铁超载对大鼠认知功能及海马神经元自噬的影响

Effects of iron overload on cognitive function and autophagy of hippocampal neurons in rats

目的探讨铁超载对大鼠认知功能的影响及其可能的内在机制。方法将30只8周龄雄性SPF级Srague Dawley(SD)大鼠随机分为两组,铁超载组(IO组)及空白对照组(Sham组),每组n=15。IO组大鼠采用右旋糖酐铁腹腔注射100 mg/(kg·d)持续28 d。利用Morris水迷宫方法检测两组大鼠的认知功能;Western blot法检测两组大鼠海马内的转铁蛋白受体1(transferrin receptor 1,TfR1)、自噬相关蛋白p-AMP-蛋白激酶(p-AMP-activated protein kinase,p-AMPK)、微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、B细胞淋巴瘤-2相互作用蛋白1(Bcl-2 interacting protein 1,Beclin1)的变化;免疫荧光观察大鼠海马内LC3、Beclin1的表达;HE染色观察各组大鼠海马内的神经元形态变化。透射电镜法观察大鼠海马区神经元内自噬小体数量及内质网形态的改变。所得数据应用SPSS 20.0统计软件进行重复测量方差分析和t检验。结果Morris水迷宫定位航行实验结果显示,两组大鼠逃避潜伏期的组别和训练时间交互作用显著(F=3.55,P<0.01)。简单效应分析显示,与Sham组[(28.09±18.41)s、(21.42±15.53)s、(16.96±8.35)s、(10.24±3.75)s]相比,IO组大鼠在第2~5天的平均潜伏期[(56.68±30.65)s、(58.21±36.09)s、(36.58±13.54)s、(27.29±14.30)s]明显延长(t=8.57,6.81,9.51,7.12,均P<0.01)。在第6天撤掉平台的空间探索实验中,与Sham组[(41.89±3.89)%]相比,IO组在平台象限内停留的时间百分比[(25.46±3.56)%]明显降低(t=24.06,P<0.01)。Western blot结果显示,IO组大鼠海马内TfR1(2.10±0.48)、p-AMPK(0.74±0.10)、LC3(1.11±0.40)、Beclin1(1.05±0.20)的相对表达水平较Sham组[TfR1(0.11±0.18)、p-AMPK(0.19±0.02)、LC3(0.22±0.11)、Beclin1(0.17±0.02)]明显升高(t=1.58,14.58,10.06,20.65,均P<0.01)。HE染色结果显示,与Sham组相比,IO组大鼠海马内神经元细胞排列稀疏、形态不规整、细胞数明显减少。透射电镜结果显示,与Sham组相比,IO组大鼠海马内神经元内自噬小体数量增多。结论铁超载可能通过提高海马区内的自噬水平发挥其神经毒性作用,引发认知功能障碍。

Objective To explore the effect of iron overload on the cognitive function of rats and its possible internal mechanism.Methods Thirty 8-week-old male Sprague Dawley rats of SPF degree were randomly divided into 2 groups,iron overload group(IO group)and control group(Sham group),with 15 in each group.The rats in IO group were injected intraperitoneally iron dextran(100 mg/(kg·d))for 28 days.The cognitive function of rats was detected by Morris water maze method.Western blot method was used to detect the expression of TfR1 and autophagy-related protein p-AMPK,LC3 and Beclin1 in the hippocampus of rats.Immunofluorescence was used to observe the expression of LC3 and Beclin1 in the hippocampus of rats.HE staining was used to observe the morphological changes of neurons in the hippocampus.Transmission electron microscopy was used to observe the number of autophagosomes and the morphology of endoplasmic reticulum in hippocampus.The software of SPSS 20.0 was used for repeated measurement ANOVA and t-test.Results Morris water maze test showed that there were significant interaction between the group factor and training time factor of escape latency(F=3.55,P<0.01).And the simple effect analysis showed that compared with the Sham group((28.09±18.41)s,(21.42±15.53)s,(16.96±8.35)s,(10.24±3.75)s),the average escape latency of rats(2nd-5th day)in IO group((56.68±30.65)s,(58.21±36.09)s,(36.58±13.54)s,(27.29±14.30)s)were significantly longer(t=8.57,6.81,9.51,7.12,P<0.01).The platform was removed on 6th day of the space exploration experiment,compared with the Sham group((41.89±3.89)%),the percentage of time spent in the target quadrant of IO group((25.46±3.56)%)was significantly decreased(t=24.06,P<0.01).Western blot showed that the relative expression levels of(TfR1(2.10±0.48),p-AMPK(0.74±0.10),LC3(1.11±0.40),Beclin1(1.05±0.20))in IO group in the hippocampus of the rats were significantly higher than those of the Sham group(TfR1(0.11±0.18),p-AMPK(0.19±0.02),LC3(0.22±0.11),Beclin1(0.17±0.02))(t=1.58,14.58,10.06,20.65,P<0.01).HE staining showed that compared with the Sham group,the neuron in the hippocampus of the IO group were sparsely arranged,morphologically irregular,and the number of the neurons was significantly reduced.Transmission electron microscopy showed that compared with the Sham group,the number of autophagosomes in the hippocampus of IO group was increased.Conclusion Iron overload may exert its neurotoxic effect by increasing the level of autophagy in the hippocampus,causing cognitive dysfunction.