雌激素缺乏加重痴呆小鼠脑内炎症反应的机制初探

Mechanisms of estrogen deficiency-induced inflammatory reaction aggravation in the brain of dementia mice

目的研究雌激素缺乏不同时间段APP/PS1双转基因小鼠脑组织细胞内线粒体结构、线粒体雌激素受体(mitochondrial estrogen receptorβ,mt-ERβ)、胰岛素生长因子1受体(insulin-like growth factor 1 receptor,IGF-1R)、星胶细胞(astrocyte, AS)以及神经炎性因子的变化,以明确雌激素缺乏促进阿尔茨海默病(Alzheimer’s disease,AD)神经炎症发生的可能分子机制。方法对雌性3月龄APP/PS1双转基因AD小鼠行双侧卵巢切除(AD-OVX),以假手术AD小鼠(AD-Sham)及同月龄正常野生型小鼠(WT)为对照,于术后1周(模拟绝经早期)和3月(模拟绝经中晚期),采用免疫荧光、透射电镜、RT-PCR和Western blot,分别检测卵巢切除(ovariectomy,OVX)后不同时间段APP/PS1小鼠脑内胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)阳性细胞、炎症因子白介素-6(interleukin-6,IL-6)和肿瘤坏死因子α(tumornecrosis factor-α,TNF-α)以及脑组织细胞内线粒体超微结构、mt-ERβ和IGF-1R的变化。结果与正常野生型小鼠相比,无论OVX后1周还是3个月,AD-OVX组和ADSham组小鼠脑内GFAP阳性细胞数均显著增加;相应地,AD小鼠脑内炎症因子IL-6、TNF-α的mRNA水平也较正常小鼠明显增高(P<0.001);超微结构显示,与正常小鼠相比,AD脑组织细胞内线粒体肿胀明显,mt-ERβ和IGF-1R蛋白表达呈下调趋势(P<0.001)。在APP/PS1双转基因AD小鼠中,OVX后1周,AD-OVX组小鼠脑内GFAP阳性细胞数量较AD-Sham组显著减少(P<0.001),且脑内炎症因子IL-6、TNF-α的mRNA水平显著降低(P<0.001),但线粒体结构、mt-ERβ和IGF-1R蛋白表达无明显差异(P>0.05);而OVX后3个月,AD-OVX组脑内GFAP阳性细胞数量较AD-Sham组显著增多(P<0.001),且脑内炎症因子IL-6、TNF-α明显升高(P<0.001),线粒体肿胀明显,mt-ERβ蛋白表达有上调趋势(P<0.05),但仍低于正常小鼠;IGF-1R蛋白表达呈下调趋势(P<0.05)。结论痴呆小鼠脑内存在星胶细胞活化及炎症反应增强,雌激素缺乏早期可反应性的减少星胶细胞的增生及炎症反应,但随着雌激素缺乏时间的延长,痴呆小鼠脑内星胶细胞的活化及神经炎症反应进行性加重,该作用可能通过mt-ERβ介导的IGF-1R信号通路相关。

To clarify the molecular mechanisms of estrogen deficiency-induced inflammatory reaction aggravation in brain of mice with dementia,we investigate the changes of mitochondrial structure,mitochondrialestrogen receptorβ(mt-ERβ),insulin-like growth factor 1 receptor(IGF-1 R),astrocyte(AS)andneuroinflammation factors in the brain of APP/PS1 double transgenic mice.Female 3-month-old APP/PS1 double transgenic AD mice underwent bilateralovariectomy(AD-OVX)were set as experiment group,while sham-operated AD mice(AD-sham)and wild-type mice(WT)of the same age were used as controls.Immunofluorescence,RT-PCR,transmission electronmicroscopy and Western blot(WB)were used to detect the changes of glial fibrillary acidic protein(GFAP),inflammatory factors(IL-6 and TNF-α),mitochondrial ultrastructure,mt-ERβand IGF-1 R at week 1(simulating earlymenopause)and month 3(simulating middle and late menopause)post-surgery.Compared with wild-type mice,thenumber of GFAP-positive cells in the brain of AD-OVX group and AD-sham group was increased significantly in 1 week or 3 months after OVX,and correspondingly,the mRNA levels of IL-6 and TNF-αwere also increasedsignificantly(P<0.001);the mitochondrial swelling was evident in AD brain,while mt-ERβand IGF-1 Rexpressions demonstrated a downward trend(P<0.001);the number of GFAP positive cells in AD-OVX group wassignificantly lower than that of AD-Sham group at 1 week after OVX(P<0.001),and the mRNA levels of IL-6 andTNF-αwere decreased significantly in AD-OVX group(P<0.001),but there were no significant difference inmitochondrial structure,mt-ERβand IGF-1 R protein expression(P>0.05).However,3 months after OVX,thenumber of GFAP-positive cells in AD-OVX group was higher than that in AD-sham group(P<0.001),and theinflammatory factors were increased significantly(P<0.001),the mitochondrial swelling was obvious,the expressionof mt-ERβwas up-regulated(P<0.05),and the expression of IGF-1 R was decreased significantly(P<0.05).Inconclusion,early estrogen deficiency reduces the proliferation of astrocytes and the inflammation reactivity in thebrain of dementia mice,but with the prolongation of estrogen deficiency,the activation of astrocytes andneuroinflammatory response are progressively aggravated.This effect may be related to the mt-ERβmediated IGF-1 R signaling pathway.