Supplementary Material for: Effects of oral administration of the probiotic Lactobacillus rhamnosus GG on the proteomic profiles of cerebrospinal fluid and immunoregulatory signaling in the hippocampus of adult male rats

Introduction: The microbiome-gut-brain axis, by modulating bidirectional immune, metabolic, and neural signaling pathways in the host, has emerged as a target for the prevention and treatment of psychiatric and neurological disorders. Oral administration of the probiotic bacterium Lactobacillus rhamnosus GG (LGG; ATCC 53103) exhibits anti-inflammatory effects, although the precise mechanisms by which LGG benefits host physiology and behavior are not known. The goal of this study was to explore the general effects of LGG on the cerebrospinal fluid (CSF) proteome and a biological signature of anti-inflammatory signaling in the central nervous system (CNS) of undisturbed, adult male rats. Methods: Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics were conducted using CSF samples collected after 21 days of oral treatment with live LGG (3.34 x 107 colony-forming units (CFU)/mL in the drinking water (resulting in an estimated delivery of ~1.17 x 109 CFU/day/rat) or water vehicle. Gene enrichment analysis (using DAVID, v. 6.8) and protein-protein interactions (using STRING, v. 11) were used to explore physiological network changes in CSF. Real time reverse transcription polymerase chain reaction (real time RT-PCR) was performed to assess gene expression changes of anti-inflammatory cytokines in the hippocampus. Genes associated with anti-inflammatory signaling that were analyzed included Il10, Tgfb1, Il4, and IL-4-responsive genes, Cd200, Cd200r1, and Mrc1 (Cd206). Results: Oral LGG administration altered the abundance of CSF proteins, increasing the abundance of five proteins (cochlin, NPTXR, reelin, Sez6l, and VPS13C) and decreasing the abundance of two proteins (CPQ, IGFBP-7) in the CSF. Simultaneously, LGG increased the expression of Il10 mRNA, encoding the anti-inflammatory cytokine interleukin 10, in the hippocampus. Conclusion: Oral LGG altered the abundance of CSF proteins associated with extracellular scaffolding, synaptic plasticity, and glutamatergic signaling. These data are consistent with the hypothesis that oral administration of LGG may improve memory and cognition, and may promote a physiological resilience to neurodegenerative disease, by increasing glutamatergic signaling and promoting an anti-inflammatory environment in the brain.引言：肠道菌群-脑轴（microbiome-gut-brain axis）通过调控宿主内双向免疫、代谢与神经信号通路，已成为精神疾病与神经退行性疾病防治的新兴靶点。鼠李糖乳杆菌GG（Lactobacillus rhamnosus GG, LGG；ATCC 53103）作为益生菌，经口服给药后可发挥抗炎作用，但其改善宿主生理状态与行为表现的确切机制仍未阐明。本研究旨在探究LGG对未受干预的成年雄性大鼠脑脊液（cerebrospinal fluid, CSF）蛋白质组，以及中枢神经系统（central nervous system, CNS）内抗炎信号通路生物标志物的整体影响。 方法：本研究采用液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）蛋白质组学技术，对经21天口服活LGG处理（饮用水中添加3.34×10^7菌落形成单位（colony-forming unit, CFU）/mL，预估每只大鼠每日给药量约为1.17×10^9 CFU）或空白溶剂饮用水的大鼠采集脑脊液样本进行分析。通过基因富集分析（采用DAVID v6.8版本）与蛋白质相互作用分析（采用STRING v11版本），探究脑脊液内的生理网络变化。采用实时逆转录聚合酶链反应（real time reverse transcription polymerase chain reaction, real-time RT-PCR）检测海马体中抗炎细胞因子的基因表达变化。本次分析涉及的抗炎信号通路相关基因包括Il10、Tgfb1、Il4，以及IL-4应答基因Cd200、Cd200r1与Mrc1（Cd206）。 结果：口服LGG可改变脑脊液内蛋白质丰度：5种蛋白质（cochlin、NPTXR、reelin、Sez6l与VPS13C）的丰度显著升高，另有2种蛋白质（CPQ与IGFBP-7）的丰度显著降低。与此同时，LGG可升高海马体中编码抗炎细胞因子白细胞介素10的Il10 mRNA表达水平。 结论：口服LGG可改变与细胞外支架、突触可塑性及谷氨酸能信号通路相关的脑脊液蛋白质丰度。本研究结果与下述假说一致：口服LGG可通过增强谷氨酸能信号通路、促进脑内抗炎微环境，改善记忆与认知功能，并提升机体对神经退行性疾病的生理抵抗能力。