Metformin Treatment Prevents Amyloid Plaque Deposition And Memory Impairment In APP/PS1 Mice

BackgroundAlzheimer’s disease(AD)is a neurodegenerative disorder that causes memory loss and impaired cognitive ability.Themajor histopathological hallmarks of AD is amyloid-β(Aβ)deposition,neurofibrillary tangles,and neuronal loss,accompanied by neuroinflammation.A large number of researches have been reported that neuroinflammation was responsible for the occurrence of AD.Abnormal activation of glial cells causes the release of pro-inflammatory cytokines,such as IL-1β and TNF-α,and studies show that these pro-inflammatory factors in animal models can in turn strengthen the expression of APP(amyloid precursor protein)and the formation of Aβ.Aβ is mainly deposited in the hippocampus and cortex,causing damage to these regions and causing neurodegeneration.Metformin is a first-line glucose-lowering drug with anti-inflammatory and immunosuppressive effects.Its anti-inflammatory effect is reported in many experimental models in vivo and in vitro.Metformin is an activator of AMPK.It is reported that AMPK activation can inhibit the deposition of Aβ,improve mitochondrial function and inhibit oxidative stress,and play a beneficial role in the mouse brain.Research shows that metformin can improve AD related nerve pathological changes,and alleviatecognitive impairment,however,there are also reports that the use of metformin can aggravate Aβ deposition,more interestingly,studies have found that metformin is harmful to male mice but is good for the female mice.This suggests that perhaps gender is an important factor in dealing with Alzheimer’s disease.In addition,metformin can improve the apoptosis of the primary hippocampal neurons caused by Aβ,and has a neuroprotective effect.Multiple studies have found that the plaque is surrounded by glial cells.There are also reports that metformin strengthens the phagocytosis of microglia by dependent on AMPK.APP/PS1 mice can simulate Alzheimer’s disease very well and amyloid plaques can be seen in its hippocampus and cortical areas,and their cognitive function is significantly lower.Therefore,the model is widely used in the study of Alzheimer’s disease.However,the role of metformin in this model is still unknown.Aims:Whethermetforminbeneficiallyaffectscognitivefunction and attenuate inflammatory reaction in the APP/PS1 miceas well asunderlyingmechanisms.Methods:Part one:Using water maze experiment to test the learning and memory of each group of mice,the immunohistochemical staining and ELISA were used to observe the expression of Aβ in mice in each group.Part two:The expression of NeuN protein was detected by Western Blot,and BRDU,NeuN and DCX were detected by immunohistochemical methods to check the neurogenesis,and the neural apoptosis in the hippocampus was detected by using the Tunnel kit.Part three:Western Blot and RT-qPCR were used to detect the expression of inflammatory factors,and immunohistochemical method was used to observe the morphology and number ofglial cells in hippocampal and cortical and their positionrelationship with Aβ.Part four:Western Blot was used to detect the expression of pathway proteins,and immunohistochemistry was used to observe the relationship between various cells in the hippocampus and AMPK.Results:Part one:In the water maze test,the results showed that the APP/PS1 mice showed significant spatial learning impairment compared with WT mice.However,the escapelatency in metformin-treated APP/PS1 mice was significantlyshortened compared to the saline-treated APP/PS1 mice with the increase of training days.In addition,swimming speed remained unaltered among the three groups.These results show that metformin treatment can improve the spatial learning ability of APP/PS1 mice.Compared with WT mice,the APP/PS1 mice had less time to search in the target quadrant and less time to cross the platform.However,the metformin treatment APP/PS1 mice significantly increased the search time in the target quadrant and the frequency of crossing the platform compared with the APP/PS1 mice.The results of immunohistochemical experiments showed that the coverage of Aβ 40 and Aβ 42 in the hippocampal and cortex of APP/PS1 mice was significantly higher than that in WT group.In metformin-treated APP/PS1 mice,the density of Aβ 40-positive and Aβ 42-positive plaques was significantly reduced compared to APP/PS1 mice.The results of ELISA showed that the soluble Aβ 40 and Aβ 42 in the APP/PS1 mice was significantly higher than that in the WT group.Treatment with metformin dramatically reduced soluble Aβ 40 and Aβ 42 levels in the hippocampus and cortex comparison to APP/PS1 group,respectively.Part two:The results of immunohistochemistry showed that the number of NeuN positive cells in the hippocampus of the APP/PS1 mice after metformin treatment was significantly increased compared with that in the control group of APP/PS1.At the same time,the WB results also showed that the expression of NeuN protein in the hippocampus of the APP/PS1 mice treated with metformin was significantly increased compared with that in the control group of the APP/PS1.Consistent with the above results,TUNEL results also showed that metformin significantly reduced the apoptosis of neurons in the hippocampal CA1 region of the APP/PS1 mice.These results showed that metformin was protective for the neurons in the hippocampus of APP/PS1 mice.The results of BRDU and DCX markers showed that the BRDU and DCX positive cells in the hippocampal dentate gyrate of the mice in the metformin treatment group were significantly more than those in the APP/PS1 model group.Part three:The immunohistochemical results showed that more activated astrocytes and microglia were observed in the hippocampus and cortex of the APP/PS1 mice compared with the WT control group.Moreover,in the metformin treatment APP/PS1 mice,activated astrocytes and microglial hyperplasia in the hippocampus and cortex were inhibited and the number decreased compared with the APP/PS1 control mice.To see the activated microglia and astrocytes around the Aβ,we use Iba1 or GFAP and Aβ for the double stain,it is found that a large amount of activated astrocytes and microglia gathered around Aβ in the APP/PS1 mice brain,however,metformin treatment group were relatively reduced.These results indicate that metformin can effectively reduce the activation of microglia and astrocytes in the APP/PS1 mice.Our ELISA results showed that the expression levels of IL-1β and TNF-αin the hippocampus and cortex of the APP/PS1 model group were significantly increased compared with those in the WT control group,while IL-4 showed no significant change.Interestingly,there was a dramatic decrease in IL-1β and TNF-αin the hippocampal and cortex of the metformin treatment group APP/PS1,while IL-4 increased compared with the mice in the APP/PS1 model group.And the results of RT-qPCR were also consistent with those of ELISA.Part four:WB results showed that p-ampk decreased significantly in the hippocampus of the APP/PS1 mice compared with the WT mice.However,the expression of p-mTOR,p-s6 k,p-p65NFκB and Bace1 were increased.It is worth noting that the expression of p-ampk in the hippocampal of the APP/PS1 after metformin treatment was significantly increased while p-mtor,p-s6 k,p-p65NFκB and Bace1 decreased.These results indicate that the changes of these proteins may be associated with the neuroprotective effect of metformin.The results of immunofluorescence were consistent with our WB results,and p-ampk was relatively less expressed in the hippocampus of the APP/PS1 mice,however,there was an increase in the hippocampus of the APP/PS1 treated by metformin.In the hippocampus of metformin treatment APP/PS1,the majority of p-ampk staining was in Iba1 positive microglia and GFAP positive astrocytes.Conclusion:1)Metformin can reduce the expression of Aβ40 and Aβ42 and improve spatial learning and memory damage in APP/PS1 mice.2)Metformin can promote adult hippocampal neurogenesis and inhibit neuronal apoptosis in the APP/PS1 mice.3)Metformin can inhibit neuroinflammation in the APP/PS1 mice.4)Metformin plays a neuroprotective role through the AMPK/mTOR signaling pathway and plays a role in inhibiting inflammation through AMPK/P65-NFκB signaling pathway.