Effect Of Fuzheng Xiefang Recipe On Cognition And Autophagy Function Of APP/PS1 Transgenic Mic

Alzheimer’s disease(AD)is the most common type of chronic neurodegenerative disease.Clinical symptoms mainly include cognitive impairment,memory loss,and changes in mental behavior.The pathological features of AD are senile plaques formed by the deposition of extracellular β amyloid(Aβ)and neurofibrillary tangles(NFTs)resulting from hyperphosphorylation of intracellular tau protein.At present,AD has no effective interventions to reverse its progress.Macroautophagy(henceforth called autophagy)is a major intracytoplasmic protein degradation pathway whereby cytoplasmic contents are delivered,by double-membraned vesicles called autophagosomes,to the lysosome for degradation.Macromolecules are recycled back into the cytosol to be reused as substrates for ATP generation or for protein synthesis.Autophagy therefore plays a critical housekeeping role in maintaining cytoplasmic homeostasis through the clearance of damaged proteins and organelles.Autophagy dysfunction will lead to the misfolded protein deposite in the cell.As a result,neuronal cell function defects and even death.Autophagy dysfunction has been implicated in various human pathologies including neurodegenerative diseases such as AD.As a promising target,autophagy is involved in many diseases in humans.Autophagy can be regulated by a variety of traditional Chinese medicines or herbal extracts,indicating that autophagy regulation may be an important mechanism for TCM treatment of disease.The mTOR signaling pathway is the most important regulator of autophagy.In the early stages of AD,excessive activation of the mTOR signaling can be detected.Studies have shown that the autophagy dysfunction in the brain of AD patients,and misfold protein degradation dysfunction and abnormal deposition,which may be an important pathological basis for the development of AD.In this study,APP/PS1 double transgenic mice were used as AD models to observe the regulation effect of Fuzheng Quxie Decoction(FQD)on the autophagy function of APP/PS1 transgenic mice and to explore its mechanism.Provide pharmacological basis for the clinical treatment of AD.Objectives:To investgate the effectiveness of FQD on regulation of autophagy in AD model APP/PS1 transgenic mice,and further detect the mechanisms of this function.Methods:40 3-month-old male APP/PS1 transgenic mice were randomly divided into five groups:Model group,Rapa group,and FQD high dose Group(FQD-H),FQD middle dose Group(FQD-M),and FQD low dose Group(FQD-L).Twelve male C57BL/6 mice with the same genetic background as 3 months of age were used as model control(Control).Each group of mice was given 24 weeks of gavage intervention.Control and Model groups were given equal volumes of distilled water,and the other groups were given the appropriate drug intervention.Then the effects of FQD on learning and memory ability of APP/PS 1 transgenic mice were assessed by New Object Recognition(NOR)and Morris Water Maze(MWM).HE staining and Nissl staining were used to observe the brain pathological morphology of APP/PS1 transgenic mice.Immunohistochemistry was used to detect Aβ deposition of APP/PS1 transgenic mice.Transmission electron microscopy was used to observe the ultrastructural such as autophagosomes and mitochondria in mouse hippocampal neurons in each group.The expression of LC3B,p62,Beclinl,mTOR,p-mTOR,p70S6K and 4EBP1 were detected by Western blot.Real-time PCR was used to detect the expression of related mRNAs expression.Results:1 The recognition memory of 9-month-old APP/PS1 transgenic mice can be improved by high doses of FQD.The results of NOR text showed that the discrimination index to the new object of APP/PS1 transgenic mice were less than the Control mice(p<0.01),indicating that the 9-month-old APP/PS1 transgenic mice had recognition memory deficit.After intragastric administration,the discrimination index of new object in the APP/PS1 transgenic mice of FQD-H group was significantly increased(p<0.05).2 FQD can improve the spatial learning and memory of 9-month-old APP/PS1 transgenic mice.In the orientation navigation experiment of the Morris water maze,comparing with the first day,the escape latency of all mice was shortened,indicating that all mice have a certain learning and memory ability.The results of repeated-measurement analysis of variance showed that:comparing with the control group,the escape latency of the model group was significantly longer(p<0.01),suggesting that the learning ability of the transgenic mice was impaired.Compared with the model group,the escape latency of Rapa and FQD-H group was significantly lower(p<0.05,p<0.01,respectively),suggesting that rapamycin and high doses of FQD could improve learning ability.For the same group,campared with the first day the escape latency at the fifth day was shortened(p<0.05 or p<0.01).Specifically,control group showed obvious learning ability on the third day(p<0.05).Rapa and FQD-H groups showed a significant improvement in learning ability at the second day(p<0.05),however,the escape latency were longer than control group.FQD-L group showed the trendency in the fourth day of training(P<0.05).During the same day,compared with control group,the escape latency of model group on the second day were longer(P<0.01).Compared with model group,Rapa and FQD-H group respectively,on the fourth day(p<0.05)and fifth day(p<0.01).In space probe test:Compared with control group,the crossing times,the percentage of swimming distance and swimming time in the target quadrant of the model group were downregulated(p<0.01).Compared with the model group,the crossing times of Rapa and FQD-H group were increased(p<0.05).The percentage of swimming distance and swimming time in the target quadrant of the FQD-L group were signficantly higher in(p<0.01,p<0.05).The percentage of swimming time was increased(p<0.05).3 The observation of hippocampus pathomorphology.In the control group,neurons in the hippocampal CA1 region were arranged neatly and orderly with clearly boundary,regularly morphology,clearly nucleoli and numberous Nissl bodies.While the model group lined scattered with wider intercellular surge and less neuron layers,even some nuclei disappeared.The Nissl bodies within the cytoplasm disintegrate finely granular and extended outward or even disapper,which made the cytoplasm stained light.After Rapa and FQD treatment,the neurons in CA1 region showed different degrees of improvement neatly arranged with more cell layers and more Nissl bodies with cytoplasm stained dark blue.4 FQD degraded Aβ1-42 deposition in hippocampus of 9-month-old APP/PS1 transgenic mice.In the model group,the Aβ precipitated plaques in the mouse brain tissue were dense,with deep coloration and clear boundaries.The amyloid plaques accounted for 2.45%of the hippocampal area;compared with the control group,the expression level of Aβ1-42 was significantly increased,and the plaque area and the ratio plaque were increased(p<0.01).In the Rapa group mice and FQD mice,the plaques in the brain tissue were loose,the amyloid plaques in the Rapa group accounted for 1.48%of the hippocampal area,and the amyloid plaques in FQD-L,FQD-M,and FQD-H accounted for the hippocampus area were 1.90%,1.55%,and 1.51%,respectively.Compared with the mice in the model group,the plaque area and the total plaque area ratio in the Rapa,FQD-M,and FQD-H groups were significantly reduced(p<0.05).After FQD intervention,the expression of Aβ1-42 decreased with increasing drug concentration(p<0.05).5 FQD promotes autophagy in hippocampus of APP/PS1 transgenic mice.The expression of Beclin 1 protein and mRNA in APP/PS1 transgenic mice was significantly lower than that in the control group.The level of p62 protein and the expression of p62 mRNA were significantly higher than that of the control mice.After FQD administration,the expression of Beclin 1 protein and mRNA was up-regulated;the conversion of LC3B I to LC3B II was significantly increased,the level of p62 protein and the expression of p62 mRNA was significantly decreased.6 FQD inhibits the activation of mTOR.The phosphorylation of mTOR at Ser2448 site and expression of mTOR mRNA in APP/PS1 transgenic mice increased,and the expression of 4EBP1 protein and mRNA was significantly lower than that in nontransgenic mice,indicating that mTOR in double transgenic mice was overactivated.The expression of p70S6k protein and mRNA was not significantly increased.After intervention with FQD,the phosphorylation and mRNA expression levels of mTOR in APP/PS1 transgenic mice were decreased.The expression of 4EBP1 protein and mRNA was significantly increased,and the expression level of p70S6k mRNA was lower than that before drug intervention.Conclutions:FQD activates autophagy in an mTOR-dependent manner.Induction of autophagy by FQD enhances the clearance of Aβ,improves the learning and cognitive behaviors in APP/PS1 transgenic mice.