Effect Of Dietary Restriction On Cognitive Impairment In APP/PS1 Double Transgenic Mice And Its Mechanism

BackgroundAlzheimer’s disease（AD）is the most common type of dementia in the elderly,in which progressive cognitive dysfunction and abnormal mental behavior are the main clinical manifestations.β-Amyloid plaques accumulate outside the cells to form senile plaques is one of its main pathological features.So far,the Aβ cascade is still an important theoretical basis for explaining the pathological mechanism of AD.Studies have found that the neurotoxicity caused by abnormal aggregation and accumulation of Aβ in the brain can cause neuronal and cognitive decline,then stimulate abnormal activation of glial cells,promote the expression of pro-inflammatory factor as IL-1β,TNF-α etal,and up-regulated the neuroinflammation.Furthermore,pro-inflammatory factors can up-regulate the expression of amyloid precursor protein（APP）,which increase the production and deposition of Aβ,forming a vicious circle.In addition,the pro-inflammatory factors stimulated by Aβ can also up-regulate the redox signals generated by reactive oxygen species（ROS）to further promote the occurrence of neuroinflammation and the death of neural cells.At the same time,the imbalance of intestinal flora may also play an important role in the progression of AD.Dietary Restriction（DR）is a way of dietary regulation that reduces daily food intake in a planned and regular manner without causing malnutrition in the body.Studies have shown that certain forms of dietary restriction can effectively prolong the life span of species,reduce AD-related pathological changes and improve cognitive function.However,studies have also reported that some forms of dietary restrictions can aggravate cognitive impairment in rats.It still remains inconclusice whether dietary restriction could improve cognitive function,which kind of dietary restriction should be chosen and when the intervening process should be starte,as by far the mechanism dietary restriction researches are not yet in-depth research on the mechanism of cognitive intervention.These are problems that needs to be solved urgently.Interestingly,studies found that some forms of dietary restriction play a positive role in reducing inflammatory response and oxidative stress injury,and optimizing the structure of intestinal flora.However,these positive effects of dietary restriction have not been fully evaluated in AD animal models.AimsTo study whether 30%Caloric Restriction（CR）diet can effectively improve the cognitive function of APP/PS1 mice,and preliminarily explore its mechanism,including protecting neuronal morphology,reducing Aβ deposition in the brain,alleviating neuroinflammation and optimizing the structure of intestinal flora.MethodsThe 7.5-month-old APP/PS1 mice were randomly divided into ad libitum（AL）AD model group（AD-AL group,n=7）and diet-restricted AD model group（AD-CR group,n=7）;and the same litter group was 7.5 Month-old wild-type（WT）mice were randomly divided into ad libitum wild-type group（WT-AL group,n=7）and diet-restricted wild-type group（WT-CR group,n=7）.At the same time,the AD-AL and WT-AL groups are the respective control groups of the AD-CR and WT-CR groups.After the start of the experiment,mice in the AD-CR group and WT-CR group were fed 70%of the average free food intake and continued to intervene for 6 weeks.The remaining mice in the control group eat freely.Stool samples were collected on the last day of the sixth week to study the structure of intestinal flora.The water maze experiment was started on the first day of the seventh week,and the number of days was counted as the first day of the 7th week.After the water maze test,the brain tissue was taken for pathological and neuroinflammatory studies.The following procedures including three parts:Part one:Studies on behavior and pathology:Morris water maze test was used to detect the learning and memory of of each group of mice;HE staining was used to observe the morphology of neurons in brain tissue of mice in each group;at the same time,the expression of Aβ in hippocampus and cortex was observed by ELISA and immunohi stochemi stry.Part two:Study on Neuroinflammation:The expression of IL-1β,TNF-α and ROS in hippocampus and cortical tissue were detected by ELISA.Part three:Study on the structure of intestinal flora:The fecal samples of each group were detected by 16S rDNA high-throughput sequencing.ResultsPart one:Behavioral and pathological results（1）Morris water maze experiment results:In the first 5 days of the positioning navigation training experiment,the incubation period of each group of mice decreases as the training days increase.From the second day of the experiment,the latency of AD-AL group mice was longer than that of WT-AL group mice（P<0.05）,indicating that the spatial learning ability of APP/PS1 mice decreased;the incubation period of AD-CR group mice began to shorten（P<0.05）;However,the incubation period of WT-CR group mice had no significant change（P>0.05）.In the space exploration experiment on the sixth day,AD-AL group mice had fewer times of appearing in the target quadrant,shorter running distance in the target quadrant and fewer times of crossing the platform than WT-AL group mice（P<0.05）;compared with the control group,the times of target quadrant appeared and crossing platform increased in AD-CR group mice（P<0.05）;there was no significant change in WT-CR group mice（P>0.05）.（2）HE results:The neuron cells in the hippocampus and cortex of all WT mice and in the cortex of all APP/PS1 mice were more complete,arranged relatively tightly and neatly,and the amount of neurons was also more;the number of neurons in the hippocampus of AD-AL group mice is reduced,the intercellular space is enlarged,and Nucleus pyknosis and vacuole-like degeneration can be seen;while the result of neuron number,cell morphology and arrangement in the hippocampus of AD-CR group mice was better.（3）Detect the expression of soluble Aβ40 and Aβ42 in hippocampus and cortex tissue by ELISA:The expression levels of soluble Aβ40 and Aβ42 in the hippocampus of AD-AL group mice were higher than WT-AL group mice（P<0.05）;compared to the control group,they were less in the hippocampus of AD-CR group mice（P<0.05）,there was no significant difference in the hippocampus of WT-CR group mice（P>0.05）;there was no significant difference in the cerebral cortex tissue of mice in each group（P>0.05）.（4）The percentage of Aβ-positive plaque coverage in hippocampus and cortex tissue was detected by immunohistochemistry:The percentage of area covered by Aβ-positive plaques was higher in the hippocampus of AD-AL group mice than WT-AL group mice（P<0.05）;compared to the control group,it was less in the hippocampus of AD-CR group mice（P<0.05）,there was no significant change in the hippocampus tissue of WT--CR group mice（P>0.05）;there was no significant difference in the cerebral cortex tissue of mice in each group（P>0.05）.Part two:Results of neuroinflammationThe expression of proinflammatory factors IL-1β,TNF-α and ROS were detected by ELISA:The expression of pro-inflammatory factors IL-1β,TNF-α and ROS in the hippocampus of AD-AL group mice was higher than WT-AL group mice（P<0.05）;compared to the control group,they were reduced in the hippocampus of AD-CR group mice（P<0.05）,but there was no significant change in the hippocampus of WT-CR group mice（P>0.05）;there was no significant difference in the cerebral cortex tissue of mice in each group（P>0.05）.Part three:Research results related to the structure of intestinal floraStool samples of each group of mice were taken on the last day of the 6th week of the experimental intervention,and 16S rDNA high-throughput sequencing was used for detection.The results are as follows:1.In terms of flora abundance and diversity:The Chao1 and Observed species values of AD-AL group mice were lower than WT-AL group mice（P<0.05）.Compared with the control group,the Chao1,Observed species,shannon,and Simpson values were not significantly different in the AD-CR group mice（P>0.05）,but decreased in the WT-CR group mice（P<0.05）.2.In terms of bacterial composition:（1）At the phylum level,the relative abundance is dominated by Bacteroidetes and Firmicutes in each group.Compared with the control group,the relative abundances of Deferribacteres,Patescibacteria,Tenericutes and Verrucomicrobia of AD-CR group mice decreased（P<0.05）.（2）At the genus level,compared with the control group,the relative abundances of Coriobacteriaceae_unclassified,Dubosiella and Faecalibaculum in the intestine of AD-CR group mice increased（P<0.05）,the relative abundance of Lactobacillus and Bifidobacterium slightly increased,but the difference was not significant（P>0.05）,the relative abundances of Ruminococcaceae_UCG-014,Alistipes,Streptococcus,Erysipelatoclostridium and Ruminococcus₁P decreased（P<0.05）.The relative abundance of Lactobacillus and Bifidobacterium in the intestines of WT-CR group mice increased（P<0.05）;the relative abundance of Ruminococcaceae_UCG-014 and Alipipes decreased（P<0.05）.Conclusion:1)Diet restriction can improve cognitive impairment in APP/PS1 mice,protect hippocampal neuronal cell morphology,and reduce Aβ deposition in hippocampus.2)Diet restriction can reduce the levels of pro-inflammatory factors（IL-1β,TNFα）and reactive oxygen species in the hippocampus of APP/PS1 mice,and alleviate neuroinflammation.3)Diet restriction can optimize the structure of the intestinal flora of APP/PS1 mice,reduce the relative abundance of harmful intestinal flora,and promote probiotics Enriched.