地黄饮子改善AD小鼠星形胶质细胞损伤调节突触结构功能的作用机制

Mechanism of Dihuang Yinzi in Improving Astrocyte Injury and Regulating Synaptic Structure and Function in AD Mice

目的:探讨地黄饮子改善AD小鼠脑中星形胶质细胞损伤及保护突触结构功能的作用机制。方法:40只4月龄雄性APP/PS1转基因小鼠随机分为模型组、模型+地黄饮子(2.5 g·kg^(-1))组,每组20只;40只同背景、同月龄C57BL/6J小鼠,随机分为正常组、正常+地黄饮子(2.5 g·kg^(-1))组,每组20只。正常+地黄饮子组和模型+地黄饮子组给予地黄饮子灌胃,正常组和模型组均给予等量无菌生理盐水,每天灌胃1次,连续给药150 d。采用小鼠避暗箱实验和Y迷宫自由交替实验测试小鼠学习记忆能力。采用液质联用质谱仪(LC-MS)测定样品中谷氨酸(Glu)和谷氨酰胺(Gln)含量;长时程增强(LTP)实验检测脑组织的突触可塑性;蛋白免疫印迹法(Western blot)检测小鼠脑组织兴奋性氨基酸转运蛋白2(EAAT2),突触后致密物-95(PSD95)及突触素(SYN)的表达水平;免疫荧光法评估EAAT2的定位及表达;比色法检测小鼠脑组织Na^(+)-K^(+)ATP酶活性。结果:与正常组比较,模型组小鼠在避暗箱实验中停留潜伏期显著缩短(P<0.01),错误次数显著增多(P<0.01),Y迷宫实验中自发交替反应率显著减少(P<0.01),进臂总次数差异无统计学意义,EAAT2,PSD95和Synaptophysin表达量显著降低(P<0.01),Na^(+)-K^(+)ATP酶活性显著下降(P<0.01),Glu水平显著升高(P<0.01),Gln水平显著降低(P<0.01),相对群峰电位(PS)幅值和兴奋性突触后电位(EPSP)斜率明显降低(P<0.05,P<0.01),而正常+地黄饮子组小鼠的上述实验指标差异没有统计学意义。与模型组比较,模型+地黄饮子组小鼠在避暗箱实验中停留潜伏期明显增加(P<0.05),错误次数显著减少(P<0.01),Y迷宫实验中自发交替反应率显著升高(P<0.01),进臂总次数差异无统计学意义,EAAT2,PSD95和Synaptophysin表达量显著升高(P<0.01),Na^(+)-K^(+)ATP酶活性显著增加(P<0.01),Glu水平显著降低(P<0.01),Gln水平显著升高(P<0.01),相对PS幅值和EPSP斜率显著升高(P<0.01)。结论:地黄饮子通过保护星形胶质细胞,增加Glu摄取,减少其异常累积,保护突触结构与功能,改善AD小鼠认知功能障碍。

Objective:To investigate the mechanism of Dihuang Yinzi in improving astrocyte injury and protecting synaptic structure and function in the brain of Alzheimer’s disease(AD)mice.Method:Forty male APP/PS1 transgenic mice aged four months were randomly divided into a model group and a model+Dihuang Yinzi(0.25 g·kg^(-1))group,with 20 mice in each group.Forty C57BL/6J mice with the same background and same age were randomly divided into a control group and a control+Dihuang Yinzi(0.25 g·kg^(-1))group,with 20 mice in each group.The mice in the control+Dihuang Yinzi group and the model+Dihuang Yinzi group were administered with Dihuang Yinzi by gavage,and those in the control group and the model group received an equal volume of sterilized normal saline,once a day for 150 days.The learning and memory ability of mice was tested by the light-dark box test and Y-maze spontaneous alternation test.The content of glutamate(Glu)and glutamine(Gln)was measured by liquid chromatography-tandem mass spectrometry(LC-MS).Long-term potentiation(LTP)assay was used to detect synaptic plasticity in brain tissues.The protein expression levels of excitatory amino acid transporter 2(EAAT2),postsynaptic density protein95(PSD95),and synaptophysin(SYN)in brain tissues were measured by Western blot.Immunofluorescence was used to assess the localization and expression of EAAT2.Colorimetry was performed to detect Na^(+)-K^(+)ATPase activity in mouse brain tissues.Result:As compared with the control group,the model group showed shortened residence latency(P<0.01),increased number of errors(P<0.01)in the light-dark box test,reduced spontaneous alternation behaviors(P<0.01),no significant difference in the total number of arm entries in the Y-maze spontaneous alternation test,down-regulated expression of EAAT2,PSD95,and SYN(P<0.01),blunted activity of Na^(+)-K^(+)ATPase(P<0.01),up-regulated Glu level(P<0.01),down-regulated Gln level(P<0.01),and reduced relative population spike(PS)amplitude and the slope of excitatory postsynaptic potential(EPSP)(P<0.05,P<0.01),while the above experimental indexes were not significantly different in the control+Dihuang Yinzi group.Compared with the model group,the model+Dihuang Yinzi group displayed prolonged residence latency(P<0.05),decreased number of errors(P<0.01)in the light-dark box test,increased spontaneous alternation behaviors(P<0.01),no significant difference in the total number of arm entries in the Y-maze spontaneous alternation test,up-regulated expression of EAAT2,PSD95,and SYN(P<0.01),potentiated activity of Na^(+)-K^(+)ATPase(P<0.01),reduced Glu level(P<0.01),up-regulated Gln level(P<0.01),and increased PS amplitude and EPSP slope(P<0.01).Conclusion:Dihuang Yinzi can improve cognitive dysfunction in AD mice by protecting astrocytes,increasing Glu uptake to reduce its abnormal accumulation,and protecting synaptic structure and function.