Effects Of Banxia Xiexin Decoction On Early Autophagy In Hippocampal Neurons Of APP/PS1 Double Transgenic Mice

Alzheimer’s disease(AD)is an age-related neurodegenerative disease.In addition to the loss of synaptic neurons,the most typical pathological features of AD are abnormal protein expression,processing and co-deposition inside and outside the cell.It is worth noting that autophagy deficiency is one of the key problems leading to abnormal protein deposition,which plays a crucial role in the typical pathological changes of AD,and improving autophagy function has become one of the goals of AD treatment.Autophagy deficiency can lead to a variety of neurodegenerative diseases,including AD,and dysfunction of the autophagy system is a recognized early neuropathological feature of AD.Classical downstream target mTOR kinase of PI3K/Akt/mTOR pathway directly regulates the process of autophagosome formation and is a key factor affecting the autophagy process.PI3K-Akt can activate mTOR mediated biosynthesis,and the aberrant expression of mTOR signal transduction in neurons is the early pathogenesis of AD.Our previous study found that Banxia Xiexin Decoction,a compound traditional Chinese medicine "treated from spleen",can improve the cognitive regulation of central insulin signaling pathway and brain glucose metabolism in AD mice,and significantly increase the expression levels of PI3K and Akt proteins upstream of the classic PI3K/Akt/mTOR autophagy pathway.In addition,berberine,an effective component of Banxia Xiexin decoction,can improve the structure and plasticity of autophagy-protected neuronal synapses,and ginsenoside intervention in Aβ-treated PC 12 cells can significantly reduce the level of apoptosis by activating autophagy PI3K/Akt signaling pathway.ObjectiveThe ability to acquire and maintain memory,explore new environment and explore memory were tested by behavioral pharmacological methods.Ultrastructural changes of hippocampal neurons and autophagosomes were detected by transmission electron microscopy.The expression and distribution of mTOR,p-mTOR,Beclinl,LC3 and Aβ42 proteins and mRNA expression of downstream proteins in hippocampus were detected by molecular biological methods.To explore the effect of Baxia Xiexin Decoction on mTOR,A key autophagy signal pathway downstream of autophagy classical pathway,and detect the expression of autophagy-related proteins and Aβ in APP/PS1 double transgenic mice,so as to understand its influence on autophagy in early AD.Methods1 Groups and Administration:The experimental animals were 45 APP/PS1 double transgenic male mice at the age of 3 months and 15 C57BL/6J mice at the same age and background.APP/PS1 double transgene mice were randomly divided into three groups:model group(constant volume of 0.5%sodium carboxymethyl cellulose),donepezil group(0.92mg/kg/d)and Banxia Xiexin decoction group(6.7mg/kg/d);C57BL/6J mice were divided into normal group(same as model group),and were given continuous intragastric administration for 3 months.2 Behavioral Test:Diving platform,Y maze and Morris water maze test were conducted after intragastric administration.The ability to acquire and maintain memory was tested by the platform test,the ability to explore the new environment was tested by the Y maze test,and the ability to locate,navigate and explore the memory of spatial position under the guidance of direction judgment was tested by the Morris water maze test.3 Electron Microscopy Observation:The ultrastructural changes of hippocampal neurons and autophagosomes were detected by transmission electron microscopy after the behavior experiment.4 Immunohistochemistry and Western-blot Analysis:The expression and distribution of mTOR and p-mTOR,as well as Beclinl,LC3 and Aβ42 of autophagy were detected.5 RT-PCR Detection:Expression of BeclinlmRNA and LC3mRNA in hippocampus of autophagy downstream related proteins.Results1 Behavior Test:In the diving platform experiment,compared with the normal group,the incubation period of the model group was significantly shortened and the number of errors was significantly increased(P<0.01).Compared with the model group,the incubation period of the drug group was increased(P<0.05)and the number of errors was significantly decreased(P<0.01).In the Y labyrinth experiment,compared with the normal group,the mice in the model group had significantly fewer times of entering the new arm(P<0.01),and the mice in the drug group had a significantly increased number of entering the new arm compared with the model group(P<0.01).In the Morris water maze experiment,compared with the normal group,the escape latency and swimming distance in the model group were increased from day 1 to 5(P<0.01 or P<0.05),and the number of crossing the platform was significantly decreased after the withdrawal from the platform on day 6(P<0.01).Compared with model group,the escape latency and swimming distance of mice in drug group were shortened from day 1 to 5(P<0.01 or P<0.05),and the times of crossing the platform were increased after withdrawal from the platform on day 6(P<0.01 or P<0.05).2 Ultrastructural Detection:Under transmission electron microscopy,nucleolus degradation,chromatin pyknosis,mitochondrial structure destruction,cavitation and internal cristae fracture were observed in the neurons of model group mice,and more autophagy vesicles were observed in the neurons.Some neurons in the drug group were swollen and pyknotic.Large autophagosomes and a few autophagosomes were found in the neurons of the donepezil group,and autophagosomes including mitochondria were found in neurons of the Banxia Xiexin decoction group.3 Autophagy Protein and mRNA Expression:Immunohistochemical detection showed that compared with the normal group,the positive expression percentages of Aβ42 and mTOR in the hippocampus of model group were significantly increased,while the positive expression percentages of Beclinl and LC3 were significantly decreased(P<0.01).Compared with model group,the positive percentage of Aβ42 and mTOR in the drug group was significantly decreased(P<0.05 or P<0.01),while the positive percentage of Beclinl and LC3 was significantly increased(P<0.01).The number of mTOR,Beclinl and LC3 positive cells was consistent with the percentage of positive expression area.Western-blot analysis showed that the protein expressions of Aβ42,mTOR and p-mTOR in hippocampus of model group were significantly increased compared with normal group,while the protein expression of Beclinl and LC3II/LC3I were significantly decreased(P<0.05 or P<0.01).Protein expressions of Aβ42,mTOR and p-mTOR in the drug group were decreased,and p-mTOR was significantly decreased(P<0.05 or P<0.01),while the protein expression of Beclinl and LC3II/LC3I were increased.Beclinl and LC3mRNA detection showed that compared with the normal group,Beclinl and LC3mRNA of the model group were significantly decreased(P<0.01).Compared with the model group,the expression of the drug group showed an increased trend,and LC3mRNA of the donepezil group was significantly increased(P<0.05).ConclusionBanxia Xiexin decoction improved the cognitive level of APP/PS1 transgenic mice,which may be through acting on the classic autophagy pathway mTOR pathway,affecting the expression of downstream mTOR protein and autophagy protein LC3 and Beclinl,reducing the accumulation of Aβ42 and playing an early neuroprotective role.