Datasets of Figure 1-4 and 16S rRNA sequencing raw data

Datasets for our study: Metformin inhibits pro-inflammatory macrophages in atherosclerotic apolipoprotein E-/- mice by suppressing microbial LPS/TLR4/NF-κB axis via restoring gut microbiota. Atherosclerosis (AS) is closely associated with chronic low-grade inflammation and gut dysbiosis. Metformin (MET) presents pleiotropic benefits for the control of chronic metabolic diseases. However, the exact impact and associated mechanism of MET intervention on gut bacteria and inflammation of atherosclerosis remains largely unknown. In the present study, ApoE deficient (ApoE-/-) mice were randomly allocated into atherosclerosis model group (AS) and AS+MET group. After 11 weeks of intervention, mice were euthanized and associated microbial community and inflammation indications were respectively investigated. Results showed that pathological plague lesion in aorta was attenuated after MET administration using oil red O staining, Masson’s trichrome staining and hematoxylin and eosin (HE) staining. Moreover, MET intervention rectified gut dysbiosis by reducing the relative abundance of Firmicutes, the ratio of Firmicutes to Bacteroidetes, Proteobacteria, Romboutsia, as well as elevating Bacteroidetes, Akkermansia and Bifidobacterium levels. Subsequently, microbial plasma lipopolysaccharide (LPS) level was lower in AS+MET group. Pro-inflammatory tumor necrosis factor (TNF)-α and interleukin (IL)-6 were decreased, while IL-1β and anti-inflammatory IL-10 showed no significant difference. Moreover, MET treatment suppressed F4/80+ infiltrated macrophages and lowered associated TLR-4, NF-κB and TNF-α expression in aorta. 1. Datasets of Figure 1-4. Fig. 1. Metformin ameliorated pathological lesion in atherosclerotic ApoE-/- mice. (A) The schematic diagram of the study. (B) Representative sections of the valve area of the aortic root of the heart were stained with Oil Red O, Masson’s trichrome and Hematoxylin and Eosin; quantitative analysis as lesion area/total area (%) shown in Oil red O staining. Fig. 2. Metformin rectified gut dysbiosis and improved associated plasma LPS level. (A) Observed-species index and PCoA analysis showing difference in terms of species in fecal samples. (B) Relative abundance of microbial species at the phylum and genus levels. (C) Determination of plasma lipopolysaccharide (LPS) levels. Fig. 3. Metformin reduced plasma pro-inflammatory cytokines. Plasma of mice from two groups were collected respectively for detection of TNF-α (A), IL-1β (B), IL-6 (C) and IL-10 (D) concentrations using flow CBA kit. Fig. 4. Metfomin inhibited inflammatory macrophages in atherosclerosis. (A) After obtaining cell suspensions from aorta root of atheroclerotic ApoE-/- mice, F4/80+ macrophages were detected by flow cytometry. (B) After isolation of macrophages, TNF-ɑ, NF-κB and TLR-4 expression were determined by western blot. **P<0.001, ***P<0.0001. 2. 16S rRNA sequencing raw data: 5 samples in AS group and 5 samples in AS+MET group

本研究所用数据集：二甲双胍通过恢复肠道菌群，抑制微生物脂多糖（lipopolysaccharide, LPS）/Toll样受体4（Toll-like receptor 4, TLR4）/核因子κB（nuclear factor kappa-B, NF-κB）通路，从而抑制动脉粥样硬化载脂蛋白E敲除（apolipoprotein E-/-, ApoE-/-）小鼠的促炎巨噬细胞。
动脉粥样硬化（atherosclerosis, AS）与慢性低度炎症及肠道菌群失调密切相关。二甲双胍（metformin, MET）对慢性代谢性疾病的管控具有多效益处。然而，二甲双胍干预对动脉粥样硬化小鼠肠道菌群及炎症的具体影响与相关机制仍未完全阐明。
本研究中，载脂蛋白E缺陷型（ApoE-/-）小鼠被随机分为动脉粥样硬化模型组（AS组）与AS+二甲双胍干预组（AS+MET组）。干预11周后安乐处死小鼠，分别对其肠道微生物群落与炎症指标进行检测。
结果显示，经油红O染色、Masson三色染色及苏木精-伊红（HE）染色后，二甲双胍给药组小鼠主动脉的病理性斑块负荷显著减轻。此外，二甲双胍干预可通过降低厚壁菌门（Firmicutes）相对丰度、厚壁菌门与拟杆菌门（Bacteroidetes）的丰度比值，以及变形菌门（Proteobacteria）、罗姆布茨菌属（Romboutsia）的相对丰度，同时提升拟杆菌门、阿克曼菌属（Akkermansia）与双歧杆菌属（Bifidobacterium）的水平，从而纠正肠道菌群失调。
后续检测发现，AS+MET组小鼠血浆脂多糖（LPS）水平显著降低。促炎因子肿瘤坏死因子（tumor necrosis factor, TNF）-α与白细胞介素（interleukin, IL）-6的表达量显著下降，而IL-1β与抗炎因子IL-10则无显著差异。此外，二甲双胍处理可抑制主动脉内F4/80+浸润巨噬细胞，并降低主动脉组织中TLR-4、NF-κB及TNF-α的表达水平。
1. 图1至图4对应数据集
图1 二甲双胍改善动脉粥样硬化ApoE-/-小鼠的病理性损伤。(A) 本研究实验流程示意图。(B) 心脏主动脉根瓣膜区域的代表性切片经油红O、Masson三色及苏木精-伊红染色；油红O染色结果以病变面积/总面积（%）进行定量分析。
图2 二甲双胍纠正肠道菌群失调并改善血浆LPS水平。(A) 观测物种指数与主坐标分析（Principal Coordinates Analysis, PCoA）结果显示粪便样本中物种组成差异。(B) 门水平与属水平的微生物物种相对丰度。(C) 血浆脂多糖（LPS）水平检测结果。
图3 二甲双胍降低血浆促炎细胞因子水平。分别收集两组小鼠血浆，采用流式细胞术微球阵列（CBA）检测试剂盒检测TNF-α(A)、IL-1β(B)、IL-6(C)及IL-10(D)的浓度。
图4 二甲双胍抑制动脉粥样硬化中的炎症巨噬细胞。(A) 从动脉粥样硬化ApoE-/-小鼠的主动脉根制备细胞悬液后，通过流式细胞术检测F4/80+巨噬细胞。(B) 分离巨噬细胞后，采用蛋白质印迹法检测TNF-α、NF-κB及TLR-4的表达。**P<0.001，***P<0.0001。
2. 16S rRNA测序原始数据：AS组与AS+MET组各5份样本。