Ferroptosis, a new type of cell death has been found to aggravate intestinal ischemia/reperfusion (I/R) injury. However, little is known about the changes of gut microbiota and metabolites in intestinal I/R and the role of gut microbiota metabolites on ferroptosis-induced intestinal I/R injury. This study aimed to establish a mouse intestinal I/R model and ileum organoid hypoxia/reoxygenation (H/R) model to explore the changes of the gut microbiota and metabolites during intestinal I/R and protective ability of capsiate (CAT) against ferroptosis-dependent intestinal I/R injury. Intestinal I/R induced disturbance of gut microbiota and significant changes in metabolites. We found that CAT is a metabolite of the gut microbiota and that CAT levels in the preoperative stool of patients undergoing cardiopulmonary bypass were negatively correlated with intestinal I/R injury. Furthermore, CAT reduced ferroptosis-dependent intestinal I/R injury in vivo and in vitro. However, the protective effects of CAT against ferroptosis-dependent intestinal I/R injury were abolished by RSL3, an inhibitor of glutathione peroxidase 4 (Gpx4), which is a negative regulator of ferroptosis. We also found that the ability of CAT to promote Gpx4 expression and inhibit ferroptosis-dependent intestinal I/R injury was abrogated by JNJ-17203212, an antagonist of transient receptor potential cation channel subfamily V member 1 (TRPV1). This study suggests that the gut microbiota metabolite CAT enhances Gpx4 expression and inhibits ferroptosis by activating TRPV1 in intestinal I/R injury, providing a potential avenue for the management of intestinal I/R injury.

Background Intestinal ischemia/reperfusion (I/R) injury has high morbidity and mortality rates. Gut microbiota is a potential key factor affecting intestinal I/R injury. Populations exhibit different sensitivities to intestinal I/R injury; however, whether this interpopulation difference is related to variation in gut microbiota is unclear. Here, to elucidate the interaction between the gut microbiome and intestinal I/R injury, we performed 16S DNA sequencing on the preoperative feces of C57BL/6 mice and fecal microbiota transplantation (FMT) experiments in germ-free mice. The transwell co-culture system of small intestinal organoids extracted from control mice and macrophages extracted from control mice or Toll-like receptor 2 (TLR2)-deficient mice or interleukin-10 (IL-10)-deficient mice were established separately to explore the potential mechanism of reducing intestinal I/R injury. Results Intestinal I/R-sensitive (Sen) and intestinal I/R-resistant (Res) mice were first defined according to different survival outcomes of mice suffering from intestinal I/R. Fecal microbiota composition and diversity prior to intestinal ischemia differed between Sen and Res mice. The relative abundance of Lactobacillus murinus (L. murinus) at the species level was drastically higher in Res than that in Sen mice. Clinically, the abundance of L. murinus in preoperative feces of patients undergoing cardiopulmonary bypass surgery was closely related to the degree of intestinal I/R injury after surgery. Treatment with L. murinus significantly prevented intestinal I/R-induced intestinal injury and improved mouse survival, which depended on macrophages involvement. Further, in vitro experiments indicated that promoting the release of IL-10 from macrophages through TLR2 may be a potential mechanism for L. murinus to reduce intestinal I/R injury. Conclusion The gut microbiome is involved in the postoperative outcome of intestinal I/R. Lactobacillus murinus alleviates mice intestinal I/R injury through macrophages, and promoting the release of IL-10 from macrophages through TLR2 may be a potential mechanism for L. murinus to reduce intestinal I/R injury. This study revealed a novel mechanism of intestinal I/R injury and a new therapeutic strategy for clinical practice.

The lack of systematic studies on the stability of ethanol's non-oxidative metabolites in postmortem specimens restricts their use in forensic cases. This study aimed to compare the stability of ethyl glucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanols (PEths) and fatty acid ethyl esters (FAEEs) in postmortem human blood. Three groups were established based on the level and source of ethanol: the blank group, the ethanol-spiked group and the ethanol-positive group. Each group contained six blood samples from different corpses. The samples in each group were placed at 37, 25, 4 and -20°C. Every 24 h for 7 days, 50 μL was collected from each sample. The levels of EtG, EtS, PEths and FAEEs were determined by liquid chromatography-mass spectrometry, and their stability was evaluated. EtG was not detected in the blank group, but it was found in samples in the ethanol-spiked group placed at 37°C, and it was degraded in the ethanol-positive group at 37 and 25°C. EtS showed no change in any of the groups. PEths were not detected in the blank group, but formation was found in the ethanol-spiked group at all temperatures. In the ethanol-positive group, PEth levels fluctuated at 37°C, decreased at 25°C and increased at -20°C. FAEEs were generated in the blank group and in the ethanol-spiked group at all temperatures. In the ethanol-positive group, FAEEs were degraded at 37 and 25°C but were generated at 4 and -20°C. EtS is a reliable biomarker of ethanol consumption, and EtG could be used as a biomarker at low temperatures (4 and -20°C), but PEths and FAEEs are not appropriate biomarkers of ethanol consumption.