Microbiota-derived tryptophan metabolites indole-3-lactic acid is associated with intestinal ischemia/reperfusion injury via positive regulation of YAP and Nrf2

Abstract

Background: Lactobacillus has been demonstrated to serve a protective role in intestinal injury. However, the relationship between Lactobacillus murinus (L. murinus)-derived tryptophan metabolites and intestinal ischemia/reperfusion (I/R) injury yet to be investigated. This study aimed to evaluate the role of L. murinus-derived tryptophan metabolites in intestinal I/R injury and the underlying molecular mechanism.

Methods: Liquid chromatograph mass spectrometry analysis was used to measure the fecal content of tryptophan metabolites in mice undergoing intestinal I/R injury and in patients undergoing cardiopulmonary bypass (CPB) surgery. Immunofluorescence, quantitative RT-PCR, Western blot, and ELISA were performed to explore the inflammation protective mechanism of tryptophan metabolites in WT and Nrf2-deficient mice undergoing intestinal I/R, hypoxia-reoxygenation (H/R) induced intestinal organoids.

Results: By comparing the fecal contents of three L. murinus-derived tryptophan metabolites in mice undergoing intestinal I/R injury and in patients undergoing cardiopulmonary bypass (CPB) surgery. We found that the high abundance of indole-3-lactic acid (ILA) in the preoperative feces was associated with better postoperative intestinal function, as evidenced by the correlation of fecal metabolites with postoperative gastrointestinal function, serum I-FABP and D-Lactate levels. Furthermore, ILA administration improved epithelial cell damage, accelerated the proliferation of intestinal stem cells, and alleviated the oxidative stress of epithelial cells. Mechanistically, ILA improved the expression of Yes Associated Protein (YAP) and Nuclear Factor erythroid 2-Related Factor 2 (Nrf2) after intestinal I/R. The YAP inhibitor verteporfin (VP) reversed the anti-inflammatory effect of ILA, both in vivo and in vitro. Additionally, we found that ILA failed to protect epithelial cells from oxidative stress in Nrf2 knockout mice under I/R injury.

Conclusions: The content of tryptophan metabolite ILA in the preoperative feces of patients is negatively correlated with intestinal function damage under CPB surgery. Administration of ILA alleviates intestinal I/R injury via the regulation of YAP and Nrf2. This study revealed a novel therapeutic metabolite and promising candidate targets for intestinal I/R injury treatment.

Keywords: Indole-3-lactic acid; Intestinal ischemia/reperfusion injury; Nrf2; Oxidative stress; YAP.

Fig. 1

Correlation between ILA, IAA, IAld levels with intestinal I/R injury. A-C The level of ILA (A), IAA (B), IAld (C) in cecal contents from the sham mice and mice undergoing intestinal I/R injury (n = 4). D-F Correlation analysis between preoperatively fecal contents of ILA (D), IAA (E), IAld (F) levels and LIFE score in CPB surgery patients (n = 12). G-I Correlation analysis between preoperatively fecal contents of ILA (G), IAA (H), IAld (I) levels and serum I-FABP levels of patients after surgery (T1) as compared to preoperatively (T0) (n = 12). J-L Correlation analysis between pre-operative fecal content of ILA (J), IAA (K), IAld (L) levels and serum D-lactate levels of patients after surgery (T1) as compared to preoperatively (T0) (n = 12). Results were presented as mean ± SEM. The statistical tests used included: two-tailed student’s t-test in A–C and Spearman’s correlation coefficients in D–L. ** p < 0.01

Fig. 2

ILA improved the survival rate and inhibited apoptosis of enterocytes under I/R and H/R injury. A Survival rate between sham, I/R and I/R + ILA groups (n = 16/group). B-C H&E staining (B) and quantification (C) of the histopathology changes of intestinal tissue sections (n = 3/group). D Relative serum LDH levels in mice under intestinal I/R injury (n = 4/group). E The effect of different ILA concentration on organoids’ cell viability under H/R injury (n = 5/group). F Representative images of morphological changes and PI staining in organoids. G Quantification of PI staining (n = 3/group). H Relative LDH levels in the organoids culture supernatant (n = 3–4/group). I AhR and CYP1A1 mRNA levels in intestinal tissues (n = 5/group). J YAP mRNA levels in organoids (n = 5/group). K Western blot for the protein expression of YAP in intestinal tissues (3 representative cases in each group). L YAP mRNA levels in intestinal tissues (n = 4/group). M YAP mRNA levels in organoids (n = 5/group). Results are presented as mean ± SEM. The statistical tests employed included: two-tailed log-rank test in (A), two-tailed student’s t-test in C–E, G–I, L and M and one-way ANOVA followed by the Tukey test for multiple comparisons in J. * p < 0.05, ** p < 0.01, **** p < 0 .0001

Fig. 3

YAP mediated the protective role of ILA in intestinal I/R injury. A-B Gene expression level (A) (n = 4–7/group) and protein expression level (B) (Four independent experiments) in intestinal tissues. C YAP downstream gene Cyr61, Ctgf expression in the intestinal tissues (n = 4–7/group). D H&E staining of the histopathological changes of intestinal tissues, immunohistochemistry staining of intestinal barrier tight junction protein (ZO-1 and Occludin), TUNEL staining of enterocyte apoptosis. E-H Quantification of pathological scores (E), relative levels of Occludin (F) and ZO-1 (G) proteins and the proportion of TUNEL⁺ cells (H) (n = 4–7/group). Results are presented as mean ± SEM. The statistical tests employed included: one-way ANOVA followed by the Tukey test for multiple comparisons in A, C and E–H. * p < 0.05, ** p < 0.01, *** p < 0 .001, **** p < 0 .0001

Fig. 4

ILA alleviated intestinal inflammation and promoted intestinal regeneration by promoting YAP expression. A Gene expression levels of TNF-α, IL-6 and IL-1β in intestinal tissue (n = 4–7/group). B Cytokines TNF-α and IL-6 levels in serum (n = 4–7/group). C Relative LDH levels in the serum (n = 4–7/group). D-F Immunofluorescence of Ki67 (D), lysozyme (E) and Muc2 (F) in intestinal tissue sections. Results are presented as mean ± SEM. The statistical tests employed included: one-way ANOVA followed by the Tukey test for multiple comparisons in A–C. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 5

ILA protected intestinal organoids against H/R injury in vitro. A YAP mRNA expression in intestinal organoids (n = 6/group). B Representative images of protein levels in intestinal organoids (Four independent experiments). C-D YAP downstream gene Cyr61 (C), Ctgf (D) expression in intestinal organoids (n = 6/group). E ELISA analysis of the cytokine TNF-α and IL-6 in the supernatant medium (n = 4/group). F Relative LDH levels in the supernatant medium (n = 4–5/group). G Immunofluorescence of YAP and Ki67 in intestinal organoids frozen sections, TUNEL staining in intestinal organoids frozen sections. H-J Quantification of YAP protein expression (H), Ki67⁺ cells (I) and TUNEL⁺ cells (J) in organoids (n = 3/group). Results are presented as mean ± SEM. The statistical tests employed included: one-way ANOVA followed by the Tukey test for multiple comparisons in A, C, D–F and H–J. * p < 0.05, ** p < 0.01, *** p < 0 .001, **** p < 0 .0001

Fig. 6

ILA reduced excessive inflammation and oxidative stress in enterocytes both in vivo and in vitro. A-B Relative GSH level (A) and MDA level (B) in the serum (n = 4/group). C Representative images and quantification of the protein level of Nrf2 in intestinal tissue (3 representative cases in each group). D Relative LDH level in the serum (n = 4/group). E Relative gene expression of TNFα, IL-1β and IL-6 in intestinal tissue (n = 4/group). F-H Representative images and quantification of HE staining (F), immunohistochemical staining of Occludin (G) and ZO-1 (H) protein expression in the intestine tissues (n = 3–4/group). I Representative images and quantification of TUNEL⁺ cells in intestinal organoids frozen sections. J-L Relative LDH level (J), GSH level (K) and MDA level (L) in the culture supernatants of organoids (n = 4/group). Results are presented as mean ± SEM. The statistical tests employed included: two-tailed student’s t-test in A–C, two-way ANOVA followed by the Tukey test for multiple comparisons in D–L. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001