Gut Microbiota-Derived Acetate Ameliorates Endometriosis via JAK1/STAT3-Mediated M1 Macrophage Polarisation

Abstract

Endometriosis (EMs) is a common inflammatory disorder in women of reproductive age, severely impacting patients' quality of life and fertility. Current hormonal therapies offer limited efficacy, and surgical interventions often fail to prevent recurrence. Recent studies suggest a close association between gut microbiota and the pathophysiology of EMs, though the precise mechanisms remain unclear. To investigate the influence of gut microbiota on EMs, this study established an EMs mouse model and performed faecal microbiota transplantation (FMT) using samples from healthy donors (AH group) and EMs patients (AE group) into the model mice. Results demonstrated that compared to the model group (M group), FMT from healthy donors (AH group) significantly reduced ectopic lesion volume (658.3 ± 116.1 vs. 167.2 ± 112.8 mm³, p < 0.01) and weight (0.7420 ± 0.1233 vs. 0.1885 ± 0.1239 mg, p < 0.01). Conversely, FMT from EMs patients exacerbated disease progression. Mechanistic studies revealed that healthy donor FMT attenuated EMs by remodelling the gut microbial composition (enhancing α-diversity and Lactobacillus abundance while suppressing Bacteroidetes), significantly elevating acetate levels in faeces and ectopic lesions, activating the JAK1/STAT3 signalling pathway within lesions, and thereby driving macrophage polarisation toward the M1 phenotype (by increased iNOS/CD86 expression and decreased Arg1/CD206 expression). Simultaneously, healthy donor FMT enhanced intestinal barrier integrity by upregulating tight junction proteins (ZO-1, Occludin, Claudin-1/5) and reducing levels of intestinal permeability markers (DAO, IFABP). In contrast, AE group FMT disrupted gut microbial ecology, reduced acetate production, failed to activate the JAK1/STAT3 pathway, promoted M2 macrophage polarisation and impaired intestinal barrier function. Collectively, this study elucidates for the first time that acetate, as a key gut microbiota metabolite, exerts anti-EMs effects by activating the JAK1/STAT3 signalling pathway to drive macrophage reprogramming toward the M1 phenotype, thereby positioning gut microbiota reconstruction as a novel therapeutic strategy for endometriosis.

Keywords: JAK–STAT signalling; acetate; endometriosis; faecal microbiota transplantation; gut microbiota; macrophage polarisation; short‐chain fatty acids (SCFAs).

FIGURE 1

Effects of FMT on endometriosis progression in mice. (A) Experimental design: endometriosis was modelled in 6–8‐week‐old female BALB/c mice by surgically transplanting endometrial fragments and administering oestrogen injections. Mice were intragastrically treated with Abx for 1 week, followed by experimental intervention with 200 μL faecal suspension via oral gavage every 2 days for 4 weeks. (B) Representative images of endometriotic lesions in each group. (C) Volume of lesions. (D) Weight of lesions. (E) Representative images of HE stains and immunohistochemical staining for Ki67 and Iba‐1 in lesions from each group. Scale bar: 20 μm. AE, FMT from endometriosis patients; AH, FMT from healthy women; M, model group. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2

FMT on gut microbiota diversity in a mouse model of endometriosis. (A) Chao1 index. (B) Good's coverage index. (C) Simpson index. (D) Pielou's evenness index. (E) Observed species index. (F) Observed_species index. (G) Principal coordinate analysis (PCoA). (H) Non‐metric multidimensional scaling (NMDS). (I) Uniform Manifold Approximation and Projection (UMAP) analysis. AE, FMT from endometriosis patients; AH, FMT from healthy women; M, model group. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3

Effects of FMT on gut microbiota composition in a mouse model of endometriosis. (A) Relative abundance of gut microbiota at the phylum level. (B) Relative abundance of Bacteroidetes. (C) Relative abundance of Firmicutes. (D) Relative abundance of gut microbiota at the genus level. (E) Relative abundance of Lactobacillus. (F) Relative abundance of Oscillospira. (G) Venn diagram of microbial taxa distribution. (H) LEfSe analysis highlighting differentially abundant taxa. (I) Feature importance heatmap of gut microbiota at the genus level. AE, FMT from endometriosis patients; AH: FMT from healthy women; M, model group. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4

Healthy gut microbiota transplantation improves intestinal barrier function by upregulating tight junction proteins. (A) Representative photographs of colons from each group. (B) Colon length across groups. (C) Histological examination of colonic tissues via H&E staining (left) and immunohistochemical staining for ZO—1 (middle) and Occludin (right), with a scale bar of 50 μm under 20× magnification; Relative mRNA expression levels of tight junction—associated genes in colonic tissues: ZO—1 (D), Occludin (E) Claudin—1 (F), Claudin—5 (G). (H, I) Concentrations of DAO (H) and IFABP (I) in serum, indicators of intestinal mucosal injury. Scale bar: 20 μm. AE, FMT from endometriosis patients; AH, FMT from healthy women; M, model group. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5

Healthy Donor FMT Promotes M1 Macrophage Polarisation via JAK‐1/Stat3 Signalling Axis in Endometriosis Lesions. (A–D) Relative mRNA expression levels of macrophage polarisation—associated marker genes in colonic tissues: ARG—1 (A), CD206 (B), iNOS (C), and CD86 (D). (E–I) Relative mRNA expression levels of genes involved in pathways regulating macrophage polarisation in Endometriotic tissues: JAK1 (E), STAT3 (F), AMPK (G), ERK1 (H), ERK2 (I). (J) Immunofluorescence staining of colonic tissues for macrophage markers: DAPI for nuclei, CD68 for total macrophages, CD206 for M2—type macrophages, CD86 for M1—type macrophages, and merged images. Scale bar: 20 μm. AE, FMT from endometriosis patients; AH, FMT from healthy women; M, model group. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6

Faecal microbiota transplantation from healthy donors specifically elevates acetate levels in faeces and ectopic lesions of mice. (A) Heatmap of SCFA relative levels in faeces among groups. (B–G) Quantification of faecal SCFAs: Propionic acid (B), Butyric acid (C), Isobutyric acid (D), Valeric acid (E), Caproic acid (F), Isovaleric acid (G). (H) Heatmap of SCFA relative levels in endometriotic lesions among groups. (I–N) Quantification of SCFAs in endometriotic lesions: Acetic acid (I), Propionic acid (J), Butyric acid (K), Isobutyric acid (L), Valeric acid (M), Caproic acid (N). AE, FMT from endometriosis patients; AH, FMT from healthy women; M, model group. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 7

Healthy donor FMT ameliorates endometriosis by promoting M1 macrophage polarisation via JAK1/STAT3 pathway activation. (A, C) Relative protein levels of iNOS (A) and Arg1 (C) in endometriotic tissues, detected by Western blotting. (B) Representative Western blot images of iNOS, Arg1, and β—actin in endometriosis tissues. (D) Representative Western blot images of P—Jak1, Jak1, P—Stat3, Stat3, And β—Actin in endometriosis tissues. (E–J) Quantitative analysis of protein levels: Jak1 (E), P—Jak1/Jak1 Ratio (F), P—Jak1 (G), Stat3 (H), P—Stat3/Stat3 Ratio (I), P—Stat3 (J). AE, FMT from endometriosis patients; AH, FMT from healthy women; M, model group. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 8

Mechanism of healthy donor faecal microbiota transplantation (FMT) in ameliorating endometriosis via acetate‐mediated JAK1/STAT3 activation and macrophage reprogramming. FMT from healthy donors enriches beneficial gut microbiota (increased Firmicutes, increased Lactobacillus), specifically elevating acetate levels in the gut and ectopic lesions. Acetate enhances intestinal barrier integrity by upregulating tight junction proteins (increased ZO‐1, Occludin, Claudin‐1 and Claudin‐5) and reducing intestinal permeability markers (DAO and IFABP). Within lesions, acetate activates the JAK1/STAT3 signalling pathway, driving macrophage polarisation toward the M1 phenotype (increased iNOS and CD86) while suppressing M2 polarisation (decreased Arg1 and CD206), ultimately inhibiting ectopic lesion growth. Conversely, FMT from endometriosis patients exacerbates dysbiosis (decreased Firmicutes and Lactobacillus), reduces acetate production, impairs barrier function, fails to activate JAK1/STAT3, and promotes M2‐dominant inflammation, accelerating disease progression.