Table 2_Metformin-associated gut microbiota remodeling correlates with reinvigorated splenic immunity in aged mice: microbiome-immune crosstalk via the gut-spleen axis.xlsx

Background and aimImmunosenescence involves age-related immune decline and chronic inflammation, with the spleen serving as a critical hub for immune dysregulation. While gut microbiota influences systemic immunity, its specific role and the potential existence of a gut-spleen axis in mediating splenic aging remains unclear. Therefore, we investigated whether metformin, a microbiota-modulating geroprotective drug, alleviates splenic immunosenescence in aged mice, specifically exploring the link between gut microbiota remodeling and splenic immune rejuvenation.
MethodsAged C57BL/6 mice (15-month-old) received oral metformin (300 mg/kg/day) or vehicle for 5 months. Systemic toxicity and metabolism were monitored. Splenic immune subsets were analyzed using flow cytometry and immunohistochemistry. Gut microbiota composition (16S rRNA sequencing), cytokine levels (RT-qPCR), and functional pathways were assessed.
ResultsMetformin caused no hepatorenal toxicity or weight changes. Treated mice exhibited increased cytotoxic T cells (Tc) and macrophages in the spleen, with reduced Th/Tc ratios and M1/M2 polarization. Pro-inflammatory cytokines (Ifng, Il17a, Il1b, Il6) decreased, while anti-inflammatory markers (Arg1, Tgfb1) rose. Gut microbiota showed enriched Akkermansia, Muribaculum, and Duncaniella, but reduced Lactobacillus. Akkermansia/Muribaculum negatively correlated with pro-inflammatory cytokines, whereas Lactobacillus and Lachnospiraceae linked to pro-inflammatory responses. Functional prediction analysis based on 16S rRNA sequencing data indicated upregulation of bile acid metabolism and oxidative phosphorylation pathways.
ConclusionMetformin reshapes the gut microbiota, which is associated with mitigation of age-associated splenic immune dysregulation, favoring anti-inflammatory macrophage polarization and cytotoxic T cell expansion. Critically, our findings establish the gut-spleen axis as a key mediator of splenic immunosenescence and a novel therapeutic target, which positions metformin as a promising microbiota-directed geroprotective agent. Future research should prioritize mechanistic dissection of gut-spleen communication and clinical validation of metformin’s geroprotective efficacy in human populations.

研究背景与目的：免疫衰老(immunosenescence)指与年龄相关的免疫衰退与慢性炎症状态，脾脏是免疫失调的关键枢纽。尽管肠道菌群可影响全身免疫，但其在脾脏衰老调控中的具体作用，以及肠道-脾脏轴(gut-spleen axis)是否介导脾脏衰老的潜在机制仍不明确。因此，本研究探讨调节菌群的抗衰老药物二甲双胍(metformin)是否可缓解衰老小鼠的脾脏免疫衰老，并重点探索肠道菌群重塑与脾脏免疫年轻化之间的关联。
实验方法：选取15月龄的衰老C57BL/6小鼠，连续5个月灌胃给予二甲双胍(300 mg/kg/天)或赋形剂。监测小鼠的全身毒性与代谢状态。采用流式细胞术(flow cytometry)与免疫组织化学(immunohistochemistry)分析脾脏免疫细胞亚群。检测肠道菌群组成(16S rRNA测序)、细胞因子水平(逆转录定量聚合酶链反应(RT-qPCR))及功能通路。
实验结果：二甲双胍未引起肝肾毒性或体重变化。给药组小鼠脾脏内细胞毒性T细胞(Tc, cytotoxic T cells)与巨噬细胞数量增加，辅助性T细胞/细胞毒性T细胞(Th/Tc)比值及M1/M2极化(M1/M2 polarization)水平降低。促炎细胞因子(Ifng、Il17a、Il1b、Il6)表达下调，而抗炎标志物(Arg1、Tgfb1)表达上调。肠道菌群分析显示，阿克曼菌属(Akkermansia)、小杆菌属(Muribaculum)及邓氏菌属(Duncaniella)丰度升高，乳酸杆菌属(Lactobacillus)丰度降低。阿克曼菌属/小杆菌属与促炎细胞因子呈负相关，而乳酸杆菌属与毛螺菌科(Lachnospiraceae)则与促炎反应相关。基于16S rRNA测序数据的功能预测分析显示，胆汁酸代谢与氧化磷酸化通路显著上调。
研究结论：二甲双胍可重塑肠道菌群结构，这与年龄相关的脾脏免疫失调得到缓解、抗炎巨噬细胞极化及细胞毒性T细胞扩增相关。尤为重要的是，本研究证实肠道-脾脏轴是调控脾脏免疫衰老的关键介质，同时也是全新的治疗靶点，表明二甲双胍是一种极具潜力的靶向菌群的抗衰老药物。未来研究应重点解析肠道-脾脏的通信机制，并开展临床研究验证二甲双胍在人群中的抗衰老功效。