脂多糖诱导孤独症样大鼠的肠道通透性及肠道黏膜免疫系统的变化

Changes of intestinal permeability and intestinal mucosal immune system in rat model of autism induced by lipopolysaccharide

目的探讨孕期LPS诱导的孤独症大鼠肠黏膜通透性及肠道区域免疫相关细胞比例的变化。方法 SD大鼠妊娠第9.5天时,腹腔注射脂多糖(lipopolysac-charide,LPS)构建LPS诱导的子代孤独症大鼠模型。生后第7、14、21、28、35天监测仔鼠的体质量。三箱社交实验、旷场实验、嗅觉适应/去适应实验、水迷宫实验检测子代大鼠孤独症样行为。ELISA检测子代大鼠血清DAO、zonulin、IL-6、IL-10、TNF-α、IFN-γ的含量;流式细胞仪检测脾、肠系膜淋巴结(MLN)、Peyer’s结(PP结)中B细胞、树突状细胞(DCs)、Treg细胞、T细胞、肠上皮间淋巴细胞(IELs)及肠道黏膜固有层淋巴细胞(LPLs)的比例。结果 LPS处理组子代大鼠可出现社会交互障碍、交流障碍、在陌生环境中的自主探索行为减少、空间学习记忆功能降低等孤独症相关的行为改变。LPS组的仔鼠血清DAO(P<0.01)、zonulin(P<0.05)含量较未处理组显著升高。同时发现LPS组仔鼠脾脏、MLN中的Treg细胞比例较未处理组显著减少(P<0.05)。在MLN及PP结中,LPS组仔鼠的B细胞、树突状细胞、T细胞、CD4^+T细胞、CD8^+T细胞比例显著升高(P<0.05)。而LPS组仔鼠脾脏中的B细胞、树突状细胞、T淋巴细胞、CD4^+T细胞、CD8^+T细胞的比例却与未处理组无显著差异。LPS组仔鼠IELs中的CD8^+TCRγδ^+T细胞的比例较未处理组明显升高(P<0.05),而LPLs中的CD4^+TCRαβ^+T细胞、CD8^+TCRαβ^+T细胞的比例却显著降低(P<0.05)。LPS组仔鼠血清中的细胞因子IL-6、TNF-α、IFN-γ较PBS组增高,而IL-10较PBS组降低。结论孕期LPS诱导可导致大鼠孤独症样行为,同时引起肠道通透性增加,肠黏膜集合淋巴组织(MLN、PP结)淋巴细胞比例增加,IELs比例升高,而LPLs中的T细胞比例降低,分泌促炎因子的T细胞功能增强,CD25^+Foxp3^+Treg细胞的抗炎功能被抑制。提示LPS诱导的孤独症模型大鼠可能会同时伴有肠道黏膜免疫异常应答现象。

To investigate the changes of intestinal permeability and the proportion of intestinal mucosalimmune-related cells in pregnancy autisic rats induced by lipopolysaccharide (LPS). Rats were injectedintraperitoneally with 100 μg / kg LPS at the gestational day 9.5 to construct autisic model in offspring rat. Theweights of the pups were monitored at 7, 14, 21, 28, and 35 days after birth. Autism behavior variations of offspringwere detected by three-chamber sociability test, open field test, olfactory habituation/dishabituation test and Morriswater test. ELISA kits were used to determine theconcentrations of serum diamine oxidase (DAO),zonulin, IL-10, IL-6, TNF- α and IFN- γ ; flowcytometry was used to detect the percentages of B cells,dendritic cells (DCs), Treg cells and T cells in thespleen, mesenteric lymph node (MLN) and peyer'spatches, intestinal epithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). Data showed that theoffsprings rats induced by LPS displayed obvious social interaction and communication disorders, decreasedexploration ability and impaired learning-memory function. The serum DAO (P<0.01) and zonulin (P<0.05) levels ofLPS group were significantly higher than those of PBS group. The ratio of Treg cells in both spleen and MLN weresignificantly lower (P<0.05) in LPS group. Meanwhile, the percentages of B cells, dendritic cells, T cells, CD4^+Tcells and CD8^+T cells in spleen and peyer 's patches were significant increased in the offsprings of LPS group (P<0.05). However, in the spleen, the percentage of B cells, DCs, T lymphocytes, CD4^+T cells and CD8^+T cells showedno significant differences compared with those of the PBS group. The ratio of CD8+TCRγδ^+T cells in the IELs wasstatistically increased (P<0.05), while the CD4^+TCRαβ^+T cells and CD8^+TCRαβ^+T cells in the LPLs were decreasedsignificantly (P<0.05). Furthermore, the serum levels of IL-6, TNF-α and IFN-γ in LPS group were higher but theIL-10 was lower than those in the PBS group. These data reveal that prenatal LPS exposure could cause rat'sautistic behavior, which accompanied with intestinal permeability increase, lymphocytes proportion upregulation inintestinal mucosa (MLN, PP), IELs ratio increase, and T cells proportion decrease in LPLs. The proinflammatorycytokines from T cells were enhanced, and the anti-inflammatory function of CD25+Foxp3+ Treg cells was inhibited.It is demonstrated that the gestation of LPS-induced autism model rats may be accompanied by abnormal intestinalmucosal immune response.