The Mechanism Of Sulforaphane Improving Learning And Memory,Emotional Behavior And Hippocampal Synaptic Plasticity In APP/PS1 Transgenic Mice

Objective:Alzheimer’s disease(AD)is the most common in neurodegenerative diseases,is also the main cause of dementia.At present,There are many types of the pathogenesis of AD hypothesis,including Aβ hypothesis,Tau hypothesis,metal ion hypothesis and so on.These pathogenic factors are all related to oxidative stress and a vicious circle with oxidative stress.Studies continue to show that oxidative stress is an important contributor to aging and neurodegenerative diseases,including AD.Nrf2(NF-E2-related factor 2)is the main regulator in response to changes in cellular microenvironment in the body.Its essence is a transcription factor.Nrf2 can induce and enhance the expression of a series of detoxification enzymes and antioxidant enzymes,thus promoting the antioxidant defense ability and maintaining the oxidative balance of the body.Sulforaphane(Sulforaphane,SFN)is one of the strongest activators of Nrf2.SFN can improve the emotional abnormality and learning and memory ability of AD animal model,and the improvement of learning and memory ability can not be improved without synaptic changes.At present,the mechanism of this improvement is not clear.Therefore,in this study,we first observed the effects of SFN on emotional and cognitive behavior of APP/PS1 mice through behavioral experiments,and then recorded in vivo hippocampal long-term potentiation of(LTP)to reflect the changes of synaptic plasticity.Finally,the expression levels of synaptic related proteins SYP and PSD-95 were detected,the VII changes of dendritic number and dendritic spine density of hippocampal neurons were observed by Golgi staining,and the mechanism of synaptic changes in AD mice induced by SFN was systematically explored.Materials and Methods:10-month-old male APPswe / PS1DE9(APP/PS1)double-transgenic mice and wild-type(WT)control mice were divided into four groups: WT + Saline,WT + SFN,APP/PS1 + Saline and APP/PS1 + SFN,with 15 mice in each group.After intraperitoneal injection of SFN for one month,the improvement of cognitive behavior and emotional abnormality was observed.After the behavioral test,the levels of oxidative stress in the brain were measured and changes in the hippocampal synapses were observed.(1)The open field experiment,every mouse was placed in an open field of 50 cm in length,width and height for 5 min,and the walking track was recorded synchronously with a camera.The ratio of total distance and residence time in the middle of the open field of the mice was analyzed.(2)Elevated plus maze test,the four arms have two open arms without edge protection and two closed arms with edge protection,every mouse was placed in the same position in the central square zone to freely active for 5 min,and the walking trajectories were synchronously recorded with a camera to analyze the total number of entries and the percentage of open arm retention time.(3)New object recognition experiment: In the exploration stage,two identical objects were placed in the open field,and the exploration time of the mice around the two objects was recorded.After 6 h,in the test stage,one of the objects in the exploration stage was changed into a new object with different color and shape.The exploration time and test time were both10 min,and the exploration time between different objects was observed.New object recognition index = time to explore new object /(time to explore new object + time to explore old object).(4)Y maze experiment: The total number of arm entry and the correct rate of spontaneous alternation were recorded within 8 min,which showed the level of mice’s short-term working memory.(5)Morris water maze test: The first five days locating sailing experiment induces mice in each group to learn and remember the location of the platform,the sixth day withdraw the platform for spatial exploration,detect the escape latency of mice in each group in the locating sailing phase and the target quadrant time proportion in the empty exploration phase,which reflects the long-term spatial learning and memory ability of mice.(6)Conditioned fear experiment: In the training stage,the mice moved freely in the box for 30 s,followed by sound stimulation for 30 s,and then DC stimulation was applied to the sole of the foot in the last 2 s.Sound wave + DC stimulation was repeated for 5 times,each time at an interval of 90 s.24 hours after the end,the mice were tested,and the mice were placed in a training box with a different color from that during the training period,waiting for the mice to move for 150 s,and then given 150 s of sound stimulation,namely conditioned stimulation(conditioningstimulus,CS).The stiffness ratios of mice in 150 s before and during conditioned stimulation were recorded,and the stiffness ratios in different periods were compared.(7)ELISA: After anesthetizing the mice,the brains were removed and the cortex and hippocampus were rapidly separated on ice.Every 1 g of tissue was added with 9 ml aseptic PBS.The homogenate was thoroughly homogenized,centrifuged at3000 rpm,and the supernatant was collected.Then add samples in turn,and all the other steps are in strict accordance with the instructions of the ELISA kit from Jiangsu Enzyme Immunoassay Industry Co,Ltd.(8)In vivo hippocampal LTP electrophysiological experiment: after anesthetizing and fixing the mice,locating the hippocampal Schaffer lateral branch-CA1 area pathway of synaptic transmission,drilling the skull and inserting the stimulation electrode and recording electrode,adjusting the depth of the stimulation electrode and recording electrode to the bestposition,recordingthe field excitatory postsynaptic potential(field excitatory postsynaptic potential,f EPSP)of 30 min,and then three pairs of paired pulse stimulation induced double pulse alienation(paired-pulse facilitation,PPF).Then high frequency stimulation(HFS)was given to induce long-term potentiation(LTP)to maintain the stable record of 60 min.(9)Western blot: After LTP experiments,the mice were anesthetized and perfused the heart.The hippocampal brain tissue was rapidly removed and the protein was extracted to measure the concentration.The protein was extracted and the concentration was detected,and then the sample was put on and the glue was transferred to film.And the first antibody and second antibody were incubated after sealing.Finally,the bands were exposed by chemiluminescence method under the action of ECL luminescent solution.(10)Golgi-staining: Deeply anesthetized mice,after decapitation,the intact brain was quickly and carefully removed,and triple distilled water was used to wash the surface blood.Soak the brain in AB mixture and C solution according to FD Rapid Golgi Stain TM Kit instructions,fix the brain on the frozen section machine with three distilled water,the thickness is 50 mm,slices were dyed,washed and dehydrated with gradient alcohol.Finally,the slices were transparent with xylene and sealed with neutral resin.Results:(1)In the open field experiment,SFN did not alter the motor ability of the mice,but the percentage of central residence time of APP/PS1 mice was lower than that of WT mice(P < 0.05).After SFN treatment,the percentage of APP/PS1 mice’s residence time in the central area of open field increased(P < 0.05),indicating that SFN can improve the autonomous exploration behavior of APP/PS1 double-transfer mice.(2)The results of elevated cross maze test showed the proportion of residence time in open arm in WT mice was significantly higher than that in APP/PS1 mice(P < 0.001).After SFN administration,the percentage of residence time in the open arm of APP/PS1 mice increased(P < 0.001),indicating that SFN could alleviate the anxie-like behavior of APP/PS1 mice.(3)In the new object recognition test,exploration period after 6 hours,the NOI of APP/PS1 mice was significantly lower than that of WT mice(P < 0.05),but NOI was significantly increased after SFN treatment(P < 0.05),which indicated that SFN could effectively reverse the impairment of scene recognition memory ability of APP/PS1 mice.(4)Y-maze test,Genotype and drug intervention had no effect on the exercise ability of mice.The accuracy of spontaneous alternation in WT control group was higher than that in APP/PS1 group(P < 0.001),while the accuracy of spontaneous alternation in APP/PS1 mice was increased after SFN intervention(P < 0.01).SFN could improve the accuracy of spontaneous alternation and reduce the damage of transient working memory without affecting the voluntary motor behavior of mice.(5)In the Morris water maze test,with the increase of training days,the escape latency of mice in the four groups was shortened.Among them,the time spent finding the platform in the APP/PS1+Saline group was significantly longer than that in the WT + Saline group on the fourth day(P < 0.01)and the fifth day(P < 0.001).The escape latency of APP/PS1 mice after SFN intervention was also significantly shortened(P < 0.05).In the positioning navigation test on the 6th day,the retention time in the target quadrant of APP/PS1 group was significantly lower than that of WT group(P < 0.05),while the proportion of APP/PS1+SFN group was increased(P < 0.05),indicating that the memory of platform location of APP/PS1 mice was enhanced after SFN treatment.(6)During the training period,after five times of combined stimulation of sound and electrical stimulation,the four groups of mice completed the learning and memory of fear,and there was no significant difference in the stiffness ratio(P > 0.05).On the second day of the test,the stiffness ratio of APP/PS1+Saline group was significantly lower than that of WT + Saline group(P < 0.01).After SFN treatment,the rigidity ratio of APP/PS1 mice increased(P < 0.05).SFN can improve the fear memory loss of APP/PS1 mice.(7)ELISA results showed that the SOD level of APP/PS1 mice decreased in the hippocampus(P < 0.001)and cortex(P < 0.01),and recovered after SFN treatment(hippocampus: P < 0.01;cortex: P < 0.01).Compared with WT mice,MDA levels of APP/PS1 mice were increased in hippocampus(P <0.01)and cortex(P < 0.05),but decreased after SFN treatment(hippocampus: P < 0.05;cortex: P < 0.01),indicating that SFN can reduce the oxidative stress level of APP/PS1 mice.(8)The percentage of f EPSP slope of 30 min and 60 min in APP/PS1+Saline group were significantly lower than those in WT + Saline group(P < 0.05),while those in APP/PS1 + SFN group after SFN treatment were significantly lower than those in APP/PS1 + SFN group(P < 0.05).There was no difference in PPF among each group,which indicated that SFN reversed LTP depression in APP/PS1 mice through postsynaptic mechanism.(9)The expression levels of PSD-95 and SYP in the APP/PS1 + Saline group were both lower than those in the WT+ Saline group(P < 0.001),while the expression levels in the APP/PS1+SFN group were significantly increased(P < 0.001),suggesting that the mechanism of SFN enhancing the cognition of APP/PS1 mice may be related to the improvement of synaptic function.(10)The results of Golgi staining showed that the number of dendrites in APP/PS1 mice was significantly less than that in WT mice,and the number of dendritic branches in APP/PS1 mice was significantly less than that in WT mice(P < 0.05).After SFN treatment,the number of dendrites in APP/PS1 mice was significantly increased(P < 0.05).The density of dendritic spines in the APP/PS1 normal saline group was significantly lower than that in WT mice(P < 0.001),and recovered somewhat after treatment(P < 0.01).Conclusion:SFN can effectively improve the cognitive behavior and emotional abnormality of APP/PS1 mice.The mechanism may be that reducing oxidative stress injury can enhance the synaptic plasticity of hippocampus,increase the expression of synaptic related proteins SYP and PSD-95,and increase the number of dendrites and the density of dendritic spines in hippocampal CA1 region to improve structural plasticity.Therefore,this study focused on oxidative stress,to observe the protective mechanism of SFN on synaptic plasticity in AD,and to provide a new strategy for the treatment of AD.