen-like and anti-oxidation of DZH. Iron reducing assays nitric oxide scavenging assay and NaNO2-induced hypoxia in mice were carried out to evaluate the anti-oxidant properties of DZH; The estrogen-like activity of DZH was evaluated using indices of uterus growth in juvenile mic; AChE activity in brains and plasma of normal mice were measured to evaluate the characteristic of inhibit AChE and distribute in the brain or blood of DZH in mice.2. The effects of compound DZH on working memory deficits induced by single factorMouse memory disorder induced by intraperitoneal injection of 3 mg/kg scopolamine, or intragastric administration of 0.01 ml/g 40% ethanol, and mice dementia were induced by injected with 2 μg/mouse aggregated Aβ1-42 into the right lateral hippocampal using a stereotaxic apparatus. The memory capacity of mouse were measured by a modification of step-down passive avoidance test or Morris water maze test, meanwhile the levels of SOD, AChE and other indicators in brain were detected in mice.3. The effects of compound DZH on amentia animal models induced by multi-factors(1) Thirty days after bilaterslly ovariectomy, all females mice were intrahippocampal infusions of neurotoxins (2 μg Aβ1-42,1 μg IBO). The next day, they were randomized divided into 8 groups and received drugs once a day for 4 weeks. At the end of the thirdly week the mice were investigated the ability of learning and memory by step-down passive avoidance test and Morris water maze, and 24 hours after the last treatment, The activity of SOD and other indicators in brain tissue homogenates were detected using commercial kits following the manufacturer’s instructions.(2) Five weeks after bilaterslly ovariectomy, all rats were bilaterally intrahippocampal infusions of neurotoxins (4 μg Aβ1-42,1μg IBO) or normal saline. The next day, they were randomized divided into 7 groups and received drugs once a day for 5 weeks. And working memory performance was assessed using Morris water maze test and Y maze. Meanwhile the ultrastructure of hippocampus examined with Transmission Electron Microscope and the western blotting assay was used to determine the iNOS protein expression in the brain tissue.Results:1. DZH had antioxidant activity and estrogen-like effects and inhibited the activity of AChE.DZH could scavenge nitric oxide (IC50=356.79 μM). DZH had a more reductive potential (IC50=126.69 μM) than did Trolox (IC50=653.04 μM). DZH prolonged the survival time of NaNO2-treated mice, decreased AChE activity significantly in the brain in mice (P< 0.05), but had no effect on the AChE activity in the plasma of normal mice, significantly increased uterine indices in juvenile mice (P< 0.05).2. DZH improved working memory deficits induced by single factorTreated with scopolamine or ethanol alone increased the step-down error times and shortened the step-down latency significantly in the step-down passive avoidance test in mice (P< 0.05 or P<0.01). Treated with DZH decreased the error times and prolonged the latency time in scopolamine -treated and ethanol-treated mice significantly(P< 0.05 or P< 0.01). The results of Morris water maze test showed that intrahippocampal infusions of Aβ decreased the escape latencies and the frequency of passing through the target quadrant in mice. It also decreased levels of ACh, ChAT, SOD and inceased the levels of MDA, AChE, NO in brain tissue in mice (P<0.01or P< 0.05).Treated with DZH can significantly ameliorate these indicators and histopathology under the transmission electron microscope.3. DZH could prevent, halt amentia induced by multi-factor(1) Combined infusion of Aβ1-42 2 μg and ibotenic acid 1 μg IBO in intrahippocampus increased the error times and shorten the latency time in step-down passive avoidance test, significantly prolonged the escape latencies, frequency of passing through the platform, standing time on the platform, and decreased the move distance in target quadrant during the Morris water maze test (P< 0.01 or P< 0.05), it also significantly decreased levels of ACh, SOD and increased the levels of MDA, AChE in brain tissue in ovariectomized mice (P< 0.01or P< 0.05). Treatment of DZH 9 mg/kg,3 mg/kg can significantly ameliorate these indicators.(2) Combined infusion of Aβ1-42 4 μg and ibotenic acid 1 μg IBO in intrahippocampus significantly reduce the correct ratio of learning and the correct ratio of testing in Y maze, significantly prolonged the escape latencies, frequency of passing through the platform, standing time on the platform, and decreased the move distance in target quadrant during the Morris water maze test (P< 0.01 or P< 0.05), it also significantly decreased levels of ACh, SOD and increased the levels of MDA, AChE in brain tissue in ovariectomized rats. Treatment of DZH 6 mg/kg,2 mg/kg can significantly ameliorate these indicators and histopathology under the transmission electron microscope.Conclusion:Treatment of DZH could obviously improve the function of learning and memory of amentia animals, this may partly due to the improvement of cholinergic system in brain, estrogen-like activity, good anti-oxidative activity and protect brain from the injury induced by Aβ.