Study Of Traditional Chinese Formula FZQX On Improving Learning And Memory Ability Of SAMP8 Mice And Affecting The Protein Expression Of NMDAR/PP2A/tau

Alzheimer’s disease (AD) is a neurodegenerative disease, characterized by two pathological manifestations-intracellular neurofibrillary tangles(NFTs), and extracellular senile plaques deposition. Recently, failure in Ap immunotherapy allows researchers to put more eyes on tau protein. The dynamic changes of tau pathology from entorhinal cortex to cerebral cortex are in consistency of the severity of AD. And hyperphosphorylation of tau protein plays a main role in its toxic effects. At present the treatment on tau hyperphosphorylation by traditional Chinese formula is not deep enough. In our study a traditional Chinese formula named Fuzhengquxiefang (FZQX) is used for AD treatment, because it is based on the traditional Chinese medicine pathogenesis yuanqi deficiency, blood stasis in microvascular of brain and toxin accumulation in the brain. What’s more, this formula has a good preliminary experimental basis and a theoretical foundation of Chinese medicine. It deserves a depth study as a potential drug for AD prevention and treatment. As traditional Chinese medicine treatment has the advantages of being valid for multiple targets and playing an active role in preventing disease, in this study FZQX is used for early intervention in Senescence-Accelerated Mice Prone 8(SAMP8), which can manifest AD characteristic lesions (hyperphosphorylation of tau protein, etc) at its early age, to observe the effect of AD prevention and treatment and explore the underlying mechanism. All are for the future clinical application of AD.Objective:FZQX is used as an early intervention in SAMP8 mice to observe its influence on behavior test, hippocampus pathomorphological changes and the protein expression in NMDAR/PP2A/tau pathway, so as to evaluate how much FZQX can play a role in preventing SAMP8 mice from cognitive impairment, and to explore the possible underlying mechanisms.Methods:40 3-month-old SAMP8 mice were randomly divided into four groups:SAMP8 control group (PCG), memantine control group (MG), FZQX low-dose group (FLG) and FZQX high-dose group (FHG). And a SAMR1 control group (RCG) at same month was also in the study. All of 50 mice were accepted gavage for 3 months. Groups of MG, FLG and FHG were given appropriate drug intervention, and the PCG and RCG groups received an equal volume of distilled water.3 months later all of them will have behavior tests like Morris water maze (MWM), some of them will have brain tissue pathological observation and detection of protein expression in NMDAR/PP2A/tau pathway. The first part is MWM behavior test, which consists of two parts. Spatial navigation was conducted in first 5 days at the same time to evaluate the spatial learning ability of all the mice. Spatial probe trial was conducted in last day to test their spatial memory ability. The second part is the observation of brain tissue pathomorphology after the behavior tests.5 mice of each group were randomly selected. They were anesthetized with chloral hydrate and decapitated. The brains were taken out of the skull quickly, and one side of the brain was fixed in 10% formalin for the later use of pathological section, which would be stain by Hematoxylin-Eosin (HE) staining and Nissl staining for the observation of hippocampal CA1 region. The hippocampus in other side was separated from other part of the brain, cryopreserved in liquid nitrogen, used for protein detection in NMDAR/PP2A/tau pathway by western-blot. HE staining was used for the observation of entire change of the number and morphology of neurons in hippocampal CA1 region. Nissl staining was used to observe the change of nissl bodies in their number morphology and distribution in hippocampal CA1 region to speculate the activity of hippocampal neurons. And compare the differences between groups. The third part is the detection of protein expression in NMDAR/PP2A/tau pathway by western-blot. Compare the expression of protein PP2Ac, P-PP2A, P-TAU (S396), TAU-5, NR2A and NR2B in different groups. And explore the correlationship between each protein and explain how they play a role in AD progression, so as to speculate the mechanism of how FZQX improve the memory impairment.Results:1 The influence of FZQX early intervention on SAMP8 mice behavior test. Place navigation results:At the first day, escape latency difference between groups was not statistically significant (P>0.05).For the same group, compared with the first day the escape latency at the fifth day was shortened (P<0.05 or P<0.01).Specifically, groups RCG and FHG showed an significant improvement (P<0.05 or P<0.01) in learning ability at the second day. FLG showed the tendency in the fourth day of training (P<0.05), while the MG and PCG showed the shortened escape latency statistically significant at the fifth day (P<0.05 or P<0.01). During the same day, compared with RCG, PCG escape latency in the fourth and the fifth day were longer (P<0.01). Compared with PCG, MG, GLG, FHG respectively, in the fifth day{P <0.05), fourth day (P<0.01) and the second day (P<0.05) showed a shortened escape latency with significant difference. Moreover, the strategies for each group mice to find the platform in the fifth day were different from that in the first day. Space exploration results:Compared with RCG, the number of crossing the platform (P <0.01), swimming time (P<0.05) and swimming distance of PCG at the first quadrant was decreased (P O.01); swimming distance of MG and FLG at the first quadrant was shorter than RCG significantly (P<0.05); but the data between RCG and FHG were of no significant differences (P>0.05). Compared with PCG, the groups with drug intervention performed better at the number of crossing the platform, the swim time and distance at the first quadrant, specifically MG perform better to cross the paltform(P<0.05); FHG perform better at swimming time(P<0.05).2 The observation of hippocampus pathomorphology.HE staining showed:for RCG, neurons in hippocampal CA1 showed clear boundaries, lined neatly and compactly, and had more neuron layers. While PCG lined scattered with wider intercellular surge and less neuron layers, even some nuclei disappeared, and became red neurons (tombstone cell). The neurons in CA1 region of MG, FLG, FHG showed different degrees of improvement neatly arranged with more cell layers and less red neurons.Nissl staining showed:for RCG, there were a larger number of Nissl bodies within the cytoplasm which made the cytoplasm stained dark blue, and the nuclei was stained pale blue. While for PCG, Nissl bodies within the cytoplasm disintegrate finely granular and extended outward or even disappear, which made the cytoplasm stained light. MG, FLG and FHG showed increased Nissl bodies within the cytoplasm, and darker staining3 The protein expression in NMDAR/PP2A/tau pathway. Compared with PCG, the expression of P-PP2A was increased significantly in groups RCG、MG、FLG and FHG(P<0.01). Compared with PCG, only the group RCG showed an increase for PP2A c expression(P<0.05), but there showed no significant differences between groups RCG、MG、FLG and FHG(P>0.05). Compared with PCG, TAU-5 and P-TAU (S396) expression of RCG showed no significant differences (P>0.05), but TAU-5 and P-TAU (S396) expression in MG, FHG significantly reduced (P<0.05 or P<0.01). Compared with PCG, NR2A expression of RCG, MG, FLG and FHG were significantly increased (P<0.05 or P<0.01). At last, the expression of NR2B of different groups showed no significant differences (P>0.05).Conclusions:6-month-old SAMP8 mice had shown a significant spatial memory disorder, the number of the neurons in hippocampal CA1 region was decreased significantly, and even dead, indicating the degradation of neuronal function. These lesions may be caused by the disorder expressions of its synaptic receptors and decreased expression of PP2A, resulting in hyperphosphorylation of tau protein. And the toxic effects caused by abnormal hyperphosphorylation of tau protein and synapse disorder injured hippocampal neurons in mice, thereby affecting the ability of memory storage and retrieval. FZQX may protect nerve cells via the NMDAR/PP2A/tau pathway to improve learning and memory ability in mice. The early intervention of FZQX on the SAMP8 mice has a positive effect on preventing the development of cognitive impairment.