前脑神经元芳香酶敲除对自然衰老雌性大鼠海马CA1区胶质细胞分化的影响

Effect of forebrain neuronal aromatase knockout on glial cell differentiation in CA1region of hippocampus in naturally aging female rats

目的 芳香化酶(Aromatase)为雌激素合成关键酶,本研究采用前脑神经元芳香化酶敲除雌性大鼠(FBN-Aro-KO, KO),观察随衰老海马CA1区神经干细胞向星形胶质细胞、小胶质细胞分化的变化,揭示脑源雌激素的神经保护作用,为神经退行性脑病的防治提供重要靶点。方法 动物分组为(1)自然衰老雌性大鼠:1、3、6、14和18Mon;(2)6、18Mon KO大鼠和年龄匹配野生型大鼠(WT)。免疫荧光染色检测神经干细胞标志蛋白(SOX2)、星形胶质细胞标志蛋白(GFAP)、小胶质细胞标志蛋白(Iba1)、轴突和树突骨架蛋白(MBP和MAP2)水平。结果 在自然衰老组,1、3、6Mon大鼠SOX2+细胞数差异无统计学意义(P>0.05);与之比较,14Mon和18Mon组SOX2+的细胞数显著降低;GFAP荧光强度、SOX2+与GFAP+的共定位表达以及SOX2+/GFAP+细胞的增殖率升高(P<0.05)。在18Mon组,与WT组比较,KO组GFAP荧光强度、SOX2+细胞数、SOX2+与GFAP+共定位细胞数以及GFAP+增殖率均显著升高(P<0.01);且与WT组比较,KO组星形胶质细胞的总体积显著增大(P<0.05)。在自然衰老组,与1Mon组比较,14和18Mon组Iba1免疫荧光强度显著增强(P<0.0001);与18Mon WT组比较,KO组SOX2+与Iba1+的共定位表达、Iba1荧光强度、小胶质细胞的总体积显著增多(P<0.05)。在6Mon组,与WT组比较,KO组SOX2+与GFAP+的共定位表达、GFAP的荧光强度及增殖率均显著增高(P<0.01);但MAP2和MBP的荧光强度显著降低(P<0.01)。结论 自然衰老过程中,雌性大鼠海马CA1区神经发生在成年后显著较少,中年后细胞增殖降低;但胶质细胞增殖率增加。前脑神经元脑源雌激素长期缺失(敲除合成关键酶Aromatase),增加海马CA1区神经干细胞增殖和胶质细胞增殖、活化,促进神经炎症,最终导致神经元骨架蛋白水平降低。

Objective Aromatase,a key enzyme in estrogen synthesis,plays a critical role in neuroprotection.This study employed a forebrain neuron-specific aromatase knockout female rat model(FBN-Aro-KO,KO)to observe changes in the differentiation of neural stem cells into astrocytes and microglia in the hippocampal CA1region with aging.The aim was to unveil the neuroprotective effects of brain-derived estrogen and provide important targets for the prevention and treatment of neurodegenerative brain diseases.Methods Animal groups included①naturally aging female rats at 1,3,6,14,and 18months(Mon);②6and 18Mon KO rats and their age-matched wild-type(WT)counterparts.Immunofluorescence staining detected levels of SOX2(neural stem cell marker protein),GFAP(astrocyte marker protein),Iba1(microglia marker protein),MBP,and MAP2(axon and dendrite cytoskeletal proteins).Results In natural aging group,there was no significant difference in the number of SOX2+cells at 1,3,and 6Mon(P>0.05);in comparison,a significant decrease in SOX2+cells occurred at 14and 18Mon.The fluorescence intensity of GFAP,co-localization expression of SOX2+and GFAP+,and the proliferation rate of SOX2+/GFAP+cells increased(P<0.05).In 18Mon group,compared to WT group,KO group showed significant increases in GFAP fluorescence intensity,the number of SOX2+cells,co-localized SOX2+and GFAP+cells and the proliferation rate of GFAP+cells(P<0.01);the total volume of astrocytes in KO group was significantly larger than that of WT group(P<0.05).In natural aging group,compared to 1Mon group,the Iba1immunofluorescence intensity significantly increased at 14and 18Mon(P<0.0001);compared to 18Mon WT group,colocalization expression of SOX2+with Iba1+,Iba1fluorescence intensity,and the total volume of microglia significantly increased in the KO group(P<0.05).In 6Mon group,compared to WT group,KO group showed a significant increase in co-localization expression of SOX2+with GFAP+,GFAP fluorescence intensity,and proliferation rate(P<0.01);however,fluorescence intensity of MAP2and MBP significantly decreased(P<0.01).Conclusion Throughout the natural aging process,hippocampal CA1region neurogenesis in female rats significantly declines after adulthood,with reduced cell proliferation in middle age;however,the proliferation rate of glial cells increases.Long-term absence of forebrain neuron brain-derived estrogen(due to knockout of the key synthesis enzyme Aromatase)increases proliferation and activation of neural stem cells and glial cells in the hippocampal CA1region,promotes neuroinflammation,and ultimately leads to reduced levels of neuronal cytoskeletal proteins.