Integrated multi-omics reveal important roles of gut contents in intestinal ischemia–reperfusion induced injuries in rats

Abstract

Intestinal ischemia–reperfusion (IIR) is a life-threatening clinical event with damaging signals whose origin and contents are unclear. Here we observe that IIR significantly affect the metabolic profiles of most organs by unbiased organ-wide metabolic analysis of gut contents, blood, and fifteen organs in rats (n = 29). Remarkably, correlations between gut content metabolic profiles and those of other organs are the most significant. Gut contents are also the only ones to show dynamic correlations during IIR. Additionally, according to targeted metabolomics analysis, several neurotransmitters are considerably altered in the gut during IIR, and displayed noteworthy correlations with remote organs. Likewise, metagenomics analysis (n = 35) confirm the effects of IIR on gut microbiota, and identify key species fundamental to the changes in gut metabolites, particularly neurotransmitters. Our multi-omics results establish key roles of gut contents in IIR induced remote injury and provide clues for future exploration.

Introduction

Intestinal ischemia-reperfusion (IIR) is a life-threatening clinical event in many diseases that affect infants, children, and adults^1,2,3,4. It is a result of both surgical and non-surgical approaches such as abdominal aortic aneurysm surgery⁵, cardiopulmonary bypass⁵, small bowel transplantation⁶, acute mesenteric ischemia⁷, neonatal necrotizing enterocolitis⁸, trauma⁶, and septic shock⁹. The primary clinical characteristic of these diseases is the interruption of blood supply, which causes ischemic impairment¹⁰. Paradoxically, restoring of blood flow does not alleviate the symptoms of the injury; on the contrary, it magnifies the damage drastically. Most astonishingly, IIR substantially impacts the integrity and function of remote organs (the non-ischemic organs), and if severe enough, it can clinically result in systemic inflammatory response syndrome and multiple organ dysfunction syndrome⁴, which account for 30–40% of in-hospital mortality¹¹.

In recent years, researchers have advanced several hypotheses on the origin and/or nature of the damaging signals to remote organs during IIR, including intestinal barrier dysfunction^12,13, inflammatory cytokines release, oxygen-free radical formation¹⁴, complement activation, microvasculature damage, and neutrophil infiltration^3,15,16. Several research groups have also investigated the roles of gut microbiota in IIR^17,18,19. Per ref. ¹⁷ found that depleting gut commensal bacteria attenuates IIR-induced injury, while ref. ¹⁸ established the essential role of commensal bacteria in epithelial restitution after IIR. Collectively, many factors, including Toll-like receptors, oxidative stress and nitric oxide, have been reported to be associated with intestinal bacteria during IIR¹⁹. However, a few questions remain to be answered: (1) what are the primary source and contents of the damaging signal(s) to remote organs during and after IIR? (2) what route do these signals take to remote organs? (3) what roles do the signals play after they reach remote organs?

To answer these questions, we performed a systemic, time-resolved multi-omic analysis of gut contents, blood, bronchoalveolar lavage fluid (BALF) and fifteen organs during IIR using a rat model. Our results support a gut translocation model in which gut contents leaked into peripheral blood because of a compromised gut barrier during IIR and then to remote organs, causing inflammatory responses. Additionally, we found that several neurotransmitter-related gut metabolites changed significantly during IIR, which could influence remote organs through the enteric nervous system. We also profiled the gut microbiota during IIR, and identified a few key species that underly the changes in metabolites and neurotransmitters. In summary, we provide evidence that gut contents, as the major source of IIR, caused damage to remote organs and established their putative routes. Our results warrant further research on the impact of gut-derived metabolites, neurotransmitters, and microbes.

Results

Gut contents leaked into remote organs during IIR because of a compromised intestinal barrier

To explore the key roles gut contents play in IIR-induced injury, we carried out a series of multi-layer and multi-angle experiments. Our overall experimental design is shown in Fig. 1a. We first explored unbiased organ-wide metabolic profiling for gut contents, blood, BALF and 15 organs in rats (n = 29) to understand the impact of IIR on gut contents and remote organs (Fig. 1b, c). Then we used metagenomics analysis (n = 35) to explore the taxonomic and functional characterization alteration of the gut microbiome during IIR.

Fig. 1: Combination of metagenomic analysis and global metabolite profiling of rat blood/tissues/cecal content reveals common and tissue-specific metabolic signatures during intestinal ischemia and reperfusion (IIR) period.

To confirm previous findings that IIR increases gut permeability by compromising the intestinal barrier^12,13, we fed Fluorescein isothiocyanate (FITC)-dextran to rats through gavage and quantified its levels in serum and a few tissues during IIR. FITC-dextran is widely used as a marker of mucosal barrier dysfunction and paracellular transport²⁰. We also quantified the concentrations of D-lactic acid, fatty acid-binding proteins (IFABPs), citrulline and endotoxins in blood during IIR. D-lactic acid is a fermentation product of various gut bacteria. It has been shown to enter the bloodstream when intestinal permeability increased²¹. IFABPs are small cytosolic water-soluble proteins present in mature enterocytes and reportedly reflect the physiological turnover rate of enterocytes, the increase of which is linked to intestinal epithelial cell damage²². Circulating citrulline, an amino acid not incorporated in proteins is mainly produced by enterocytes and is frequently suggested as a biomarker of remnant small bowel mass and function²³. Endotoxins are constituents of the outer membrane of gram-negative bacteria²⁴; systemic endotoxemia is also associated with increased intestinal permeability. These selected substances are representatives of the broad spectrum of gut contents and thus ideal markers for intestinal damage and gut translocation.

As expected, we detected significant changes in the concentrations (p values < 0.05, Wilcoxon test) of most of these substances in the blood and a few of them in several tissues (see Fig. 1a for the overall experimental design and Fig. 1d and Supplementary Fig. 1 for the results), suggesting that IIR caused damage to the intestinal barrier, resulting in gut contents leaking into the blood and remote organs. Reduced plasma citrulline levels have only been recognized recently as a biomarker of significantly reduced enterocyte mass and function in different disease states in humans²³. Here, we detected reduced levels of citrulline in IIR6h compared to the Sham Group, though with no significance. We further confirmed damage to the intestinal barrier (Supplementary Fig. 2) and remote organs (Supplementary Figs. 3–5) using haematoxylin and eosin staining. Levels of the inflammation marker IL-6 increased in the blood (Fig. 1d). We also revealed that several oxidative stress indices, including malondialdehyde, superoxide dismutase and myeloperoxidase, increased in remote organs (Supplementary Fig. 1). Together, our results suggest that gut contents can leak into the blood and remote organs due to compromised intestinal barrier and contribute to IIR-induced damages.

IIR altered the metabolic profiles of gut contents and remote organs

To provide an unbiased view of body-wide metabolic changes during IIR, we performed global metabolomics profiling using both liquid chromatography-mass spectrometry (LC/TOF-MS) and gas chromatography-mass spectrometry (GC/MS) on blood, gut (cecal) contents, BALF and 15 organs including liver, kidney, adrenal glands, thymus, heart, jejunum, ileum, colon, rectum, brain, epididymal fat (EpiFat), lung, lymph, spleen and subcutaneous fat (SubFat) (Fig. 1a). To check whether the overall metabolic profiles (i.e., features and their relative abundances) of these organs significantly changed during IIR, we applied a multiblock method^25,26,27 to integrate metabolomic profiles from both LC/MS and GC/MS. And a PCA2Tree tool²⁸ was used to visualize the relationships between the metabolic profiles at four different time points for all organs. PCA2Tree generates a dendrogram from scores in principal component analysis (PCA) using Mahalanobis distances and reports p values for the null hypothesis at all internal branches; p values less than 0.05 indicated a statistically significant difference.

We found that IIR indeed induced metabolic changes in remote organs and identified three distinct patterns (Fig. 2). First, cecal contents and seven organs, including the liver, kidney, rectum, adrenal, thymus, colon, and brain, displayed a stress-recovery pattern. As shown in Supplementary Fig. 6, the metabolic profiles of these samples changed significantly at IR0h (2 h after ischemia) and/or IR6h (6 h after reperfusion) compared to Sham (control group; P < 0.01) and were restored to normal at IR72h (72 h after reperfusion). Colon and brain also belonged to this pattern with a different mode (Supplementary Fig. 7). Second, blood and two organs, including the heart and epididymal, showed a stress-non-recovery pattern (Supplementary Fig. 7): their metabolic profiles differed significantly between Sham and IIR72h, indicating a long-lasting effect on these organs.

**Fig. 2: IIR affected metabolomic profiles of intestinal and remote organs that showed three distinct patterns during IIR (selected representatives: liver, heart, lymph).**

Third, BALF and six organs, including jejunum, ileum, lung, lymph, spleen, and subcutaneous fat showed no noteworthy changes in metabolic profiles during IIR (Supplementary Fig. 8). These results suggest that our methods could capture metabolic dynamics during IIR. In the subsequent analysis, we focused on the nine organs (liver, kidney, rectum, adrenal glands, thymus, colon, brain, heart, and epididymal), blood and cecal contents that experienced drastic changes in their metabolic profiles (those in the stress-recovery pattern and stress-non-recovery pattern) during IIR and excluded the other six organs (jejunum, ileum, lung, lymph, spleen, and subcutaneous fat) and BALF that showed no notable changes in metabolic profiles during IIR.

We focused on the features (metabolic fragments, Supplementary Data 1) that displayed significant differences in the multiblock analysis (selected using the selectVar function in the partial least squares discriminant analysis (sPLS-DA)²⁶) and identified their corresponding metabolites (see Methods). We identified 132 metabolites in total from the abovementioned nine organs, blood, and cecal contents, including 32 amino acids, peptides, and analogs; 5 benzenoids; 7 bile acids, alcohols, and derivatives; 5 Eicosanoids; 18 lipids and lipid-like molecules; 25 lysophosphatidylcholine or lysophosphatidylethanolamine or phosphatidylcholine or phosphatidylethanolamine; 5 nucleosides, nucleotides, and analogs; 25 organic compounds and others and 10 unsaturated fatty acid (Supplementary Fig. 9 and Supplementary Data 1; see Methods for details). The liver presented the most significantly changed metabolites, followed by adrenal glands, with the epididymal fat displaying the least altered metabolites (Supplementary Fig. 10a). The class distributions of the identified differential metabolites were similar across tissues except brain, gut content and epididymal fat (Supplementary Fig. 10b, c). The metabolic contents of an organ, i.e. the metabolite classes and their relative abundances, often showed distinct characteristics from others and changed during IIR (see Fig. 1c and Supplementary Data 2 for the data). Specifically, amino acids and their derivatives increased during ischemia/reperfusion injury in colon, gut content, rectum, liver, thymus, and kidney, but not in the adrenal glands. Owing to the limitations of metabolomics technology and the peculiarity of epididymal fat samples, we identified only four metabolites as significant ones. Therefore, amino acids may not be the dominant metabolites in this tissue. Similarly, unsaturated fatty acids decreased during damage (IIR0h and IIR6h) in gut content and many tissues, like colon, rectum and thymus, whereas increased in adrenal glands, liver and heart. Correlations between metabolites can reflect the synchronization of individual cells in tissues. Metabolites with positive correlations possibly belong to associated pathways, having common functions or origins, whereas negative correlations between metabolites probably reflect incompatible pathways²⁹. Astonishingly, the inter-tissue correlation analysis showed extensive negative correlations between unsaturated fatty acids and amino acids (especially tyrosine, phenylalanine, tryptophan, leucine and lysine) in gut content, blood, and most organs (adrenal glands, kidney, thymus, colon, and rectum) (Supplementary Fig. 11).

Pathway enrichment analysis showed that pathways involved in the biosynthesis of amino acids, 2-oxocarboxylic acid metabolism, biosynthesis of unsaturated fatty acids, bile secretion, phenylalanine, tyrosine and tryptophan biosynthesis ranked top (Fisher's exact test) in most organs (Supplementary Figs. 12, 13). Of these, phenylalanine, tyrosine, and tryptophan are aromatic amino acids that can serve as precursors for neurotransmitters, such as serotonin, dopamine, and norepinephrine^30,31. Our results suggest that IIR can trigger changes in neurotransmitter metabolism of remote organs. Other inflammation-related pathways, like the mechanistic target of the rapamycin signaling pathway, inflammation mediator regulation of transient receptor potential channels, and eicosanoids changed markedly in several remote organs (Supplementary Figs. 12,13), indicating that inflammatory processes were modulated during IIR. In summary, multiple pathways in intestinal tissues, gut contents, and remote organs were altered during IIR.

Gut contents play critical roles in IIR-induced damages to remote organs

We next compared metabolic profiles between organs and sought to identify the primary source of IIR-caused damaging signals. We speculated that the metabolic profiles of the source organ would correlate most with other organs, and these correlations would show dynamic changes during IIR.

We first calculated pairwise correlations of the metabolic profiles across organs (Fig. 3a) using the R package "mixOmics"²⁶. Accordingly, gut contents displayed the most significant overall correlation co-efficiencies with other organs (Fig. 3b), followed by kidney, adrenal glands, liver, and thymus. We then calculated the correlations as a function of time during the IIR experiments and found that gut contents were the only one that showed dynamic changes in its correlation co-efficiencies with other tissues (Fig. 3c, d). These correlations increased considerably at IR0h compared to Sham and rose further at IR6h, consistent with our observation that gut contents leaked into remote organs during IIR and decreased to comparable levels to that of the Sham group at the end of the recovery stage (IR72h, Fig. 3c). Similar patterns were not observed in other tissues (Fig. 3d). Furthermore, we identified more correlations between gut contents and remote organs than between intestinal tissues (colon and rectum) and remote organs (Supplementary Fig. 14). Together, our results indicate that gut contents are crucial to IIR induced damages to remote organs.

**Fig. 3: Cross-tissue metabolite correlations revealed the vital role of gut content in the impact of remote organs caused by IIR.**

We then examined the metabolites underlying the correlations between gut contents and remote organs. As shown in Supplementary Fig. 15, nine gut content metabolites, including leucine, cyclic phosphatidic acid (CPA (18:1(9Z)/0:0)), phenylalanine, acetylcholine, lysine, linoleic acid, alpha-linolenic acid, leucyThreonine, and hypotaurine correlated substantially with remote organs. Surprisingly, most of them are either neurotransmitters or associated with their biosynthesis or regulations. For example, acetylcholine is a neurotransmitter³². Phenylalanine is an aromatic amino acid and a precursor for neurotransmitters, such as serotonin, dopamine, and others³⁰. Lysine is a key precursor for the de novo synthesis of glutamate, which is the most important excitatory neurotransmitter in the mammalian central nervous system³³. Linoleic acid is a long-chain polyunsaturated fatty acid, which has been verified as capable of inducing neurotransmitter release³⁴. These results suggest that gut-derived neurotransmitters also contribute to IIR-induced damages to remote organs.

Alteration in gut content metabolome during IIR points to an impairment in intestinal neuroendocrine function

Having established that gut contents contribute notably to IIR-induced damages, we further explored the impact of their alteration during IIR and their putative roles in IIR-induced injury to remote organs.

We first identified metabolites that changed notably during IIR (Fig. 4a, b and Supplementary Data 3–6), and classified them into two groups according to whether their abundances differed considerably between IR6h and Sham (referred to as the Unrecovered group) or not (referred to as the Recovered group). We further divided the Recovered metabolites into two subgroups based on whether they were up- or downregulated at IR0h, and referred to them as Recovered_UpDown and Recovered_DownUp, respectively. The first subgroup (Recovered_UpDown) included l-arginine, 4-guanidinobutyric acid, d-fructose, cellobiose, d-maltose, d-lactose, alpha-d-glucose, stachyose, maltotriose, 3-phenylpropanoic acid, and kynurenic acid (Supplementary Fig. 16), while the second (Recovered_DownUp) consisted of flavin adenine dinucleotide, uridine monophosphate, guanosine monophosphate, l-aspartate, and arachidonic acid (Supplementary Fig. 17). Most of these molecules participate in carbohydrate metabolism, the probable sources of carbon and energy for intestinal bacteria. L-arginine is also a possible carbon and nitrogen source. Their significant changes at IR0h and subsequent recovery at IR6h are likely caused by the fluctuations in the metabolic capabilities of gut microbiota in response to oxygen supplies during IIR. For example, large quantities of carbon, nitrogen, and energy accumulated during ischemia, probably due to metabolism arrest, and then were used by intestinal bacteria in the following reperfusion period.

**Fig. 4: Significantly altered metabolites and markers for neuroendocrine system in the gut during IIR.**

We also divided the UnRecovered metabolites into two subgroups based on whether they were up- or down-regulated at IR6h and labeled these groups as Unrecovered_Up and Unrecovered_Down, respectively. The UnRecovered_Down subgroup included nicotinamide adenine dinucleotide, uridine diphosphate glucose (UDP-glucose), UDP, UDP-N-acetylglucosamine, cytidine diphosphate, citrate, and isocitrate (Supplementary Fig. 18). UDP-glucose³⁵, as an activated form of glucose, is a nucleotide sugar involved in glycosyltransferase reactions in metabolism. It is a precursor of glycogen, sucrose, lipopolysaccharides and glycosphingolipids. The abnormal variations in the quantities of these molecules could reflect disturbances in carbohydrate metabolism.

The Unrecovered_Up subgroup comprised uric acid, allantoin, L-pipecolic acid, 5-aminopentanoic acid, 3-methylhistidine, histamine, acetylcholine, sphingosine, N^G,N^G-dimethyl-L-arginine (ADMA), and N-(omega)-Hydroxyarginine (Fig. 4c). Outstandingly, several of them are associated with neurotransmitters. For example, acetylcholine and histamine are neurotransmitters. ADMA is an endogenous nitric oxide synthase inhibitor,³⁶ and N-(omega)-Hydroxyarginine is an intermediate in the biosynthesis of nitric oxide from l-arginine³⁷. They are both linked to nitric oxide, which is a signaling molecule in the nervous system³⁸. 5-Aminopentanoic acid is a methylene homolog of gamma-aminobutyric acid, an inhibitory neurotransmitter in the human cerebral cortex³⁹, and sphingosine is a sphingolipid metabolite that can exert vital neuron-specific functions, such as the regulation of neurotransmitter release⁴⁰.

To further explore the influence of IIR on neurotransmitter levels and intestinal neuroendocrine function, we first performed a targeted metabolomics analysis using LC–MS/MS. Several neurotransmitters and related metabolites, including γ-aminobutyric acid, serotonin, epinephrine, and glutamine changed (Supplementary Fig. 19). Next, we utilized an immunohistochemical approach to assess the influence of IIR on the intestinal neuroendocrine system. We applied several antibodies, including nitric oxide synthase, C-Kit, synaptophysin, protein gene product 9.5, and substance P. They are commonly used molecular markers of the neuroendocrine system⁴¹ and enteric nervous system^42,43,44,45. As expected, these markers were drastically altered during IIR (Fig. 4d and Supplementary Fig. 20), indicating an impairment in intestinal neuroendocrine function.

Together, our results suggest that IIR induces complex changes in the gut metabolome. Many of the Recovered metabolites participated in carbohydrate metabolism and energy production, consistent with the stress-recover design of our experiments. However, the functions of the Uncovered metabolites, most of them being neurotransmitters, were more diverse. The latter metabolite types can interact with and subsequently induce changes to the enteric nervous system and then influence remote organs. The long-lasting changes in their abundance after reperfusion indicate that they possibly contribute to the long-term impact of IIR on remote organs. Compared to their Recovered counterparts, UnRecovered metabolites perhaps also play critical roles in physiological or pathological processes of IIR-induced impairment of remote organs and warrant further investigation.

Taxonomic and functional alterations in the gut microbiome during IIR

To further explore the alteration of intestinal flora and their associations with gut metabolome, we submitted cecal samples to metagenomic next-generation sequencing, followed by taxonomic analysis using MetaPhlAn2⁴⁶ and functional characterization using HUMAnN2⁴⁷ (see Methods for details).

We identified a total of 111 species in all samples (see Fig. 1a for the overall experimental design). We first focused on species with known functions/characteristics, whose information we obtained from large numbers of literature retrieval (listed in Supplementary Data 7). We found that obligate anaerobes increased at IR0h and decreased at IR6h (Supplementary Fig. 21a), consistent with our experimental design showing that oxygen supply decreased during ischemia but leveled up during reperfusion. There showed no significant difference in microbial alpha diversity (Shannon index and Simpson index) between groups (Supplementary Fig. 21b). And beta diversity showed a tendency of separation among microbial communities characterized by PCoA on weighted UniFrac distance (Supplementary Fig. 21b). Remarkably, the overall abundances in pro-inflammation species increased significantly at IR6h (Fig. 5a and Supplementary Data 7), some of which are known to modulate gut inflammation, suggesting that changes in gut microbiota during IIR can, in turn, influence the physiology and function of the gut. We also revealed that neurotransmitter-producing bacteria were considerably affected during IIR; more importantly, their overall abundances were notably higher at IR6h than IR0h, with an increasing trend, though with an insignificant difference, also apparent at IR6h compared to Sham, although the difference was not significant (Fig. 5a). Worth noting is that we only assigned functional characteristics to part of the total 111 species identified in this study. Our results could, therefore, be biased because of such a limitation. Additionally, we found significant differences in HDCs (host DNA contents, an indicator of a compromised intestinal barrier, Fig. 5a) between groups, indicating that intestinal barriers were compromised during IIR⁴⁸.

**Fig. 5: Taxonomic and functional analysis of gut microbiome during IIR.**

We used a linear discriminant analysis (LDA) effect size (LEfSe) analysis to identify differential taxa between time points. Five differential taxa were identified at IR0h compared to Sham (Fig. 5b and Supplementary Fig. 21c; LDA score >2.0, p < 0.05 with Kruskal–Wallis test, Supplementary Data 8). And 37 differential taxa were identified at IR6h compared to IR0h (Supplementary Data 9). Of these, several beneficial species, including Lactobacillus johnsonii and Lactobacillus sp ASF360, reduced significantly, with others, including Escherichia coli and Bacteroides vulgatus, and species in the Enterobacteriaceae and Enterococcaceae families increasing markedly (Fig. 5c and Supplementary Figs. 21c, 22).

As expected, the functional analysis of the sequencing data identified significantly more downregulated genes and pathways than upregulated ones at IR0h compared to Sham (Fig. 5e, g and Supplementary Data 10–15; see Methods for details), suggesting that cutting off blood supply to the gut suppressed the overall activities of gut microbes. Conversely, we identified significantly more upregulated genes and pathways than downregulated ones after blood supply restoration (IR6h compared to IR0h, Fig. 5e, g). Furthermore, PCA analysis established differences in both microbial beta diversities (Supplementary Fig. 21b) and their functional potentials (i.e., genes and microbial pathways, Fig. 5d, f) between IR6h and Sham. Therefore, gut microbes changed significantly during IIR, with some changes not restored after reperfusion. Additionally, functional characterization analysis of gut microbiome revealed that bacterial metabolisms associated with the biosynthesis of neurotransmitters-related metabolites, such as pathway phosphoribosyl pyrophosphate (PRPP)-PWY; histidine and tryptophan biosynthesis pathways; CITRULBIO-PWY: l-citrulline biosynthesis, were significantly augmented (Supplementary Data 13–15). PRPP is a precursor for histidine and tryptophan biosynthesis pathways, which are associated with the neurotransmitter histamine. CITRULBIO-PWY is linked to N-(omega)-Hydroxyarginine, which is related to nitric oxide, a signaling molecule in the nervous system. Remarkably, metabolomics analysis identified these neurotransmitter-related molecules to be significantly increased. Hence, we inferred that alterations in gut content metabolites could be the result of abnormal gut microbiota metabolism.

We also uncovered the principal microbial driver species for all the functional pathways using a leave-one-out algorithm⁴⁹ (Supplementary Data 16, 17). Surprisingly, E. coli, which increased sharply at IIR6h, was identified as the strongest driver specie for most of the functional pathways (Fig. 5h and Supplementary Fig. 21d). These results suggested a causal relation between E. coli and those significantly altered metabolites at IIR6h, which is further supported by several literatures, linking Enterobacteriaceae, and more specifically E. coli, to IIR associated diseases such as acute surgical abdomen⁵⁰, small bowel transplantation⁵¹, necrotizing enterocolitis^52,53,54, and sepsis⁵⁵. Because IIR-induced remote organ injury is a complex physiological process associated with many factors, systematic and comprehensive explorations are still needed to further validate whether E.coli is truly the driver of downstream IIR-instigated complications.

Inter-omics analysis links gut microbiota to metabolites during IIR

To link the altered gut metabolites during IIR to gut microbes, we performed an inter-omics Spearman correlation analysis (see Methods for details). The overall correlation pattern between microbial metabolites and gut pathways during reperfusion was distinctive from that during ischemia (Fig. 6b and Supplementary Fig. 23). We identified more connections between pathways and metabolites (Supplementary Fig. 23), particularly markedly elevated neurologic function-related molecules, like ADMA, 5-aminopentanoic acid, histamine, and acetylcholine during reperfusion. Likewise, we observed more links between gut metabolites and E. coli, L. johnsonii and several other species in genus Enterococcus during reperfusion (Fig. 6a and Supplementary Fig. 24). Furthermore, E. coli and species in the genus Lactobacillus correlated strongly with gut-derived metabolites in Model-based Integration of Metabolite Observations and Species Abundances 2 (MIMOSA2)⁵⁶ analysis (Supplementary Fig. 25). These results are consistent with the microbial driver analysis that a few key species are probably fundamental to the changes in gut metabolites.

**Fig. 6: Integrated microbiome-metabolome analysis identified key metabolic pathways and underlying key species.**

To further explore the influence of intestinal microflora on metabolites at the biotransformation level, we created a global metabolic map connecting significantly altered metabolites at IIR6h (primarily including metabolites in carbohydrate metabolism and several neurotransmitters-related ones, Fig. 6c, Supplementary Fig. 26, and Supplementary Data 13–15) based on known pathways they involved in. As Fig. 6c demonstrates, pathway phosphoribosyl pyrophosphate (PRPP)-PWY (superpathway of histidine, purine, and pyrimidine biosynthesis) played a vital role in linking carbohydrate metabolism with multiple pathways like pyrimidine, purine, and pyridine nucleotide synthesis pathway, histidine and tryptophan biosynthesis pathways. These PRPP-related pathways increased significantly at IIR6h (Supplementary Data 15), and metabolites synthesized from them, including histamine, 3-methylhistidine, uric acid, and allantoin, also rose significantly at IIR6h compared to Sham (Fig. 4c). Moreover, ADMA, N-(omega)-hydroxyarginine and sphingosine underwent significant amplification at IIR6h compared to Sham (Fig. 4c). ADMA is synthesized when arginine residues in proteins are methylated. Methylation-associated pathways increased at IIR6h in this research. N-(omega)-Hydroxyarginine is an intermediate in the biosynthesis of nitric oxide from l-arginine. The corresponding urea cycle-related pathways like CITRULBIO-PWY: l-citrulline biosynthesis were also increased at IIR6h. Sphingosine belongs to lipids, and pathways related to lipids synthesis, like PWY-5138: unsaturated, even-numbered fatty acid and beta-oxidation, widely increased at IIR6h.

In summary, we identified some key species fundamental to the changes in gut metabolites and their putative roles in regulating the metabolism of neurotransmitters. Gut metabolite alterations during reperfusion could be the result of gut bacteria metabolism activation after blood supply restoration.

Peripheral Arterial Insufficiency