The Neuroprotective Effect And Mechanism Of Combined Use Of Metformin And Memantine On Learning And Memory In Mice With Alzheimer’s Disease

Objective:The pathogenesis of Alzheimer’s disease(AD)is complex and involves multiple pathologic cascades.Currently,the most widely accepted hypothesis is the Aβ toxicity hypothesis,which holds that with the progression of AD,Aβ aggregates in the brain and forms irreversible toxic protein aggregates,leading to a series of downstream toxic reactions.Synaptic dysfunction is the most direct cause of AD.Synaptic proteins in neurons maintain synaptic function and synaptic homeostasis,and the loss of synaptic proteins will cause synaptic dysfunction and then lead to memory impairments.AD is also known as a metabolic disease.There are frequent reports of cognitive dysfunction caused by abnormal glucose metabolism and cholesterol metabolism at an early stage.Metformin is an insulin sensitizer that has the pharmacological effects of regulating blood sugar and lipid metabolism.Memantine is an NMDAR antagonist that can inhibit cell damage caused by over-activated NMDA.Therefore,in this experiment,APP/PS1 double transgenic mice were selected as model animals,and metformin,memantine and their combinations were administered to investigate the improvement of learning and memory of mice and the protective effects of memantine combined with metformin on neuronal synaptic plasticity.It was corroborated by vitro experiments.Methods:(1)4-month-old APP/PS1 double transgenic mice(the same lair mice C57BL/6J of the same age as the blank control group)were treated with memantine(2.5 mg/kg),metformin(300 mg/kg),memantine combined with metformin(2.5 mg/kg+300 mg/kg)and normal saline(blank control group and model group were treated with equal volume of normal saline)for 4 months.The Nesting experiments,Morris water maze experiments,Fearing condition experiments and New object recognition experiments were used to detect the effects of metformin and memantine combined on the learning and memory ability of APP/PS1 mice.The glucose tolerance test was used to detect the changes of glucose metabolism in mice at each month of age.The changes of T-CHO,TG,HDL-C and LDL-C in serum of mice aged each month were detected by commercial kits,and the changes of lipid metabolism of mice were observed.The activities of MDA and SOD in mice brain homogenate were detected by commercial kits.Western Blot was used to detect AMPK,p-AMPK protein levels in cortex of mice,as well as the expressions of synaptic proteins Shank3,NR2B,NR2A,NR1 and PSD95.Immunohistochemistry and Thioflavin-S staining were used to detect Aβ deposition in the brain of mice.Commercial kits were used to detect mice serum AST/GOT,AKT,ALT/GPT,BUN,CRE,and to observe whether the long-term use of metformin combined with memantine has an effect on the liver and kidney function of APP/PS1 mice.(2)Mice hippocampal neuron cells(HT22 cells)were cultured in vitro,27hydroxycholesterol was used to create cell injury model,the pre-protection concentration of metformin was screened by MTT methods,the cell morphology was observed under microscope,and the activity of metformin on LDH and SOD of cells was tested with commercial kits.Western Blot was used to detect AMPK,p-AMPK protein levels,and the expressions of synaptic protein NR2B.The pre-protection concentration of metformin combined with memantine was screened by MTT methods,and the expression of synaptic protein PSD95 was detected by Western Blot.Results:(1)APP/PS1 transgenic mice aged 7-8 months showed behavioral impairments,decreased executive abilities,decreased spatial memories,decreased fear memories and decreased episodic memories in the Nesting test,Morris water maze test,Fearing condition test and New object recognition test.Compared with the model group,the combined use of metformin and memantine can improve the nesting behavior of mice,prolong the escape latency of mice,enhance the percentage of stiffness of mice,and improve the position preference index of mice.(2)The 4-month-old APP/PS1 double transgenic mice showed abnormal glucose metabolism.The results of glucose tolerance test showed that the combination of metformin and memantine had no correction effects on abnormal blood glucose in mice.The 6-month-old APP/PS 1 double transgenic mice showed abnormal lipid metabolism,and the serum TC and TG levels of the model group were increased.Combined use of metformin and memantine can reduce the serum TC and TG levels of the mice.(3)The 8-month-old APP/PS 1 double transgenic mice were in oxidative stress injury.Compared with the control group,the SOD activity and MDA activity in the brain of the model group were decreased.Compared with the model group,the combined use of metformin and memantine can increase SOD activity and decrease MDA activity.(4)Western Blot results showed that the phosphorylation level of AMPK protein in the cerebral cortex of the model group was reduced,the expression of NR2A protein was upregulated and the expression of synaptic proteins Shank3,NR2B,NR1 and PSD95 were downregulated.Compared with the model group,the combined use of metformin and memantine can up-regulate Shank3,NR2B,NR1,PSD95 protein expression,down-regulate NR2A protein expression,and restore synaptic homeostasis.(5)The results of ICH and Thioflavin-S staining experiments showed that compared with the model group of mice,the combination of metformin and memantine can reduce Aβdeposition in the brain.(6)The test results of liver and kidney function indexes showed that compared with the control group,the serum BUN content in model group was increased.Compared with the model group,the combined use of metformin and memantine had no significant effects on AST/GOT,AKT,ALT/GPT,BUN and CRE levels.(7)1 μmol/L of 27 hydroxyl cholesterol can cause cell damage and reduce cell survival rate.1 mmol/L of metformin can increase SOD activity,reduce LDH activity,activate AMPK protein phosphorylation,up-regulate NR2B subunit,and promote the formation of neural network.1 m mol/L metformin combined with 10 μmol/L memantine can up-regulate the expression of PSD95 protein.Conclusions:(1)The abnormal glucose metabolism of APP/PS1 double transgenic mice is earlier than the abnormal cholesterol metabolism.The combined use of metformin and memantine can correct the abnormal cholesterol metabolism effectively,but has no corrective effects on the abnormal glucose metabolism.(2)The combined use of metformin and memantine can improve the learning and memory impairment and behavioral impairment in APP/PS1 double transgenic mice.(3)The combined use of metformin and memantine restored synaptic homeostasis in APP/PS1 transgenic mice by enhancing the phosphorylation level of AMPK protein in the brain,reducing the oxidative stress level in the brain,regulating the expression of synaptic protein.(4)The combined use of metformin and memantine can reduce the deposition of Aβ in the brain of APP/PS1 double transgenic mice and reduce the toxic effect of Aβ.(5)Metformin can improve the survival rate of 27-hydroxycholesterol induced cell injury,reduce LDH activity,enhance SOD activity,enhance AMPK protein phosphorylation and regulate NR2B subunit to reduce 27-hydroxycholesterol induced cell injury.The combined use of metformin and memantine can up-regulate the expression of PSD95 protein and reduce the cell damage induced by 27-hydroxycholesterol.