Oral Clostridium butyricum on mice endometritis through uterine microbiome and metabolic alternations

Abstract

Endometritis occurs frequently in humans and animals, which can negatively affect fertility and cause preterm parturition syndrome. Orally administered Clostridium butyricum, a butyrate-producing gram-positive anaerobe, exhibits anti-inflammatory effects. However, the precise mechanism by which Clostridium butyricum attenuates endometritis remains unclear. This in vivo study evaluated the anti-inflammatory effects of orally administered Clostridium butyricum on uterine tissues. In addition, we conducted uterine microbiome and lipid metabolome analyses to determine the underlying mechanisms. Female Balb/c mice were divided into the following four groups (n = 5-20): (1) mock group, (2) only operation group (mice only underwent operation to exposed uterine horns from the side), (3) control group (mice underwent the same operation with the operation group + perfusion of lipopolysaccharide solution from uterine horns), and (4) Clostridium butyricum administration group (mice underwent the same operation with the control group + oral Clostridium butyricum administration from days 0 to 9). Clostridium butyricum was administered via oral gavage. On day 10, we investigated protein expression, uterine microbiome, and lipid metabolism in uterine tissues. Consequently, orally administered Clostridium butyricum altered the uterine microbiome and induced proliferation of Lactobacillus and Limosilactobacillus species. The effects can contribute to show the anti-inflammatory effect through the interferon-β upregulation in uterine tissues. Additionally, oral Clostridium butyricum administration resulted in the upregulations of some lipid metabolites, such as ω-3 polyunsaturated fatty acid resolvin D5, in uterine tissues, and resolvin D5 showed anti-inflammatory effects. However, the orally administered Clostridium butyricum induced anti-inflammatory effect was attenuated with the deletion of G protein-coupled receptor 120 and 15-lipooxgenase inhibition. In conclusion, Clostridium butyricum in the gut has anti-inflammatory effects on uterine tissues through alterations in the uterine microbiome and lipid metabolism. This study revealed a gut-uterus axis mechanism and provided insights into the treatment and prophylaxis of endometritis.

Keywords: Clostridium butyricum; G protein-coupled receptor 120; Lactobacillus species; Limosilactobacillus species; endometritis; metabolome; microbiome; resolvin D5.

Figure 1

Anti-inflammatory effects of orally administered Clostridium butyricum in uterine tissue. (A) Balb/c mice were divided into 4 groups. Mock group received no treatments; only operation group, control group and CBM 588 group received operations on days 0 and 5. Control group and CBM 588 group conducted LPS perfusions from the uterus horns. Only CBM 588 groups received oral CBM 588 administrations from days 0 to 9. (B) Cytokine levels in uterine tissues on day 10 (n = 9 or 10). (C) Weights of uterine tissues (n = 9 or 19) and colon tissues (n = 9). (D) Representative histological images of uterine tissues on day 10 (scale bar, 100 mm, bottom right). Results are presented as mean ± standard deviation. Each dot represents a single mouse. The results were considered statistically significant when the differences were p < 0.05, as determined using one-way ANOVA (B,C). See also Supplementary Figure S1. CBM 588, Clostridium butyricum MIYAIRI 588; LPS, lipopolysaccharide.

Figure 2

Effect of oral C. butyricum on uterine microbiome. (A) Balb/c mice were divided into 2 groups. Control group (n = 4) and CBM 588 group (n = 4) received operations on days 0 and 5. Control group and CBM 588 group conducted LPS perfusions from the uterus horns. Only CBM 588 group received oral CBM 588 administrations from days 0 to 9. (B) Bacterial composition in uterine tissues at the family level. (C) Relative abundances of Lactobacillales in uterine microbiome at order level. (D) Relative abundances of Lactobacillaceae in uterine microbiome at family level. (E) Relative abundances of Lactobacillus, Ligilactobacillus, and Limosilactobacillus in uterus microbiome at genera level. Results are presented as mean ± standard deviation. Each dot represents a single mouse. Results were considered statistically significant when the differences were p < 0.05, as determined by Student’s t-test (C–E). See also Supplementary Figure S2. CBM 588, Clostridium butyricum MIYAIRI 588; LPS, lipopolysaccharide.

Figure 3

Supernatant of C. butyricum promotes Lactobacillus spp. and Limosilactobacillus spp. proliferations. (A) Lactobacillus spp. and Limosilactobacillus spp. were exposed to the supernatant of C. butyricum incubation medium (0, 1, 5, and 10%) and incubated anaerobically for 24 h. (B) Bacterial concentrations of Lactobacillus spp. (n = 4, respectively). (C) Bacterial concentrations of Limosilactobacillus spp. (n = 4, respectively). Results are presented as mean ± standard deviation. Each dot represents a single sample. The results were considered statistically significant when the differences were p < 0.05, as determined using one-way ANOVA (B,C). See also Supplementary Figure S3. CBM 588, Clostridium butyricum MIYAIRI 588.

Figure 4

Orally administered C. butyricum shows anti-inflammatory effects through the interferon-β upregulation in uterine tissues. (A) Balb/c mice were divided 3 groups. Mock group received no treatment (n = 8), control group (n = 8) and CBM 588 group (n = 8) received operations on day 0 and 5. Then, control group and CBM 588 group conducted LPS perfusions from uterus horns. Only CBM 588 group received orally CBM 588 administrations from days 0 to 9. (B) IFN-β level in uterine tissues on day 10. (C) C57BL/6 J mice were divided into control group (n = 5), CBM 588 group (n = 5) and CBM 588 (IRF-7 KO) group (n = 5). They received operations on day 0 and 5, and then, conducted LPS perfusions from uterus horns. Two groups in the back received oral CBM 588 administrations from day 0 to 9. (D) Cytokine levels in uterine tissues on day 10. (E) Weights of uterine tissues and colon on day 10. Results are presented as mean ± standard deviation. Each dot represents a single mouse. Results were considered statistically significant when the differences were p < 0.05, as determined by one-way ANOVA (B–E). See also Supplementary Figure S4. CBM 588, Clostridium butyricum MIYAIRI 588; LPS, lipopolysaccharide; IFN-β, interferon-β; IRF-7, interferon regulation factor-7.

Figure 5

Orally administered C. butyricum alters lipid metabolism in uterine tissues. (A) Balb/c mice were divided into 2 groups. The control group (n = 4) and CBM 588 group (n = 4) received operations on days 0 and 5. The control group and CBM 588 group conducted LPS perfusions from the uterus horns. Only the CBM 588 group received oral CBM 588 administrations from days 0 to 9. (B) LDA Score (Log2) of lipid metabolites in uterine tissues when compared with the control and CBM 588 administration group. (C) Peak areas of DHA, resolvin D5 and 13,14-dyhydro-15-keto PGF2α in uterine tissues. Results are presented as mean ± standard deviation. Each dot represents an individual mouse. Results were considered statistically significant when differences were p < 0.05 by Student’s t-test (C). See also Supplementary Figure S5. CBM 588, Clostridium butyricum MIYAIRI 588; LPS, lipopolysaccharide.

Figure 6

Orally administered C. butyricum shows anti-inflammatory effects through G protein-coupled receptor 120. (A) Balb/c mice were divided into 6 groups (n = 5, respectively). All groups received operations on days 0 and 5, and then, conducted LPS perfusions from the uterus horns. CBM 588 group, CBM 588 + 15-LOX inhibitor group and CBM 588 (GPR120 KO) group received oral CBM 588 administrations from days 0 to 9. ATCC 19398 group received oral C. butyricum ATCC 19398 administrations from days 0 to 9. Resolvin D5 group received resolving D5 intraabdominally from day 0 to 9. (B) TNF-α level in uterine tissues on day 10. (C) IFN-β level in uterine tissues on day 10. (D) Weights of uterus tissues, colon and mouse body. Results are presented as mean ± standard deviation. Each dot represents a single mouse. Results were considered statistically significant when the differences were p < 0.05, as determined by one-way ANOVA (B–D). CBM 588, Clostridium butyricum MIYAIRI 588; LPS, lipopolysaccharide; IFN-β, interferon-β.

Figure 7

Orally administered C. butyricum alters uterine microbiome and lipid metabolisms to attenuate inflammation in uterus tissues. Orally administered CBM 588 can contribute to show the anti-inflammation effects in uterine tissues through the IFN-β upregulation and GPR120 activation with lipid metabolites, such as resolvin D5. CBM 588, Clostridium butyricum MIYAIRI 588; IFN-β, interferon-β.