妊娠期脂多糖暴露通过激活子代小胶质细胞诱导大鼠孤独症样行为

Lipopolysaccharide exposure during gestation activates microglia to induce autism-like behavior in offspring of rats

目的:探讨妊娠期脂多糖(LPS)诱导的孤独症样行为仔鼠脑组织Toll样受体4(TLR4)及小胶质细胞分型的变化。方法:24只孕鼠随机平分为2组(n=12),分别于怀孕9.5 d时腹腔注射LPS(100μg/kg)或等体积磷酸盐缓冲液(PBS),所产子代大鼠即为LPS组(n=72)及PBS组(n=72)仔鼠。采用real-time PCR和Western blot技术测定仔鼠TLR4、离子钙结合衔接分子1(IBA-1)、诱导型一氧化氮合酶(iNOS)和精氨酸酶1(Arg-1)的表达水平。免疫荧光染色检测仔鼠前额叶皮质小胶质细胞的形态学改变。ELISA试剂盒测定孕鼠血清脂多糖结合蛋白(LBP)和肿瘤坏死因子α(TNF-α)及仔鼠脑组织TNF-α和白细胞介素10(IL-10)含量。三箱社交实验、嗅觉适应/去适应实验和旷场实验检测子代大鼠社交行为、探索行为及刻板行为。体外培养N9小胶质细胞,予以TLR4阻断剂瑞沙托维(TAK242)处理后,观察LPS诱导的N9细胞TLR4、iNOS及Arg-1表达水平的变化。结果:妊娠期LPS处理可诱导母鼠血清LBP及TNF-α显著升高(P<0.01或P<0.05),呈免疫激活态,其子代大鼠出现社会交往障碍、重复刻板行为等孤独症样行为。LPS组仔鼠前额叶皮质中TLR4、IBA-1和iNOS的mRNA及蛋白表达水平显著增加(P<0.05或P<0.01),而Arg-1的mRNA及蛋白表达水平却显著下降(P<0.05或P<0.01),M1型小胶质细胞增多,M2型减少,TNF-α表达水平升高(P<0.01),而IL-10表达水平降低(P<0.01)。形态学显示LPS组小胶质细胞分支减少,胞体增大、松散,呈激活态。体外实验结果显示,TLR4抑制剂TAK242显著减少了LPS诱导的M1型小胶质细胞转化。结论:妊娠中期LPS暴露激活仔鼠TLR4信号通路,诱导前额叶皮质小胶质细胞向M1型极化,这可能是子代大鼠孤独症样行为的一个发病风险因素。

AIM:To investigate the changes of Toll-like receptor 4(TLR4)expression level and microglial subtypes in the prefrontal cortex of autism-like pups induced by lipopolysaccharide(LPS) during pregnancy.METHODS:Pregnant rats(n=24)were randomly divided into 2 groups(n=12 in each group).The pregnancy rats were intraperitoneally injected with 100 μg/kg LPS or equal volume of phosphate-buffered saline(PBS)at the gestational day9.5,and their pups were named as LPS group(n=72)and PBS group(n=72),respectively.The expression of TLR4,ionized calcium-binding adaptor molecule-1(IBA-1),inducible nitric oxide synthase(iNOS)and arginase-1(Arg-1)at mRNA and protein levels in the prefrontal cortex was determined by real-time PCR and Western blot.The morphologic changes of microglia in the prefrontal cortex of pups were observed by immunofluorescence.The concentrations of lipopolysaccharide-binding protein(LBP)and tumor necrosis factor-α(TNF-α)in maternal serum,and TNF-α and interleukin-10(IL-10)in offspring cortex were measured by ELISA.Three-chamber sociability test,olfactory habituation/dishabituation test and open-field test were used to assess offspring’s autism-like behavior.N9 microglia were cultured in vitro,and the changes of TLR4,iNOS and Arg-1 expression levels in the N9 cells induced by LPS combined with resatorvid(TAK242)treatment were measured by real-time PCR and immunofluorescence.RESULTS:The LBP and TNF-α levels in maternal serum were significantly induced(P<0.05 or P<0.01)by LPS treatment to state of immune activation.The offspring born by the mothers with maternal immune activation appeared obvious social interaction and communication disorders,decreased exploration ability and increased repetitive behaviors.The real-time PCR and Western blot data showed that both mRNA and protein expression levels of TLR4,IBA-1 and iNOS were significantly increased(P<0.05 or P<0.01)in the prefrontal cortex of pups in LPS group compared with PBS group,while the mRNA and protein expression levels of Arg-1 were significantly decreased(P<0.05 or P<0.01).The M1 subtype of microglia was induced and M2 was reduced in the prefrontal cortex of the pups treated with LPS.The expression level of TNF-α was increased in the prefrontal cortex of pups in LPS group,while the level of IL-10 was reduced(P<0.01).The number of microglia with branches in LPS group was decreased,and the cell body was loose and enlarged.TAK242(TLR4 inhibitor)treatment in vitro suppressed M2 transformation to M1 in the N9 microglia induced by LPS.CONCLUSION:LPS exposure in mid-pregnant rats activates offspring’s TLR4 signaling pathway and induces offspring’s prefrontal cortical microglia to M1 type polarization,which may be a risk factor for autism-like behavior in offspring rats.