Xiao Lu, Yan Junyan, Feng Di, Ye Shasha, Yang Ting, Wei Hua, Li Tingyu, Sun Wuqing, Chen Jie
Chongqing Key Laboratory of Childhood Nutrition and Health, Children's Hospital of Chongqing Medical University, Chongqing, China.
Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, Chongqing, China.
Front Cell Dev Biol. 2021 Mar 4;9:634837. doi: 10.3389/fcell.2021.634837. eCollection 2021.
To investigate the role of TLR4 on the microglia activation in the pre-frontal cortex, which leads to autism-like behavior of the offspring induced by maternal lipopolysaccharide (LPS) exposure. Pregnant TLR4 (knockout, KO) and WT (wild type, WT) dams were intraperitoneally injected with LPS or PBS, respectively. The levels of TNFα, IL-1β, and IL-6 in the maternal serum and fetal brain were assessed with ELISA following LPS exposure. The gestation period, litter size and weight of the offspring were evaluated. Three-chamber sociability test, open field test and olfactory habituation/dishabituation test were used to assess the offspring's autism-like behavior at 7 weeks of age. Western blotting was performed to examine the levels of TLR4, Phospho-NFκB p65, IKKα, IBA-1, iNOS, Arg-1, C3, CR3A, NMDAR2A, and Syn-1 expression in the pre-frontal cortex. The morphological changes in the microglia, the distribution and expression of TLR4 were observed by immunofluorescence staining. Golgi-Cox staining was conducted to evaluate the dendritic length and spine density of the neurons in 2-week-old offspring. Maternal LPS stimulation increased serum TNFα and IL-6, as well as fetal brain TNFα in the WT mice. The litter size and the weight of the WT offspring were significantly reduced following maternal LPS treatment. LPS-treated WT offspring had lower social and self-exploration behavior, and greater anxiety and repetitive behaviors. The protein expression levels of TLR4 signaling pathways, including TLR4, Phospho-NFκB p65, IKKα, and IBA-1, iNOS expression were increased in the LPS-treated WT offspring, whereas Arg-1 was decreased. Maternal LPS treatment resulted in the significant reduction in the levels of the synaptic pruning-related proteins, C3 and CR3A. Moreover, the neuronal dendritic length and spine density, as well as the expression levels of the synaptic plasticity-related proteins, NMDAR2A and Syn-1 were reduced in the WT offspring; however, gestational LPS exposure had no effect on the TLR4 offspring. Activation of TLR4 signaling pathway following maternal LPS exposure induced the abnormal activation of microglia, which in turn was involved in excessive synaptic pruning to decrease synaptic plasticity in the offspring. This may be one of the reasons for the autism-like behavior in the offspring mice.
为了研究Toll样受体4（TLR4）在小胶质细胞激活中的作用，该激活导致母体暴露于脂多糖（LPS）后子代出现自闭症样行为。分别对怀孕的TLR4基因敲除（KO）和野生型（WT）母鼠腹腔注射LPS或磷酸盐缓冲液（PBS）。LPS暴露后，采用酶联免疫吸附测定（ELISA）评估母体血清和胎脑肿瘤坏死因子α（TNFα）、白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6）水平。评估子代的妊娠期、窝仔数和体重。采用三室社交性试验、旷场试验和嗅觉习惯化/去习惯化试验评估7周龄子代的自闭症样行为。采用蛋白质免疫印迹法检测前额叶皮质中TLR4、磷酸化核因子κB p65（Phospho-NFκB p65）、IκB激酶α（IKKα）、离子钙结合衔接分子1（IBA-1）、诱导型一氧化氮合酶（iNOS）、精氨酸酶1（Arg-1）、补体C3、补体受体3A（CR3A）、N-甲基-D-天冬氨酸受体2A（NMDAR2A）和突触素1（Syn-1）的表达水平。通过免疫荧光染色观察小胶质细胞的形态变化、TLR4的分布和表达。采用高尔基-考克斯染色评估2周龄子代神经元的树突长度和棘密度。母体LPS刺激增加了野生型小鼠血清TNFα和IL-6以及胎脑TNFα水平。母体LPS处理后，野生型子代的窝仔数和体重显著降低。LPS处理的野生型子代社交和自我探索行为较低，焦虑和重复行为较多。LPS处理的野生型子代中，包括TLR4、Phospho-NFκB p65、IKKα和IBA-1、iNOS表达在内的TLR4信号通路蛋白表达水平升高，而Arg-1降低。母体LPS处理导致与突触修剪相关的蛋白C3和CR3A水平显著降低。此外，野生型子代的神经元树突长度和棘密度以及与突触可塑性相关的蛋白NMDAR2A和Syn-1的表达水平降低；然而，妊娠期LPS暴露对TLR4基因敲除子代没有影响。母体LPS暴露后TLR4信号通路的激活诱导了小胶质细胞的异常激活，进而参与过度的突触修剪，以降低子代的突触可塑性。这可能是子代小鼠出现自闭症样行为的原因之一。
