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Gut microbiota-derived indole 3-propionic acid protects against radiation toxicity via retaining acyl-CoA-binding protein - PubMed

  • ️Wed Jan 01 2020

Gut microbiota-derived indole 3-propionic acid protects against radiation toxicity via retaining acyl-CoA-binding protein

Hui-Wen Xiao et al. Microbiome. 2020.

Abstract

Background: We have proved fecal microbiota transplantation (FMT) is an efficacious remedy to mitigate acute radiation syndrome (ARS); however, the mechanisms remain incompletely characterized. Here, we aimed to tease apart the gut microbiota-produced metabolites, underpin the therapeutic effects of FMT to radiation injuries, and elucidate the underlying molecular mechanisms.

Results: FMT elevated the level of microbial-derived indole 3-propionic acid (IPA) in fecal pellets from irradiated mice. IPA replenishment via oral route attenuated hematopoietic system and gastrointestinal (GI) tract injuries intertwined with radiation exposure without precipitating tumor growth in male and female mice. Specifically, IPA-treated mice represented a lower system inflammatory level, recuperative hematogenic organs, catabatic myelosuppression, improved GI function, and epithelial integrity following irradiation. 16S rRNA gene sequencing and subsequent analyses showed that irradiated mice harbored a disordered enteric bacterial pattern, which was preserved after IPA administration. Notably, iTRAQ analysis presented that IPA replenishment retained radiation-reprogrammed protein expression profile in the small intestine. Importantly, shRNA interference and hydrodynamic-based gene delivery assays further validated that pregnane X receptor (PXR)/acyl-CoA-binding protein (ACBP) signaling played pivotal roles in IPA-favored radioprotection in vitro and in vivo.

Conclusions: These evidences highlight that IPA is a key intestinal microbiota metabolite corroborating the therapeutic effects of FMT to radiation toxicity. Owing to the potential pitfalls of FMT, IPA might be employed as a safe and effective succedaneum to fight against accidental or iatrogenic ionizing ARS in clinical settings. Our findings also provide a novel insight into microbiome-based remedies toward radioactive diseases. Video abstract.

Keywords: ACBP; Acute radiation syndrome; Gastrointestinal tract toxicity; Gut microbiota; Gut microbiota metabolite; Hematopoietic toxicity; Indole 3-propionic acid; Radiotherapy.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1

IPA replenishment protects against radiation-induced mortality in vitro and in vivo. a, b The concentrations of IPA in fecal pellets from each cohort was measured at the end of receiving 10 days of FMT. The IPA levels are not significantly different between control vs TAI + FMT or TBI + FMT. Significant differences between each two cohorts are indicated: *P < 0.05 and **P < 0.01; Student’s t test. c The relative abundance of g_Clostridium was compared among control, TAI, and TAI + hydrogen-water groups through 16S rRNA sequencing analysis. d Kaplan-Meier analysis of male mice treated with the indicated irradiation and with IPA or saline. n = 24 per group. *P < 0.05 by log-rank test between 7.5 mg/ml IPA and TBI groups. e, f Body weights were compared among male mice after 7.2 Gy TBI or 12 Gy TAI, n = 24 per group; Significant differences between each two cohorts are indicated: *P < 0.05, **P < 0.01, and ***P < 0.005; Student’s t test. g–i The effects of concentration gradient IPA on the proliferation of MODE-K cells (g) and HIEC-6 cells (h–j) were assessed by CCK-8 assays and cloning formation assays, respectively. Significant differences between each two cohorts are indicated: *P < 0.05, **P < 0.01, and ***P < 0.005; Student’s t test. k Body weights were compared between FMT group and IPA group after 12 Gy TAI, n = 24 per group

Fig. 2
Fig. 2

Oral gavage of IPA ameliorates TBI-associated hematopoietic system injury. a, b Photographs (a) and weight (b) of dissected thymuses from mice in the three groups, the thymuses were obtained at day 15 after 4 Gy TBI. Mean ± SEM. Significant differences between each two cohorts are indicated: ***P < 0.005; Student’s t test, n = 12 per group. c, d Photographs (c) and weight (d) of the dissected spleens from mice in the three groups, the spleens were obtained at day 15 after 4 Gy TBI. Significant differences between each two cohorts are indicated: **P < 0.01 and ***P < 0.005; Student’s t test, n = 12 per group. e, f White blood cell (WBC) counts (e) and percentage of lymphocytes (LY%) (f) in PB were measured at day 15 after 4 Gy TBI. The data were presented as means ± SEM (n = 12 per group). Significant differences between each two cohorts are indicated: *P < 0.05 and ***P < 0.005; Student’s t test. g, h The content of IL-6 (g) and MDA (h) in PB were examined. Mean ± SEM. Significant differences between each two cohorts are indicated: **P < 0.01 and ***P < 0.005; Student’s t test, n = 6 for control group; n = 11 for TBI group; n = 12 for TBI + IPA group. i–k Representative FACS plots of HSCs, HPCs. The percentage of hematopoietic progenitor cells (HPCs) and HSC cells in lineage-negative cells were analyzed at day 15 after 4 Gy TBI. The data were presented as means ± SEM (n = 6 per group). Significant differences between each two cohorts are indicated: **P < 0.01 and ***P < 0.005; Student’s t test

Fig. 3
Fig. 3

IPA administration improves GI tract toxicity after total abdominal irradiation. a, b Photographs (a) and length (b) of dissected colon from mice in the three groups, the colon tissues were obtained at day 21 after 12 Gy TAI. Mean ± SEM. Significant differences between each two cohorts are indicated: *P < 0.05 and ***P < 0.005; Student’s t test, n = 12 per group. c The morphology of the small intestine was shown by H&E (×100 magnification; scale bar: 100 μm) and PAS (×1000 magnification; scale bar: 50 μm) staining. The small intestine tissues were obtained at day 21 after 12 Gy TAI. The arrows point to the goblet cells. d, e The content of IL-6 (d) and TNFɑ (e) in the small intestine tissues were examined by ELISA. Mean ± SEM. Significant differences between each two cohorts are indicated: *P < 0.05, **P < 0.01, and ***P < 0.005; Student’s t test, n = 6 for control group, n = 11 for TAI group, n = 12 for TAI + IPA group. f–h The expression levels of Glut1 (f), Pgk1 (g), and MDR1 (h) were examined in the small intestine tissues by qRT-PCR. The small intestine tissues were obtained at day 21 after 12 Gy TAI. Mean ± SEM. Significant differences between each two cohorts are indicated: *P < 0.05 and **P < 0.01; Student’s t test, n = 12 per group. i The FITC-dextran in PB was assessed at day 21 after 12 Gy TAI. Mean ± SEM. Significant differences between each two cohorts are indicated: **P < 0.01 and ***P < 0.005; Student’s t test, n = 12 per group. j The expression levels of Nrf2 was assessed in the small intestine tissue by qRT-PCR. The small intestine tissues were obtained at day 21 after 12 Gy TAI. Mean ± SEM. Significant differences between each two cohorts are indicated: *P < 0.05; Student’s t test, n = 12 per group. k The content of MDA in the small intestine tissues were examined. Mean ± SEM. Significant differences between each two cohorts are indicated: ***P < 0.005; Student’s t test, n = 6 for control group, n = 11 for TAI group, n = 12 for TAI + IPA group

Fig. 4
Fig. 4

IPA protects against radiation toxicity in mouse models without accelerating tumor growth. a Kaplan-Meier analysis of female mice in the three groups after 7.2 Gy TBI, n = 30 per group. *P < 0.05 by log-rank test between IPA and TBI groups. b, c Photographs of dissected the thymuses and spleens from female mice in the three groups, and the thymuses and spleens were obtained at day 15 after 4 Gy TBI. n = 12 per group. d White blood cell (WBC) counts in PB from female mice were measured at 15 days after 4 Gy TBI. The data were presented as means ± SEM (n = 12 per group). Significant differences between each two cohorts are indicated: ***P < 0.005; Student’s t test. e Photographs of dissected colon from female mice in the three groups, and the colon tissues were obtained at day 21 after 12 Gy TAI. n = 12 per group. f–h The expression levels of Pgk1 (f), IL-6 (g), and Nrf2 (h) were examined in the small intestine tissues by qRT-PCR. The small intestine tissues were obtained at day 21 after 12 Gy TAI. Mean ± SEM. Significant differences between each two cohorts are indicated: *P < 0.05, **P < 0.01, and ***P < 0.005; Student’s t test, n = 12 per group. i, j The growth images of HCT-8 and ME-180 cells in nude mice administrated with IPA and local radiation. k, l The growth curve of HCT-8 and ME-180 cells in nude mice administrated with IPA and local radiation. Data are expressed as mean ± SEM from 7 mice. Statistically significant differences between each two cohorts are indicated: ***P < 0.001; Student’s t test. m The expressions of Ki-67 were examined by immunohistochemistry staining in HCT-8 and ME-180 tumor tissues from nude mice

Fig. 5
Fig. 5

IPA preserves irradiation-shifted enteric bacterial composition at day 12 after TAI. a, b The observed species number and Chao1 diversity index of intestinal bacteria was examined by 16S rRNA high-throughput sequencing after 12 days of TAI exposure. Significant differences are indicated: Wilcoxon rank sum test. n = 6 per group. c, d PCoA and NMDS were used to measure the shift in intestinal bacterial composition profile after irradiation at day 12. e The β diversity of intestinal bacteria was compared by the weighted unifrac analysis. Significant differences are indicated: Wilcoxon rank sum test. n = 6 per group. f The alteration of intestinal bacterial patterns at the genus level was assessed by 16S rRNA sequencing, n = 6 per group. The heat map is color-based on row Z-scores. The mice with the highest and lowest bacterial level are in red and blue, respectively. g The abundances of most varied strain bacteria was assessed using 16S high-throughput sequencing after irradiation at day 12. Statistically significant differences are indicated: Wilcoxon rank sum test, n = 6 per group. h Linear discriminant analysis (LDA) effect size (LEfSe) results represented significantly different in abundance of gut bacteria between TAI and IPA groups and indicated the effect size of each differentially abundant bacterial taxon in the small intestine after irradiation at day 12, n = 6 per group. Significant differences are indicated: Wilcoxon rank sum test. i The content of IPA in fecal was examined by ELISA. Mean ± SEM. Significant differences between each two cohorts are indicated: *P < 0.05 and ***P < 0.005; Student’s t test, n = 8 per group. j, k Photographs (j) and length (k) of dissected colon from IPA gavage mice with or without antibiotics (ABX) treatment, the colon tissues were obtained at day 21 after 12 Gy TAI. Mean ± SEM. Significant differences are indicated: Student’s t test, n = 6 per group. l The morphology of the small intestine was shown by H&E (× 100 magnification; scale bar: 100 μm) and PAS (× 1000 magnification; scale bar: 50 μm) staining. The small intestine tissues were obtained at day 21 after 12 Gy TAI. The arrows point to the goblet cells

Fig. 6
Fig. 6

IPA replenishment reprograms small intestinal protein expression profile following TAI challenge. a Volcano plots of identified different proteins from the small intestine of mice with or without IPA treatment. In the volcano plots, each point represented a protein. b–d Bioinformatics analysis of different proteins in small intestine of mice in IPA gavage group compared to the TAI group through gene ontology (GO) in biological process (b), cellular component (c), and molecular function (d). Information on the number of involved proteins in a term is shown on the x-axis. e Hierarchical cluster analysis for the different proteins in the small intestine of mice in IPA gavage group compared to TAI group. f The expression level of ACBP was examined in small intestine tissues from male mice by qRT-PCR. Mean ± SEM. Significant between each two cohort differences are indicated: *P < 0.05; Student’s t test, n = 18 per group. g The relative level of ACBP were measured at the time of 0, 0.5, 1, 1.5, and 2 h with (or without) 4 Gy irradiation after IPA treatment (37.8 μg/mL) by qRT-PCR. Mean ± SEM. Significant differences between each two cohorts are indicated: ***P < 0.005; Student’s t test. h The mRNA levels of ACBP were examined in HIEC-6 and MODE-K cells which included control, 4 Gy irradiation, and 4 Gy irradiation with IPA supplement. i–k The effects of IPA (37.8 μg/mL) on the proliferation of ACBP siRNA-treated HIEC-6 cells were assessed by cloning formation (i, j) and CCK-8 assays (k), respectively. Mean ± SEM. Significant differences between each two cohorts are indicated: *P < 0.05, **P < 0.01, and ***P < 0.005; Student’s t test. l A model showed the predicted binding site for PXR at 678–668 nt and 488–478 nt of ACBP mRNA promoter named PGL3-ACBP-1 and PGL3-ACBP-2. m The effect of PXR and IPA on PGL3-ACBP-1 reporter was measured by luciferase reporter gene assays in HIEC-6 cells. Mean ± SEM. Significant differences between each two cohorts are indicated: **P < 0.01; Student’s t test. n The expression of ACBP was examined by qRT-PCR after transfection of HIEC-6 cells with si-PXR and (or) treated with IPA (37.8 μg/mL)

Fig. 7
Fig. 7

ACBP contributes to the protective function of IPA toward irradiation via PXR. a The presence of gene expression after injection in various organs, including the liver, heart, lung, colon, small intestine, and tongue, as confirmed by bioluminescent imaging. b, c The expression level of ACBP (b) and PXR (c) was examined in the small intestine tissues by qRT-PCR. Significant differences are indicated: *P < 0.05 and ***P < 0.005; Mean ± SEM. Student’s t test, n = 10 per group. d Body weights were compared among four group mice after 12Gy TAI, n = 18 per group; *P < 0.05, **P < 0.01, and ***P < 0.005 represent TAI + sh-ACBP group compared with TAI + IPA group; #P < 0.05, ##P < 0.01, and ###P < 0.005 represent TAI + sh-PXR group compared with TAI + IPA group; Student’s t test. e, f Photographs (e) and length (f) of dissected colon from mice in the four groups, the colon tissues were obtained at day 21 after 12 Gy TAI. Mean ± SEM. Significant differences between each two cohorts are indicated: *P < 0.05 and ***P < 0.005; Student’s t test, n = 5 for TAI group, n = 6 for TAI + IPA group, n = 10 for TAI + sh-ACBP group, n = 9 for TAI + sh-PXR group. g, h, j The expression levels of IL-6 (g), Nrf2 (h), and Glut1 (j) were examined in the small intestine tissues by qRT-PCR. The small intestine tissues were obtained at day 21 after 12 Gy TAI. Mean ± SEM. Significant differences between each two cohorts are indicated: *P < 0.05, **P < 0.01, and ***P < 0.005; Student’s t test, n = 6 for control group, n = 6 for TAI group, n = 6 for TAI + IPA group, n = 10 for TAI + sh-ACBP group, n = 9 for TAI + sh-PXR group. i The morphology of the small intestine was shown by H&E (×100 magnification; Scale bar: 100 μm) and PAS (×1000 magnification; Scale bar: 50 μm) staining. The small intestine tissues were obtained at day 21 after 12 Gy TAI. The arrows point to the goblet cells. k The FITC-dextran in PB was assessed at day 21 after 12 Gy TAI. Mean ± SEM. Significant differences between each two cohorts are indicated: **P < 0.01 and ***P < 0.005; Student’s t test, n = 6 for control group, n = 6 for TAI group, n = 6 for TAI + IPA group, n = 10 for TAI + sh-ACBP group, n = 9 for TAI + sh-PXR group

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