萝卜硫素对AD模型小鼠焦虑和恐惧记忆的影响及氧化应激机制

Effects of sulforaphane on anxiety and fear memory in AD mice and its oxidative stress mechanism

目的:探究Nrf2激活剂萝卜硫素(sulforaphane,SFN)改善阿尔茨海默病(Alzheimer's disease,AD)模型小鼠焦虑情绪和恐惧记忆的行为及其机制。方法:选取AD转基因小鼠和同窝对照野生型(WT)小鼠,随机分为野生型+生理盐水组(WT+NS组)、野生型+萝卜硫素组(WT+SFN组)、AD模型+生理盐水组(AD+NS组)和AD模型+萝卜硫素组(AD+SFN组),每组12只。SFN用生理盐水(0.9%NaCl溶液)溶解并配制成浓度为1 g/L的溶液,每天在固定时间根据体质量对WT+SFN组和AD+SFN组小鼠腹腔注射SFN(10 mg/kg),WT+NS组和AD+NS组小鼠腹腔注射等体积生理盐水,1次/d,连续30 d。采用旷场实验检测小鼠的自主探究能力及焦虑行为,高架十字迷宫检测小鼠的焦虑情绪,条件恐惧实验检测小鼠的恐惧记忆行为,最后通过ELISA检测小鼠海马和脑皮质中超氧化物歧化酶(superoxide dismutase,SOD)及丙二醛(malondialdehyde,MDA)的表达水平。采用Graphpad Prism 8.0.2软件进行双因素方差分析。结果:在旷场实验中,AD+SFN组小鼠在中央区域停留时间的百分比[(9.99±0.37)%]高于AD+NS组[(8.47±0.42)%](q=3.842,P<0.05);高架十字迷宫中,AD+SFN组小鼠在SFN干预后开放臂停留时间百分比[(26.2±1.6)%]高于AD+NS组[(15.8±1.0)%](q=7.452,P<0.01)。条件恐惧实验中,各组小鼠均形成了恐惧记忆,但在测试阶段AD+SFN组小鼠僵直时间百分比[(64.5±3.8)%]高于AD+NS组[(51.0±4.3)%](q=5.266,P<0.01);除此之外,AD+SFN组小鼠的海马(q=6.370,P<0.01)和皮质(q=7.858,P<0.01)组织中反映氧化应激水平的重要指标SOD表达均增高,而MDA在海马(q=5.146,P<0.05)和皮质(q=5.833,P<0.01)中的表达均出现降低。结论:SFN可能是通过Nrf2通路抑制氧化应激,从而改善了AD小鼠的焦虑情绪和恐惧记忆。

Objective To explore the effects of sulforaphane(SFN),an activator of Nrf2,on anxiety and fear memory in Alzheimer's disease(AD)model mice and mechanism.Methods The AD mice and wild type(WT)mice with the same background were randomly divided into four groups(n=12 for each group):wild type+normal saline group(WT+NS),wild type+sulforaphane(WT+SFN),AD model+normal saline group(AD+NS)and AD model+sulforaphane group(AD+SFN).SFN was dissolved in normal saline(0.9%NaCl)and prepared solution with concentration of 1 g/L.According to body weight,mice in WT+SFN group and AD+SFN group were intraperitoneally injected with SFN(10 mg/kg),and mice in WT+NS group and AD+NS group were intraperitoneally injected with the same volume of normal saline once a day for 30 days.The open field test was used to detect the autonomous exploration ability and anxious behavior of mice.The elevated cross maze was used to detect the anxiety of mice.Conditional fear test was used to test the fear memory behavior of mice.Finally,the expression of superoxide dismutase(SOD)and malondialdehyde(MDA)in the hippocampus and cerebral cortex were detected by ELISA.Two-way ANOVA analysis was performed using GraphPad Prism 8.0.2 software.Results In the open field test,the percentage of time in central region in AD+SFN group((9.99+0.37)%)was higher than that of AD+NS group((8.47+0.42)%)(q=3.842,P<0.05).In the elevated cross maze,the percentage of time in open arm of AD+SFN group((26.2±1.6)%)was higher than that in AD+NS group((15.8±1.0)%)(q=7.452,P<0.01).In the conditional fear test,all the mice of the four groups developed the fear memory,but AD+SFN group showed higher freezing time ratio((64.5±3.8)%)than AD+NS group((51.0±4.3)%)(q=5.266,P<0.01)in the testing stage.After SFN intervention,the important indicator of oxidative stress,the expression levels of SOD in hippocampus(q=6.370,P<0.01)and cortex(q=7.858,P<0.01)of AD mice increased,while the level of MDA in hippocampus(q=5.146,P<0.05)and cortex(q=5.833,P<0.01)decreased.Conclusion SFN may inhibit oxidative stress through Nrf2 pathway,thereby improving anxiety and fear memory in AD mice.