The gut microbiota in mice with erythropoietin-induced abdominal aortic aneurysm

Abstract

Background: In recent years, a novel animal abdominal aortic aneurysm (AAA) model was established by administering erythropoietin (EPO) to wild-type (WT) mice. However, the influence of EPO on the murine fecal microbiota remains uninvestigated. Therefore, this study aims to explore the potential association between gut microbiota changes and AAA development in this model.

Methods and results: Adult male C57BL/6 mice were used to establish the AAA model by intraperitoneal administration of recombinant human EPO at a dosage of 10,000 IU/kg daily for 28 consecutive days. Hematoxylin and eosin (H&E) and Elastin Van Gieson (EVG) staining revealed that EPO administration increased aortic wall thickness and diameter, accompanied by enhanced degradation of the elastic lamina. The 16S rRNA-sequencing data were deposited in the Sequence Read Archive (PRJNA1172300). LEfSe analysis revealed that Akkermansia, Lawsonibacter, Clostridium, and Neglectibacter were significantly associated with EPO-induced AAA development, while Lactobacillus, Alistipes, Limosilactobacillus, and Eisenbergiella showed significant negative correlations. Analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) prediction module revealed significant differences in metabolic pathways between the two groups, including alanine, aspartate and glutamate metabolism; cysteine and methionine metabolism; pyrimidine metabolism; carbon metabolism; ABC transporters; and oxidative phosphorylation pathways.

Conclusions: EPO-induced gut dysbiosis, particularly changes in Akkermansia, Lactobacillus, and Alistipes abundance, may contribute to AAA formation via inflammation, oxidative stress, and metabolic dysfunction. While this model advances AAA research, its limitations underscore the need for human validation and mechanistic studies. Future work should prioritize multi-omics integration and cross-model comparisons to unravel the complex microbiota-AAA axis.

Keywords: 16S rRNA-sequencing; Abdominal aortic aneurysm; Erythropoietin; Gut microbiota.

Figure 1. Identification of the EPO-induced AAA mouse model.

(A) Photographic representations of aortic specimens from the control group (con) and the EPO- treated group (AAA). (B and C) Typical H&E staining micrographs depicting the abdominal aorta from the control group (B) and EPO- treated group (C). (D) The statistical graph of the vascular diameter. Data represent the mean ± SD (n = 6 and 7 in the control and AAA groups, respectively). **P < 0.01 (Student’s t-test). (E and F) Representative EVG staining pictures of the abdominal aorta of control (E) and EPO-treated mice (F).

Figure 2. The alpha and beta diversities of the gut microbiota.

(A) Rarefaction curve. (B) ACE index. ** p < 0.01, Student’s t-test (C) Shannon diversity index. p > 0.05, Student’s t-test (D and E) 2D (D) and 3D (E) PCoA analysis. con: control group; AAA: EPO-treated group.

Figure 3. Analysis of the GMHI.

(A) Analysis of the difference of GMHI between control (con) and EPO (AAA) groups; (B) GMHI stratified control (con, n = 6) and EPO (AAA, n = 7) groups more strongly than Chao diversity. Each point in the scatter plot corresponds to a metagenomic sample (12 in total). The histogram shows the distribution of control (con, blue) and EPO (AAA, orange) samples based on the parameters of each axis.

Figure 4. Changes in gut microbiota between the control group and the EPO-treated group.

(A) Venn diagram of ASV. (B and C) Bar diagram at phylum level (B) and genus level (C). (D) Circos diagram at phylum level (left) and genus level (right). con: control group; AAA: EPO-treated group.

Figure 5. Analysis of species differences.

(A) The two groups were compared by Student’s t-test. The red bar was the control group (con) and the blue bar was the EPO-treated group (AAA); (B, C) Linear discriminant analysis (LDA) effect size (LEfSe) was used to analyze, the differences between the control group (con) and the EPO-treated group (AAA) was shown by cladogram (B) and histogram (C, at genus level). The red bar was the control group and the blue bar was the EPO-treated group, LDA > 2.

Figure 6. PICRUSt2 analysis predicted the potential role of the gut microbiota.

(A) COG function classification. (B) Difference between the control group (con) and the EPO-treated group (AAA) in COG function. *p < 0.05, Student’s T test. U: intracellular trafficking, secretion, and vesicular transport; P: inorganic ion transport and metabolism; E: amino acid transport and metabolism. (C) Heatmap of KEGG pathway level 3. (D) Difference between the EPO and control groups in KEGG pathway level 3. ***p <  0.001, **p < 0.01, *p < 0.05, Student’s T test.