基于非靶向代谢组学的普雷沃氏菌对动脉粥样硬化小鼠作用机制

Untargeted metabolomics reveals the mechanism of Prevotella copri in promoting atherosclerosis in mice

摘要:

目的 从宿主-肠道菌群-代谢的角度，探讨普雷沃氏菌(Prevotella copri)促进动脉粥样硬化(atherosclerosis, AS)的可能作用机制。方法 将ApoE^-/-小鼠随机分为4组，每组8只：对照组(Chow组)，喂以普通饲料；模型组(AS组)，喂以高脂饲料；低浓度P. copri移植组(P. copri-low组)，自高脂饮食开始的第1天起每天灌胃10⁹ CFU/mL的P. copri；高浓度P. copri移植组(P. copri-high组)，自高脂饮食开始的第1天起每天灌胃10¹¹ CFU/mL的P. copri。每周测量并记录体重变化，评估体重增长趋势。5周后，应用油红O染色评估主动脉斑块面积，结合酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)检测血脂水平，评估P. copri对AS进展的影响。同时，利用实时定量PCR (quantitative PCR, qPCR)检测P. copri在肠道中的丰度，并通过非靶向代谢组学技术分析小鼠粪便代谢物的变化。结果 AS组小鼠的体重、主动脉斑块面积显著增加，血浆低密度脂蛋白胆固醇(low-density lipoprotein cholesterol, LDL-C)、总胆固醇(total cholesterol, TC)和甘油三酯(triglycerides, TG)水平显著升高，高密度脂蛋白胆固醇(high-density lipoprotein cholesterol, HDL-C)水平显著降低，与Chow组相比均存在显著差异。P. copri-low组和P. copri-high组在肠道中的P. copri丰度无显著差异，表明两组P. copri均能在小鼠肠道成功定殖。基于此，后续实验选取P. copri-low组作为标准浓度组(P. copri组)进行分析。与AS组相比，P. copri成功定殖显著增加了小鼠的体重和主动脉斑块面积，并加重了血脂紊乱。代谢组学分析显示，P. copri移植导致多种代谢物显著升高，包括Cer(d18:1/18:1(9Z))、棕榈酰鞘氨醇(N-palmitoylsphingosine)、染料木素(genistein)、腺嘌呤(adenine)和亚油酸(linoleic acid)。KEGG通路富集分析进一步揭示，P. copri通过调控ABC转运蛋白、胆汁酸代谢和神经活性配体-受体相互作用等关键通路参与AS的发生发展。结论 P. copri通过调控鞘脂信号通路、嘌呤代谢和亚油酸代谢，可能加剧炎症和脂质代谢失衡，从而促进AS的进展。

关键词:普雷沃氏菌;动脉粥样硬化;代谢组学;ApoE^-/-小鼠

Abstract:

Objective To explore the potential mechanism by which Prevotella copri promotes atherosclerosis (AS) from the perspective of host-gut microbiota-metabolism.Methods ApoE^-/- mice were randomized into four groups (n=8): control group (Chow group, fed with a normal diet), model group (AS group, fed with a high-fat diet), low-concentration P. copri group (P. copri-low group, administrated with P. copri at 10⁹ CFU/mL by oral gavage daily from the first day of feeding with the high-fat diet), and high-concentration P. copri group (P. copri-high group, administrated with P. copri at 10¹¹ CFU/mL by oral gavage daily from the first day of feeding with the high-fat diet). The body weight was measured and recorded weekly to evaluate the weight gain trend. After 5 weeks, oil red O staining was employed to evaluate the aortic plaque area, and enzyme-linked immunosorbent assay (ELISA) was employed to measure lipid levels, on the basis of which the impact of P. copri on AS progression was assessed. Additionally, qPCR was used to detect the abundance of P. copri in the gut, and untargeted metabolomics was employed to analyze the metabolite changes in the feces of mice.Results Compared with the Chow group, the AS group showed increases in the body weight, aortic plaque area, and plasma levels of low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TG) and a decline in the high-density lipoprotein cholesterol (HDL-C) level. The abundance of P. copri in the gut showed no significant difference between the P. copri-low group and the P. copri-high group, indicating that P. copri successfully colonized the gut in both groups. Based on this, the P. copri-low group was selected as the standard concentration group (P. copri group) for further analysis. Compared with the AS group, P. copri colonization in the gut significantly increased the body weight and aortic plaque area and exacerbated dyslipidemia. Metabolomic analysis revealed that P. copri transplantation led to significant increases in the content of several metabolites, including Cer(d18:1/18:1(9Z)), N-palmitoylsphingosine, genistein, adenine, and linoleic acid. KEGG pathway enrichment analysis further indicated that P. copri might contribute to the development and progression of AS through key pathways such as the regulation of ABC transporters, bile acid metabolism, and neuroactive ligand-receptor interactions.Conclusion P. copri may exacerbate inflammation and lipid metabolism imbalance by regulating sphingolipid signaling, purine metabolism, and linoleic acid metabolism, thereby promoting the progression of AS.

Key words:Prevotella copri;atherosclerosis;metabolomics;ApoE^-/- mice