Effect Of Gubenjiannao Methods On Learning Memory Ability And Caveolin-1 Expression In Hippocampal Formation Of MCI Rats

ObjectiveFrom the theory of chinese medical science, to explore the etiological factor of Mild Cognitive Impairment (MCI) is the deficiency of spleen and kidney. Follows the chinese medical principle of "treating the asthenia-syndrome by therapy of invigoration" and "protecting the healthy body, preventing the transmission of the disease", we propose the therapy of Gubenjiannao Methods is operative on treating MCI. In the experiments, To built the three mouse models of the impairment of memory acquisition, consolidated memory disorder and recovered memory disorder caused respectively by scopolamine, NaNO2 and 40% ethanol. To built MCI model of rats by the way of intra-peritoneal injection of D-galactose and NaN02. To observe the effects of Gubenjiannao Methods on learning memory, pathological, the hippocamPal levels of Caveolin-1 (Cav-1), Synaptophysin(Syn) and Synaptosomal Associated Protein of 25 kD (SNAP-25). To provide traditional Chinese medicine with experimental accordance and clinical guidance. Methods1. The normal 90 3-month-old Kunming rats were randomly divided into three groups:normal group (A group), Gubenjinnao liquid group (B guoup)and model group(C group). there are 15 rats in every group. and each group was divided into three sub-groups (10 rats in every sub-groups). After feeding in ordinary ways for one week, the normal group and model group were fed with 0.9% physiological saline, the Gubenjiannao group were fed with the Gubenjinnao liquid (containing crude drug 1g/ml). Then 10 days later, the B1 and C1 group were prepared by injection of scopolamine model of memory deficits, B2 and C2 preparation of intraperitoneal injection of sodium nitrite memory consolidation Disorder, B3 and C3 intraperitoneal injection of 40% ethanol prepared by the memory representation Disorder. A1, A2, A3 groups with intraperitoneal injection of physiological saline of equal dosage. Test the method of learning and memory abilities of mice with step-down method experiment.2. Filtering 84 3-month-old SD rats of memory with Morris water maze(MWM), randomly dividing into six groups:normal group, MCI model group, piracetam treated group, high-dose of Gubenjiannao Liquid group (HGG for short), medium-dose of Gubenjiannao Liquid group (MGG for short), low-dose of Gubenjiannao Liquid group (LGG for short), there are 14 rats in every group. After adaptively feeding all rats for one week, injecting physiological saline into abdomen cavity of mormal guoup every morning, injecting D-galactose and NaNO2 into abdomen cavity of model group, piracetam treated group, HGG, MGG and LGG every morning,40 days continuous to copy the MCI model. From the 13th model-copying days on, Filling physiological saline with normal group and model group every afternoon, piracetam treated group, HGG, MGG and LGG of rats fed with the appropriate drugs prevention every afternoon, for consecutively 28 days. Furthermore Morris water maze(MWM) was used to observing the ability of space learning memory of each group. Use the light microscope and electron microscope to observe the changes of morphology and structure in the hippocampus. Test Cav-1 expression by immunohistochemical staining, real-time PCR method was used to detect the hippocampus Cav-1, SNAP-25 expression, hippocampus Cav-1, Syn expression was detected by Western blot.Results1. Compared with rat in the model groups of learning and memory disorder acquisition, disorder strengthening and disorder representation, the level of incubation period of the rats in the normal group are higher than that of the rest groups (P<0.01), while their level of making mistakes within 5 minutes are lower than that of the model groups (P<0.01); incubation period level of the Gubenjiannao Liquor group is higher than the other moder groups (P<0.05) and their mistake-making rate within 5 minutes is lower the rest groups (P<0.05).2. Compared with normal group, the test 5 group all show different degrees characterized by decline in water intake, food intake and activition, and fur dry shriveling, thinning and falling.3. In the Morris water maze behavior experiment, the average incubation period, percentage of quadrant in the learning ability gain, the average incubation period, percentage of quadrant, the frequency in span of platform localization in space research. The results show that has significant differences between the model group and normal group, HGG and MGG(p<0.01), compared with model guoup, LGG and piracctam treated group (p<0.05), there aren't differences between the LGG and piracctam treated group(p>0.05); there are significant differences between the HGG and piracctam treated group (p<0.01).4. By the light microscopy and electrical microscopy observation, the neuron of model group was less than that of normal group, death cell could be seen clearly, the neuron decreased and deformed in model group rats, pellet vacuole denaturation, cell nucleus lessen, the mitochondria swelling and vacuole, the endoplasmic reticulum dilatation, amount of synapse decrease, the HGG can ameliorate this pathological changes obviously, there are significant differences between the HGG and piracctam treated group.5. The results of immunohistochemical staining, the hippocampus of rats treated Cav-1 expression between the normal group and model group, which shows different signs of improvements, the HGG and the MGG being the most remarkable.6. In testing the hippocampal Cav-1, SNAP-25 expression by the Real-time quantitative PCR method, the results is that in each group they show high positive revelance. The results of the normal group is significantly higher that that of the model group (P<0.01); the hippocampus of rats treated Cav-1, SNAP-25 expression between the normal group and model group, with a notable difference over the model group (P<0.01); there is a difference between the piracctam treated group and the HGG as welll as the MGG(P<0.05); while the comparison between the LGG and the piracctam treated group has no statistical meaning (P>0.05). 7. In testing the blood serum Cav-1, Syn by the Western blotting, the results is that in each group they show high positive revelance. The result of the normal group is better than that of the model group(P<0.01); the hippocampus of rats treated Cav-1, Syn expression between the normal and model groups, with a notable improvement over the model group(P<0.01); there is a difference between the piracctam treated group and the HGG as welll as the MGG(P<0.05); while the comparison between the LGG and the piracctam treated group has no statistical meaning (P>0.05).Conclusion1. Spleen and kidney deficiency is the basic etiological factor for MCI, the therapy of Gubenjiannao Method is a effective and feasible cure.2. The Gubenjiannao Method can notably improve the modeling rats' learning and memorizing ability in the learning and memory acquisition, strengthening and representation disorder.3. By combining the D-gal intraperitoneal injection and NaNO2 intraperitoneal injection, the MCI model is comparatively easier, and better to make, with a high repetition..4. The Gubenjiannao Method can effectively improve the learning and memorizing ability of the MCI rats, lighten the pathomorphological changes of the MCI rats and better the Cav-1, Syn, SNAP-25 expression in the hippocampal sections of the MCI rats.5. The Gubenjiannao Method may increased levels of presynaptic proteins such as Syn and SNAP-25 expression, regulating neuronal plasticity by working on the Cav-1 protein, thus to lower the decline of the age-related nerve formation, to protect the nerves, better the learning and memorizing ability and to delay senility.