高龄孕鼠子代脑海马星形胶质细胞和神经元初级纤毛功能缺陷可能与LKB1/AMPK通路促进凋亡相关

Defective hippocampal astrocyte and neuronal primary cilia function in offspring brain of pregnant rats with advanced age might be associated with promoting apoptosis through LKB1/AMPK pathway

目的探讨高龄妊娠子代脑海马星形胶质细胞和神经元初级纤毛与凋亡的变化情况以及LKB1/AMPK通路在其中的作用。方法采用12月龄SD雌鼠与3月龄SD雄鼠交配产下子代作为高龄(advanced maternal age,AMA)组,3月龄SD雌鼠与3月龄SD雄鼠交配产下子代作为适龄(control,Ctl)组,于生后第14、28、60天(P14、P28、P60)对两组(各时间点每组14只,均雌雄各半)进行下列研究:苏木精-伊红(hematoxylin-eosin,HE)染色和尼氏染色观察海马神经元形态、数量及其内部尼氏体的变化;免疫荧光检测海马初级纤毛标志物抗ADP核糖基化因子相似蛋白-13B(anti-ADP-ribosylation factor-like protein 13B,ARL13B)表达,Western blot检测海马鞭毛内运输蛋白88(intraflagellar transport 88,IFT88)表达,评估初级纤毛发生功能变化;TUNEL染色及Western blot检测促凋亡基因Bax表达蛋白(BAX)、抑凋亡基因Bcl-2表达蛋白(BCL-2)水平,观察海马内细胞凋亡变化;Western blot检测肝脏激酶B1(liver kinase B1,LKB1)、单磷酸腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)及其对应的磷酸化蛋白表达水平,评估LKB1/AMPK通路变化。结果与Ctl组相比:HE染色、尼氏染色结果显示,AMA组在P14时海马神经元数量明显减少(P<0.05),AMA组在各时间点海马神经元形态和尼氏体数量、形态均存在异常变化;AMA组ARL13B阳性细胞发生率在P14(P<0.05)和P28(P<0.001)时均显著下降,ARL13B长度在P14和P28显著缩短(P均<0.05),同时,AMA组IFT88蛋白表达在P14时明显下降(P<0.01);TUNEL染色发现AMA组在P14时海马内细胞凋亡率明显增高(P<0.05);Western blot结果显示AMA组在P14时BAX蛋白表达显著升高(P<0.001),BCL-2蛋白表达显著降低(P<0.01);AMA组在P14时磷酸化LKB1表达明显下降(P<0.05),磷酸化AMPK表达水平在P28和P60时明显升高(P均<0.01),磷酸化mTOR表达在P60时明显下降(P<0.05)。结论高龄妊娠子代幼年期脑海马存在神经元损伤、初级纤毛发生缺陷和生长功能抑制,以及凋亡异常激活,同时高龄妊娠子代脑海马星形胶质细胞和神经元初级纤毛调控的LKB1/AMPK通路在各年龄期均存在异常活化。

Objective To investigate the changes in hippocampal astrocytes and neuronal primary cilia,and neuronal apoptosis in the offspring brain of advanced maternal age and the role of LKB1/AMPK pathway.MethodsThe 12-month-old SD female rats were mated with 3-month-old SD male rats to produce offspring as the advanced maternal age(AMA)group,and the 3-month-old SD female rats were mated with 3-month-old SD males to produce offspring as the appropriate age(Control,Ctl)group.The following studies were conducted on postnatal days 14,28 and 60(P14,P28 and P60)in the 2 groups(14 rats per group at each time point,half males and half females).HE staining and Nissl staining were used to observe the changes in the morphology and the number of hippocampal neurons and their internal Nissl bodies.Immunofluorescence assay was employed to detect the expression of hippocampal primary cilia marker,anti-ADP ribosylation factor-like protein-13B(ARL13B),and Western blotting was applied to measure the expression of hippocampal intraflagellar transport protein 88(IFT88)to assess functional changes in primary ciliogenesis.TUNEL staining and Western blotting were performed to detect the expression levels of pro-apoptotic BAX,and anti-apoptotic protein,BCL-2 to observe the changes of apoptosis in the hippocampus.Western blotting was also used to detect the expression levels of liver kinase B1(LKB1),adenosine monophosphate-activated protein kinase(AMPK),mammalian target of rapamycin(mTOR)and the levels of their corresponding phosphorylated proteins to assess LKB1/AMPK pathway changes.ResultsCompared with the Ctl group:HE staining and Nissl staining showed that the number of hippocampal neurons was significantly reduced in the AMA group at P14(P<0.05),and there were abnormal changes in hippocampal neuron morphology and Nissl body number and morphology in the AMA group at all time points;the incidence of ARL13B primary cilia was significantly decreased in the AMA group at both P14(P<0.05)and P28(P<0.001),and the length of ARL13B was obviously shortened in the AMA group at both P14(P<0.05)and P28(P<0.05);the hippocampal expression level of IFT88 was notably lower in the AMA group at the P14(P<0.01).TUNEL staining showed that the apoptotic rate of hippocampal neurons at P14 was remarkably higher in the AMA group(P<0.05),while Western blotting indicated that the expression of BAX was significantly higher(P<0.001)and that of BCL-2 was lower(P<0.01).In the AMA group,the expression of p-LKB1 was significantly decreased at P14(P<0.05),that of p-AMPK was statistically elevated at P28(P<0.01)and P60(P<0.01),and that of p-mTOR was notably decreased at P60(P<0.05).ConclusionNeuronal damage,defective primary ciliogenesis and growth inhibition,and abnormal activation of apoptosis are present in the hippocampus of juvenile offspring brain of advanced maternal age.Meanwhile,the LKB1/AMPK pathway,which is regulated by primary cilia in hippocampal astrocytes and neurons of advanced maternal age offspring brain,is abnormally activated at all ages.