Supplementary Materials_ Extended Barrier, Transcriptomic, Metabolomic, and Microbial Co-Metabolism Analyses in Healthy-FMT and GWI-FMT Mice

Supplementary Figure 1. Regional assessment of epithelial junctional markers in GWI-FMT mice. (A) Cecum and colon weight-to-length ratios of Healthy-FMT and GWI-FMT mice demonstrate no gross morphological differences. (B) Ocln mRNA levels in ileum are reduced in GWI-FMT mice. (C–D) Western blot and densitometric analysis of Occludin (OCLN), Claudin-2 (CLDN2), and E-cadherin (ECAD) in proximal colon lysates show no significant change between groups. Data are presented as mean ± SD; n = 4/5 mice per group; unpaired two-tailed t-test. Supplementary Figure 2. Transcript analysis of solute carriers in the intestine of Healthy- and GWI-FMT mice. (A–C) Quantitative RT-PCR analysis of Slc26a3 and other solute carriers (Slc16a1, Abcb1, Slc9a3, Slc5a8, Slc10a2, Slc26a4, Slc26a6) in the (A) ileum, (B) proximal colon, and (C) distal colon. Transcript levels were normalized to Gapdh. No significant differences were observed between groups. Data are mean ± SD; n = 5 mice per group; unpaired two-tailed t-test. Supplementary Figure 3. Metabolomic profiling in positive ion mode reveals additional discriminatory features. (A) Principal component analysis (PCA) of fecal metabolites in positive ion mode shows overlapping but distinct clustering between Healthy-FMT (green) and GWI-FMT (purple) groups. (B) Volcano plot identify significantly altered metabolites (|log₂FC| ≥ 1.3, p < 0.05); red = upregulated, blue = downregulated, grey = not significant. (C) Bar graph of representative metabolites elevated in GWI-FMT mice, including procollagen-5-hydroxy-L-lysine (5-Hyl), cholic acid, and 5-acetamidovalerate. Data are mean ± SD; n = 5 mice per group; unpaired two-tailed t-test (*p < 0.05). Supplementary Figure 4. Correlation and enrichment analyses identify metabolites and pathways associated with GWI status. (A–B) Pattern Hunter correlation analysis of the top 25 metabolites most strongly associated with GWI status in negative and positive ion modes. Red indicates positive correlation and blue negative correlation with GWI. (C) Quantitative enrichment analysis (QEA) of correlated metabolites show significant overrepresentation of pathways linked to amino acid metabolism, urea cycle, SLC-mediated transport, and bile acid/small-molecule transport. Dot size represents the number of matched metabolites; color denotes statistical significance (red = higher –log₁₀p).补充图1. GWI-FMT小鼠上皮连接标志物的区域评估。 (A) 健康-FMT与GWI-FMT小鼠的盲肠、结肠重量长度比未呈现明显大体形态学差异。 (B) GWI-FMT小鼠回肠内的闭合蛋白（Ocln）信使核糖核酸（mRNA）水平降低。 (C–D) 对近端结肠裂解液中的闭合蛋白（Occludin, OCLN）、紧密连接蛋白-2（Claudin-2, CLDN2）以及上皮钙黏蛋白（E-cadherin, ECAD）进行蛋白质印迹（Western blot）与光密度分析，结果显示两组间无显著变化。数据以平均值±标准差（mean ± SD）表示；每组n=4/5只小鼠；采用独立样本双侧t检验。 补充图2. 健康-FMT与GWI-FMT小鼠肠道内溶质转运蛋白（solute carriers）的转录组分析。 (A–C) 分别对(A)回肠、(B)近端结肠以及(C)远端结肠中的Slc26a3及其他溶质转运蛋白（包括Slc16a1、Abcb1、Slc9a3、Slc5a8、Slc10a2、Slc26a4、Slc26a6）进行实时定量逆转录聚合酶链反应（Quantitative RT-PCR）分析。转录水平以甘油醛-3-磷酸脱氢酶（Gapdh）作为内参进行归一化处理。两组间未观察到显著差异。数据以平均值±标准差表示；每组n=5只小鼠；采用独立样本双侧t检验。 补充图3. 正离子模式下的代谢组学分析揭示额外的判别特征。 (A) 正离子模式下粪便代谢物的主成分分析（Principal Component Analysis, PCA）结果显示，健康-FMT（绿色标记）与GWI-FMT（紫色标记）两组的聚类存在重叠但界限分明。 (B) 火山图（Volcano plot）可识别显著差异代谢物（|log₂FC| ≥ 1.3，p < 0.05）；红色代表上调代谢物，蓝色代表下调代谢物，灰色代表无显著差异的代谢物。 (C) 展示GWI-FMT小鼠中升高的代表性代谢物的柱状图，包括前胶原-5-羟基-L-赖氨酸（procollagen-5-hydroxy-L-lysine, 5-Hyl）、胆酸以及5-乙酰氨基戊酸。数据以平均值±标准差表示；每组n=5只小鼠；采用独立样本双侧t检验（*p < 0.05）。 补充图4. 相关性与富集分析揭示与GWI状态相关的代谢物及通路。 (A–B) 分别对负离子模式与正离子模式下与GWI状态相关性最强的前25种代谢物进行Pattern Hunter相关性分析。红色代表与GWI呈正相关，蓝色代表与GWI呈负相关。 (C) 对相关性代谢物的定量富集分析（Quantitative Enrichment Analysis, QEA）结果显示，与氨基酸代谢、尿素循环、溶质转运蛋白（SLC）介导的转运以及胆汁酸/小分子转运相关的通路显著富集。圆点大小代表匹配到的代谢物数量；颜色代表统计学显著性（红色代表更高的–log₁₀p值）。