Anti-Inflammatory Effect Of Semaglutide,a Novel Long-Acting GLP-1 Analogue,in APP/PS1/tau Transgenic Mice Of Alzheimer’s Disease

Objective:To observe whether Semaglutide,a novel long-acting GLP-1 analogue,can effectively ameliorate the cognitive impairment and the pathological features in the 3xTg-AD mouse model of Alzheimer’s disease（AD）,and further explore the possible mechanism of its neuroprotective effect at the cellular and molecular level to open up new ideas and strategies for the prevention and treatment of AD.Methods:1.In vivo transgenic mice experimentThe 9-month-old 3xTg transgenic mice and wild type C57BL/6 mice were randomly divides into four groups:WT+PBS,3xTg+PBS,WT+Semaglutide and3xTg+Semaglutide.Semaglutide（25nmol/kg）orequivalent PBS was intraperitoneally injected every two days for 30 days,and then proceeded with a series of behavioral experiments.First,the open field experiment was carried out to remove the influence of the obstacle of exercise ability and exploration ability on the experimental results.Y maze experiment was recorded the next day,and the correct spontaneous alternation rate was used as the evaluation index of working memory ability of mice.A total of 6 days of Morris water maze（MWM）experiment was carried out,in which the first five days were used to locate the navigation experiment,and the escape latency and swimming trajectory map of each group were recorded daily to evaluate the improvement of long-term spatial learning;On the sixth day,the space exploration experiment was carried out.The percentage of swimming time in the target quadrant and the times of crossing the platform were observed to evaluate the memory ability of mice.Finally,the visual platform test was conducted to detect the swimming ability and visual acuity of each group of mice,and to exclude its influence on the water maze test.At the end of the behavioral experiment,the mice were killed.Half of them was used in immunohistochemistry（IHC）for testing the distribution and amount of amyloid-βto evaluate the effect of Semaglutide on the pathological impairments of AD.Another half of mice was used in qPCR and ELISA techniques for determining the expressions of GLP-1R,CD86,CD206,IL-1,TNF-and other related inflammatory factors,the expression levels of M1 and M2 glial cell marker related proteins were analyzed by western blot,so as to illustrate the possible mechanismofthe neuroprotective effect of Semaglutide.2.In vitro cell experimentCultured BV₂ microglias were divided into groups as follow:BV₂+Vehicle,BV₂+Aβ,and BV₂+Semaglutide+Aβ.（1）Cell morphology observation、cell viability determination and apoptosis detection were used to verify the establishment of Aβ-activated BV₂ cell model.The effects of Semaglutide on Aβ-activated BV₂ cells and the release of inflammatory cytokines were measured to evaluate the neuroprotective effect of Semaglutide.（2）qPCR,ELISA and Western blot were used to investigate the effects of Semaglutide on neuroinflammatory factors in Aβ-induced BV₂ cells,and the key proteins were verified.Meanwhile,The effect of Semaglutide on GLP-1 receptor expression in BV₂cells was detected.Results:1.In vivo study（1）Open filed test:There was no statistical difference in total distance and time spend in the centre area among four groups（P>0.05）.（2）Y maze test:There was no difference in the total number of arm entries among the four groups（P>0.05）.Compared with WT+PBS control group,the percentage of spontaneous change was lower in the 3xTg+PBS group（P<0.001）,while the spontaneous change rate was higher in the 3xTg+Semaglutide group after medication（P<0.001）.（3）Classical Morris water maze test:In the 1-5 days of hidden platform test,as the number of days of training,escape latencies varying degrees of reduction.Significant difference（P<0.01）3 x Tg+PBS escape latencies with WT+PBS group of mice,and after the treatment Semaglutide 3xTg mice at 3xTg+PBS mice was shorter in the second day;On the third day,a significant difference was formed with 3xTg control mice（P<0.01or P<0.001）.The percentage of time spent in the target quadrant of the 3xTg+PBS group was significantly less than that of the WT+PBS group（P<0.001）;the percentage of swimming time in the target quadrant of the 3×Tg+Semaglutide group was more significant than that of the 3xTg+PBS group Increase（P<0.001）in space exploration experiments.There was no significant difference in the time used by the four groups of mice to find the platform（P>0.05）in the visual platform experiment.（4）qPCR,ELISA,Western blot experiments:In the 3×Tg+PBS group,the levels of CD86,IL-1β,and TNF-αin the hippocampus and cortex tissues of mice increased to varying degrees（P<0.01）,and the expression levels of CD206,IL-4,and IL-10 decreased（P<0.01）compared with the WT+PBS group,the expression levels of the above factors have changed to varying degrees（P<0.01）after treatment with Semaglutide.The expression of GLP-1R in the brain of 3×Tg mice decreased（P<0.01）,and there was no significant change after administration of Semaglutide（P>0.05）.（5）Immunohistochemical:A large amount of Aβplaques were deposited in the hippocampus of the 3×Tg+PBS group mice,which was significantly different from that of the WT+PBS group（P<0.001）;The Aβof 3×Tg mice treated with Semaglutide was significantly lower than that of 3×Tg mice without drug treatment（P<0.01）.2.In vitro study（1）CCK8 experiments:Aβhas obvious cytotoxic effect on BV₂ microglia.（2）qPCR,ELISA,Western blot experiments:In the BV₂+Aβmodel group,the levels of CD86,IL-1β,and TNF-αincreased to varying degrees（P<0.01）,and the expression levels of CD206,IL-4,and IL-10 decreased（P<0.01）compared with BV₂+vehicle Ratio;the expression levels of these factors have changed to varying degrees（P<0.01）after Semaglutide treatment.After Aβstimulation,the expression level of GLP-1R decreased,and the receptor expression level did not change significantly after treatment with Semaglutide.Conclusions:Semaglutide chronic treatment can improve the short-term working memory and long-term spatial learning and memory ability of 3xTg-AD mice,and reduce the deposition of Aβplaques in the hippocampus of mice.Without changing the expression of its action receptor,Semaglutide exerts a similar effect to GLP-1,and effectively antagonizes the cytotoxicity caused by Aβand reduces the cell damage,which may be performed by inducing the transformation of Microglia M1 type to M2 type.Therefore,we speculate that Semaglutide plays a neuroprotective role by activating GLP-1 receptors and improving the release of inflammatory mediators in the brain microenvironment.