远志皂苷改善APP/PS1转基因小鼠认知功能损伤的机制研究

Mechanism of tenuigenin ameliorating cognitive impairment of APP/PS1 transgenic mice

目的探讨远志皂苷对阿尔兹海默病(AD)模型小鼠认知损伤的改善作用及可能的机制。方法6月龄APP/PS1转基因小鼠18只,随机均分为远志皂苷治疗组(连续8周每天腹腔注射8 mg/kg远志皂苷)与生理盐水对照组(连续8周每天腹腔注射等体积生理盐水),同时选取同月龄野生型小鼠9只作为野生型对照组(连续8周每天腹腔注射等体积生理盐水)。8周后采用Morris水迷宫法检测各组小鼠的学习记忆水平,免疫荧光法检测小鼠脑内海马区突触后致密蛋白-95(PSD-95)的表达,硫黄素染色检测海马区老年斑水平,ELISA检测海马区β-淀粉样多肽(Aβ42)含量,Western blotting分析海马区PSD-95表达水平以及tau蛋白Ser231、Ser214、Ser396位点的磷酸化水平。结果在Morris水迷宫实验中,生理盐水对照组小鼠的逃避潜伏期自第2天起较野生型对照组小鼠明显延长(P<0.05);远志皂苷治疗组小鼠的逃避潜伏期自第3天起较生理盐水对照组明显缩短(P<0.05);与野生型对照组相比,远志皂苷治疗组小鼠的逃避潜伏期在前4天无明显改变(P>0.05),在第5天时逃避潜伏期明显延长(P<0.05)。空间搜索实验发现,生理盐水对照组小鼠定位目标位置花费时间较野生型对照组明显延长(P<0.05);远志皂苷治疗组小鼠定位目标位置时间较生理盐水对照组明显减少(P<0.05),而与野生型对照组相比则无明显改变(P>0.05)。生理盐水对照组小鼠穿越平台的次数较野生型对照组明显减少(P<0.05);远志皂苷治疗组小鼠穿越平台的次数大于生理盐水对照组(P<0.05),而与野生型对照组比较则无明显改变(P>0.05)。生理盐水对照组小鼠海马PSD-95相对含量低于野生型对照组(P<0.05);远志皂苷治疗组小鼠海马PSD-95相对含量高于生理盐水对照组(P<0.05),但与野生型对照组比较差异无统计学意义(P>0.05)。与生理盐水对照组相比,远志皂苷治疗组小鼠海马区内老年斑数量明显减少(P<0.05)。ELISA检测结果显示,与生理盐水对照组相比,远志皂苷治疗组小鼠海马区内Aβ42含量明显减少[(2.859±0.864)ng/mg vs.(5.154±0.735)ng/mg],差异有统计学意义(P<0.05)。生理盐水对照组小鼠海马tau蛋白Ser231、Ser214、Ser396位点磷酸化水平高于野生型对照组(0.947±0.131 vs.0.540±0.076、0.832±0.161 vs.0.305±0.088、0.819±0.053 vs.0.338±0.052),差异均有统计学意义(P<0.05);远志皂苷治疗组小鼠脑组织tau蛋白Ser231、Ser214、Ser396位点磷酸化水平低于生理盐水对照组(0.568±0.051 vs.0.947±0.131、0.472±0.094 vs.0.832±0.161、0.452±0.071 vs.0.819±0.053),差异均有统计学意义(P<0.05);远志皂苷治疗组小鼠海马tau蛋白Ser214、Ser396位点磷酸化水平高于野生型对照组,差异有统计学意义(P<0.05)。结论远志皂苷可上调AD小鼠PSD-95表达并改善认知损伤,其途径可能是通过减少Aβ沉积及tau蛋白的过度磷酸化来实现的。

Objective To explore the effect and potential mechanism of tenuigenin(TEN)treatment on ameliorating cognitive impairment of Alzheimer’s disease(AD)model mice.Methods 6-month-old APP/PS1 transgenic mice(n=18)were randomly divided into the saline group and TEN group(9 each),9 wild-type mice of the same age were selected as control group.Mice in TEN group were intraperitoneally injected with TEN at a dose of 8 mg/(kg.d)for 8 weeks,those in both saline group and control group were intraperitoneally injected with same dose of saline for 8 weeks.The Morris water maze test was carried out with all the mice of the three groups to assess the spatial memory level,and immunohistochemistry was performed to detect the distribution and expression of postsynaptic density protein 95(PSD-95)in hippocampal area of brain,and thioflavin staining was performed to check the senile plaque in hippocampus.ELISA was used to assess the Aβ42 level.Western blotting was used to detect the levels of PSD-95 and p-tau protein(Ser231,Ser214,Ser396)in brain.Results In the Morris water maze test,mice in saline group exhibited greater escape latency compared with that of mice in control group after the second day,the difference was statistically significant(P<0.05);mice in TEN group exhibited shorter escape latency compared with that of mice in saline group after the third day,the difference was statistically significant(P<0.05).However,no difference was observed in escape latency between TEN group and control group in the first four days(P>0.05);mice in TEN group exhibited longer escape latency compared with that of control group at the 5th day.Space search trial found that mice in saline group spent obviously longer time to find the original position of the platforms than those in control group[(40.428±3.408)s vs.(14.142±7.289)s,P<0.05].While the time for positioning target location was markedly shorter in TEN group than in saline group[(21.001±8.925)s vs.(40.428±3.408)s,P<0.05],but no significant difference existed between TEN group and control group(P>0.05).It was found that mice in saline group exhibited fewer times of crossing platform than those in control group(0.428±0.035 vs.2.285±1.380,P<0.05).While the mice in TEN group exhibited more times of crossing platform than those in saline group(1.714±0.756 vs.0.428±0.035,P<0.05),but no significant difference existed between TEN group and control g roup(P>0.05).The relative amount of PSD-95 was lower in mice of saline group than that in control group(0.570±0.700 vs.0.740±0.054),the difference was statistically significant(P<0.05);while the relative amount of PSD-95 was higher in TEN group than that in saline group with significant difference(0.800±0.098 vs.0.570±0.700,P<0.05),but no difference was observed when compared with control group(P>0.05).The number of plaques and Aβ42 level in hippocampus decreased significantly in TEN group than those in saline group[(8.889±1.692 vs.18.000±2.000)and(2.859±0.864)ng/mg vs.(5.154±0.735)ng/mg,P<0.05].As to the expression level of protein p-tau Ser231,Ser214 and Ser396 in mice hippocampus,they were higher significantly in saline group than those in control group(0.947±0.131 vs.0.540±0.076,0.832±0.161 vs.0.305±0.088 and 0.819±0.053 vs.0.338±0.052,P<0.05),while they were obviously lower in TEN group than in saline group(0.568±0.051 vs.0.947±0.131,0.472±0.094 vs.0.832±0.161 and 0.452±0.071 vs.0.819±0.053,P<0.05).The expression levels of protein p-tau Se214 and Ser396 in mice hippocampus of TEN group were higher than those in control group with statistical significance(0.472±0.094 vs.0.305±0.088 and 0.452±0.071 vs.0.338±0.052,P<0.05),but the expression level of p-tau Ser231 showed no significant difference between TEN group and control group(0.568±0.051 vs.0.540±0.076).Conclusion Tenuigenin may attenuate cognitive deficits by up-regulating the PSD-95 level in APP/PS1 mice,decrease Aβdeposition and excessive phosphorylation of protein p-tau,and might be a potential therapeutic agent for Alzheimer’s disease.