| ObjectiveAlzheimer' s disease(AD),a neurodegenerative disease associated with aging,is characterized by progressive loss of cognitive function,behavioral changes and reduced living ability.Although AD is the most common kind of dementia,its etiology remains unclear,thus severely limiting the pharmacological treatment of AD.The increase in human longevity,coupled with the high incidence rate of AD among the elderly,has exacerbated global costs of public health care.This study sought to determine the therapeutic effect of Zhisanzhen on cognitive dysfunction,cell morphology and related proteins in AD model rats,by the means of behavioral test and molecular biology.Base on Wnt/?-catenin signaling pathway,we investigated the possible mechanism of the effect of Zhisanzhen on the improve of cognitive function of AD rats.Considering the relationship between hippocampus and learning and memory ability,we further clarify the underlying mechanisms of Zhisanzhen in the treatment of AD,and provide experimental evidence for clinical application of Zhisanzhen in treating AD.MethodsSixty rats were divided into normal group,model group,Zhisanzhen group and non-acupoint group according to the random number table method,with 15 rats in each group.Rats of model group,Zhisanzhen group and non-acupoint group were performed bilateral intracerebroventricular injection of A?1-40 combining with intraperitoneal injection of d-galactose to induce the rat model of AD.Electroacupuncture was perform on the rats of Zhisanzhen group at DU24 and bilateral GB13 used continuous wave type,with a frequency of 30Hz and a current of 1mA.The duration of electroacupuncture was 30min.Electroacupuncture was perform on the rats of non-acupoint group at points below bilateral costal arch and 10 mm away from the iliac crest,which were not acupoints,with the same parameter as that of Zhisanzhen group.Electroacupuncture were perform once a day,6 days a week,4 weeks in total.The rats of normal group and the model group were also grasped and fixed for 30 minutes without any treatment,while the Zhisanzhen group and the non-acupoint group were receiving electroacupuncture.Behavioral tests were proceeded in the fifth week,in the order of open field test,the Morris water maze and fear condition.All rats were sacrificed the next day after the end of behavioral testing.The expressions of p-tau(s396),p-tau(s404)and A? in hippocampus were observed by Western Blot,immunohistochemistry and immunofluorescence.The expressions of the key factors of Wnt/?-catenin signaling pathway,GSK-3?,?-Catenin,LRP5 and LEF1,were observed by Western Blot.Results1.In the open field test,compared with the normal group,the horizontal activity distance between the model group and the non-acupoint group was significantly increased(P<0.05),the central grid time was significantly increased(P<0.05),and the number of crossing the central area was significantly reduced(P<0.05).The number of erects was significantly reduced(P<0.05).Compared with the model group and the non-acupoint group,the horizontal activity distance of the Zhisanzhen group was significantly reduced(P<0.05),the central grid time was significantly reduced(P<0.05),and the number of crossing the central area was significantly increased.The number of erects increased significantly(P<0.05).Compared with the normal group,the number of modification of the other three groups all decreased(P<0.05).Compared with the model group and non-acupoint group,the number of modification of the Zhisanzhen group increased slightly,but the results were not statistically significant(P>0.05).There was nosignificant difference between the non-acupoint group and the model group.2.In the Morris water maze,the average escape latency of the model group and the non-acupoint group was significantly longer(P<0.05),and the average escape distance was significantly increased in the first 5 days of the navigation test(P<0.05).Compared with the model group and the non-acupoint group,the average escape latency of the Zhisanzhen group was significantly shorter(P<0.05),and the average escape distance wassignificantly lower(P<0.05).There was no significant difference between the model group and the non-acupoint group in the average escape latency and the average escape distance(P>0.05).In the space exploration experiment on the 6th day,compared with the normal group,the rats in the model group prolonged the time of crossing the platform for the first time(P<0.05),the number of crossing platforms was significantly reduced(P<0.05),the percentage of swimming distance and swimming time in the target quadrant were significantly reduced(P<0.05).Compared with the model group and the non-acupoint group,the time of crossing the platform for the first time in the Zhisanzhen group was reduced(P<0.05),and the number of crossing the platform was significantly increased(P<0.05).The percentage of swimming distance and swimming time in the target quadrant increased significantly(P<0.05).Compared with the model group,There was no significant difference between the model group and the non-acupoint in the Morris water maze test(P>0.05).3.The results of fear condition showed that during the test of conditioned fear memory,the frozen time of the normal group showed a significant increase,while the frozen time of the model group and the non-acupoint group showed no obvious trend.The Zhisanzhen group had a slight decrease after the fourth white noise stimulation,and then increased slightly,and there was a certain upward trend.Compared with the normal group,the other three groups had significantly reduced frozen time,and the difference was statistically significant(P<0.05).Compared with the model group,the Zhisanzhen group had a higher frozen time,but there was no significant difference between the other two groups(P>0.05).4.Effects of ZhiSanZhen on p-tau(s396),p-tau(s404)and A? in hippocampal tissue of AD rats:compared with the normal group,expressions of P-tau(s396),p-tau(s404)and A? were significantly increased in the hippocampal area of the model group(P<0.05).Compared with the model group,the expression of p-tau(s396),p-tau(s404)and A?in the Zhisanzhen group was significantly reduced(p<0.05).There were no significant differences in the expression of p-tau(s396),p-tau(s404)and A? in the hippocampus between the model group and the non-acupoint group(P>0.05).5.Effects of Zhisanzhen on GSK-3?,?-catenin,LRP5 and LEF1 in the hippocampus of AD rats:Compared with the normal group,the expression of GSK-3? in the hippocampus of the model group was significantly increased(P<0.05).Compared with the model group,the expression of GSK-3? was significantly reduced in the Zhisanzhen group(P<0.05).Compared with the model group,there was no significant difference in GSK-3? expression in the hippocampus of the non-acupoint group(P>0.05).Compared with the normal group,the expressions of ?-catenin,LRP5 and LEF1 in the hippocampus of the model group were significantly decreased(P<0.05).Compared with the model group,the expressions of ?-catenin,LRP5 and LEF1 were significantly increased in the Zhisanzhen group(P<0.01).Compared with the model group,there were no significant difference in the expressions of ?-catenin,LRP5 and LEF1 proteins in the hippocampus of the non-acupoint group(P>0.05).Conclusion1.Zhisanzhen can effectively improve the behavioral abnormalities in the pathogenesis of AD model rats.It can be found in the open field test that Zhisanzhen group can effectively improve the cognitive ability,exploration behavior and excitability of AD rats in the new environment,and can reduce its abnormal activity and tension.The results of Morris water maze showed that Zhisanzhen can effectively improve the spatial learning ability and memory ability of AD rats.In the fear condition test,there was an increasing trend in the acquisition of fear memory between the Zhisanzhen group and the model group,but there was no Not statistically significant difference between them,indicating that Zhisanzhen can improve the fear memory of rats to a certain extent,but it still need to increase the samplesize to furtherstudy its effects.2.Zhisanzhen can promote the expression of Wnt/?-Catenin signaling pathway by up-regulating the expression of ?-Catenin,LRP5 and LEF1 and inhibiting the activation of GSK-3?,as well as improve the learning and memory ability of AD model rats.3.It is possible that Zhisanzhen may indirectly down-regulate the expression of p-tau(s396)and p-tau(s404)and reduce A? deposition by promoting Wnt/?-Catenin signaling pathway.Thereby reducing the damaging effects of neurotoxicity,improving the learning and memory ability of AD model rats,delaying the occurrence and development of AD. |
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