introduction: Severe acute pancreatitis (SAP) is a life-threatening condition characterized by persistent organ failure and local complications stemming from systemic inflammation and multi-organ dysfunction [1,2]. Current therapeutic approaches predominantly focus on supportive care and managing complications, yet there is a notable paucity of targeted interventions addressing the underlying mechanisms that link gut dysbiosis with systemic inflammation [3]. Intestinal barrier disruption in SAP is a critical factor, exacerbating bacterial translocation and systemic inflammation. This, in turn, fuels complications such as acute lung injury via pathways like the pancreatic-intestinal-endotoxin-lung axis [4,5]. The gut microbiota, both a victim and an amplifier of pancreatic injury, significantly influences SAP progression by modulating immune responses and metabolite profiles [6]. Emerging evidence underscores intestinal dysbiosis as a pivotal factor in SAP progression. Patients with SAP typically exhibit a reduction in beneficial probiotics and an increase in pathogenic bacteria, alongside decreased levels of short-chain fatty acids (SCFAs)—crucial metabolites that support colonic epithelial cells, enhance barrier function, and suppress pro-inflammatory cytokine production [7-9]. Such microbial and metabolic imbalances amplify inflammation and oxidative stress through pathways like NF-κB and JAK2/STAT3, perpetuating a vicious cycle of organ damage [3]. Recent multi-omics studies have unveiled that gut-derived metabolites, such as tryptophan derivatives and polyamines, interact directly with host immune cells, highlighting a microbiota-metabolite crosstalk as a promising therapeutic target [10,11]. The endocannabinoid system presents a novel therapeutic avenue in SAP management. Monoacylglycerol lipase (MAGL), an enzyme that hydrolyzes the anti-inflammatory lipid 2-arachidonoylglycerol (2-AG), emerges as a promising target. Inhibition of MAGL by JZL184 elevates 2-AG levels, leading to reduced pro-inflammatory cytokines (IL-6, TNF-α) and improved intestinal permeability in SAP models [12,13]. Notably, 2-AG not only mitigates systemic inflammation but also modulates gut motility and microbial composition, suggesting its role in harmonizing host metabolism and microbiota homeostasis [14,15]. However, the specific impact of MAGL inhibitors on the gut microbiota-metabolite network and pancreatic gene expression remains largely unexplored, representing a significant gap in our understanding of their therapeutic potential. This study endeavors to elucidate how JZL184 alleviates SAP by restoring gut microbiota balance, regulating metabolites, and impacting pancreatic gene co-expression networks. By integrating metagenomic, metabolomic, and transcriptomic analyses, we aim to unravel the interconnected "microbiota-metabolite-gene" axis. Our findings seek to provide insights into novel therapeutic strategies that concurrently target inflammation, microbial dysbiosis, and metabolic dysfunction in SAP. materials and methods: Animals All animal experiments were conducted in strict accordance with the guidelines and regulations set by the Shandong Provincial Hospital Committee on Use and Care of Animals, with ethical approval granted under the reference number NSFC: No.2024–369. Male Sprague-Dawley (SD) rats, each weighing between 200 and 230 grams, were procured from the Experimental Animal Center at Shandong University, China. These rats were housed in an environment regulated at 22°C, following a 12-hour light/dark cycle, to ensure consistent living conditions. Researchers conducting the experiments were blinded to the treatment groups to maintain objective and unbiased assessment of the outcomes. Establishment of SAP Rat Model To induce severe acute pancreatitis (SAP), we employed a reliable surgical model, following a methodology refined in previous research [16]. Rats aged 6-8 weeks were anesthetized, and a median abdominal incision allowed access to the duodenal opening. A precise retrograde puncture of the pancreaticobiliary duct was performed using a No. 5 needle near this site. Secured with non-traumatic clamps, a micropump administered a 3% sodium taurocholate solution into the pancreatic duct at 0.1 ml/min (corresponding to 0.1 ml/100 g body weight). The needle was kept in place for five minutes post-injection. Upon confirming the pancreas color change to dark red, indicative of successful induction, surgical steps were reversed, and the abdomen was closed meticulously to preserve physiological integrity. Experimental Design and Sample Collection Twenty-four hours post-surgery, SD rats were randomly distributed into three groups: the Control group (CON), the SAP group (SAP), and the SAP group receiving JZL184 treatment (JZL184). The JZL184 group received intraperitoneal administration of the MAGL inhibitor JZL184 (Cayman Europe) at 10 mg/kg, prepared in a saline/ethanol/Tween-80 mix. Control groups were given equivalent volumes of vehicle solution. After 24 hours of intervention, fecal samples were collected for subsequent analysis. 16S rDNA Microbiota Profiling DNA was extracted, quantified using the Qubit quantification kit, and integrity was confirmed via 1% agarose gel electrophoresis. The 16S rRNA gene's V3-V4 region was amplified using primers 341F and 805R to create sequencing libraries. These were sequenced using the Illumina MiSeq platform with paired-end sequencing (2×250 bp). Public Data Retrieval and Processing 16S amplicon and metabolomic profiles from Xi Chen et al. were accessed from Meneley Data (DOI: 10.17632/v8tx42gchw.1 and 10.17632/4kb39tg8gj.1). Bioinformatics Analysis Bioinformatics processing involved primer removal and quality filtering using USEARCH, with a maximum error threshold of 0.01. The Unoise3 algorithm denoised data, generating amplicon sequence variants (ASVs), excluding those under 10 reads. The Silva v123 database was utilized for species annotation via the SINTAX algorithm, with a confidence cutoff of 60%. Alpha diversity metrics were calculated using the R vegan package, while beta diversity analysis was conducted with the R phyloseq package, employing PCoA, heatmaps, and UPGMA clustering. Taxonomic differences were evaluated using the Wilcoxon rank-sum test and LEfSe, with significance thresholds at P < 0.05 and FDR < 0.2. PICRUSt2 was used for microbial functional predictions. Metabolite Information and Interaction Evidence Metabolite details, including CID and SMILES notation, were gathered using the PubChem API. ChEMBL IDs were retrieved via the UniChem API using CIDs. Interaction evidence with chemical targets was compiled from the PubChem API, and additional target data were sourced from the ChEMBL database for human-derived targets (Taxonomy ID: 9606), supplemented by predictions from the ChEMBL SEA based on SMILES. Targets identified from these sources were amalgamated into a comprehensive target list. Functional Enrichment Analysis Gene Ontology (GO) terms and KEGG pathways were identified using the clusterProfiler package (v4.6.2) to categorize gene functions. Validation of ACE Expression via qRT-PCR Pancreatic tissues from each experimental group were homogenized for RNA extraction using TRIzol. Reverse transcription was performed to synthesize cDNA, and qRT-PCR was carried out using primers specific for ACE and β-actin (reference gene). The 2−ΔΔCt method was applied to normalize expression levels across groups, and differences were statistically analyzed using one-way ANOVA (p < 0.05). results: 1. Changes in Intestinal Microbiota Induced by JZL184 in Rats with Severe Acute Pancreatitis (SAP) The overall technical framework of the study is illustrated in Figure 1A. Using Amplicon Sequence Variant (ASV) classification from 16S rRNA sequencing, we explored the shared and unique microbial taxa across three groups: CON rats, SAP rats, and JZL184-treated rats. Notably, 1,520 bacterial species were common to all groups, while 171 were unique to the CON group, 32 unique to the SAP group, and 29 specific to the JZL184 group (Figure 1B). Despite no significant differences in α-diversity indices (species-level Chao1, Shannon, ACE) among the groups (Figures 1C–E), β-diversity analysis suggested subtle distinctions between the control group and the SAP or JZL184 group (Figure 1F). These results indicate stable microbial richness and evenness across the groups. However, specific microbial compositions exhibited significant changes; at the phylum level, the reduced abundance of Tenericutes and Bacteroidetes in the SAP group was restored post-JZL184 treatment (Figure 1G). At the genus level, SAP rats showed a marked increase in Escherichia–Shigella, which was notably diminished following JZL184 treatment, alongside an increase in beneficial genera including Lactobacillus and Bacteroides (Figure 1H). 2. JZL184 Modulates Microbial Composition in SAP, Enhancing Lactobacillus and Reducing Escherichia–Shigella Through LEfSe analysis, we identified group-specific microbial taxa, coded by color and taxonomic rank prefix—phylum (p), class (c), order (o), family (f), and genus (g). The SAP group exhibited increased abundance of Rhodocyclaceae (f), Lentisphaerae (c), Globicatella (g), and Victivallales (o), while the CON group primarily featured Bacteroidetes (p). Post JZL184 treatment, the abundance of Plesiomonas and Marvinbryantia (genera) increased (Figure 2A). KEGG pathway analysis identified biological roles for these altered taxa, revealing upregulated pathways in SAP such as NOD-like receptor signaling, MAPK signaling, and PPAR pathways. Treatments with JZL184 modulated these pathways towards levels observed in healthy controls (Figure 2B). JZL184 restored normalized taxa abundances post-treatment; specifically, increased Bacteroidetes and Firmicutes, and reduced Proteobacteria and Verrucomicrobia at the phylum level, and significant rebalancing of eight key genera including decreased Escherichia–Shigella and increased Lactobacillus (Figure 2C-D), underscoring JZL184's therapeutic potential in SAP. 3. Therapeutic Efficacy of JZL184 in SAP through Microbial Metabolite Modulation JZL184's ability to increase beneficial probiotics and decrease pathogenic bacteria in SAP rats prompted further validation in an independent dataset using 16S rRNA and fecal metabolomics from SAP mice. Principal coordinates analysis distinctly separated control from SAP groups, with overlap seen between SAP and GV-971 treated mice (Figure 3A). Comparative analysis showed trends consistent with JZL184-modulated taxa, such as similar abundance changes in Escherichia–Shigella and Lactobacillus (Figure 3B-C), aligning with prior findings in SAP models and patients (Figure 3D). Metabolomics analysis identified 80 differentially abundant metabolites between SAP and control mice, including elevated polyamines and reduced short-chain fatty acids, and correlations indicated Escherichia–Shigella's pivotal role in SAP pathogenesis (Figure 3E-F). 4. Integrated Microbiota, Metabolite, and Gene Analysis Reveals JZL184's Mechanistic Pathways in SAP To understand JZL184's molecular impact, we assessed transcriptomic data (GSE161945) from SAP mouse pancreatic tissues. PCA differentiated SAP from control mice (Figure 4A), with differential expression identifying SAP-specific upregulated and downregulated genes (Figure 4B). Mapping to human orthologs and leveraging databases, we identified target genes associated with JZL184 metabolites, with functional enrichment in processes like cell adhesion and apoptosis (Figure 4C-D). A "microbiota–metabolite–gene" network linked arachidonic acid with Escherichia-Shigella and Lactobacillus, aligning with JZL184's targets, including HMOX1 (Figure 4E). Additionally, subsequent validation in pancreatic tissues showed significant upregulation of ACE in SAP rats, which was normalized following JZL184 treatment (Figure 4F). discussion: Intestinal microbial dysbiosis plays a critical role in the progression of severe acute pancreatitis (SAP). In our study, we observed a decrease in Bacteroidetes and an increase in Proteobacteria in SAP rats, consistent with other SAP models [17,18]. At the genus level, Escherichia–Shigella was notably enriched in SAP rats, corroborating its documented role in exacerbating inflammation. Treatment with JZL184, a monoacylglycerol lipase (MAGL) inhibitor, notably reduced the abundance of Escherichia–Shigella, likely curbing gut-derived inflammation and mitigating barrier dysfunction. Simultaneously, JZL184 restored the levels of Lactobacillus, a probiotic often depleted early in SAP progression. This rebalancing has profound implications, given Lactobacillus's role in pathogen inhibition [19], reduction of sepsis [20], and enhancement of barrier integrity [21]. This effect may be mediated through the competitive exclusion of pathogens and the production of anti-inflammatory metabolites, such as short-chain fatty acids [22]. The negative correlation observed between Lactobacillus and aspartic acid levels suggests a modulation of metabolic pathways, highlighting JZL184's dual therapeutic mechanism that encompasses both microbial rebalancing and metabolic reprogramming in addressing SAP. The MAPK signaling pathway is crucial in regulating inflammation. Clinical evidence suggests that MAPK inhibitors can ameliorate intestinal mucosal barrier damage in SAP patients [22]. Similarly, the NOD-like receptor family, particularly NLRP3, promotes pancreatic neutrophil infiltration, tissue necrosis, and intestinal barrier disruption, exacerbating systemic inflammation in murine models [23]. Our multi-omics analysis indicates that microbial dysbiosis in SAP rats triggers the activation of both MAPK and NOD-like receptor signaling pathways, thereby amplifying inflammatory responses. Notably, JZL184 treatment effectively mitigated the activation of these pro-inflammatory pathways, suggesting its therapeutic efficacy through a dual mechanism that addresses both microbial-driven inflammation and the restoration of intestinal barrier function. Metabolomic profiling of SAP mice revealed an increase in amino acids such as tryptophan and aspartic acid, alongside a reduction in short-chain fatty acids like butyric acid. These shifts were tightly correlated with the abundance of Escherichia–Shigella and Lactobacillus. JZL184 treatment elevated Lactobacillus levels, which inversely correlated with aspartic acid—a metabolite implicated in the disruption of pancreatic homeostasis, as observed in asparaginase-induced pancreatitis [24]. Furthermore, aspartic acid metabolism was linked to the expression of angiotensin-converting enzyme (ACE). Our qRT-PCR validation confirmed ACE upregulation in pancreatic tissues of SAP rats, which was normalized by JZL184, directly associating microbial-metabolite dynamics with ACE regulation. Angiotensin II, produced by the action of ACE, is a potent vasoconstrictor involved in inflammation, fibrosis, cell proliferation, and apoptosis [25]. Thus, ACE may significantly influence the inflammatory response in pancreatitis by modulating angiotensin II production. Although ACE inhibition involves asparagine/glutamine antagonism [25], the precise mechanism linking aspartic acid to ACE remains unclear. Our experimental and validation studies demonstrate JZL184's potential by highlighting its ability to coordinate microbial-metabolite networks and crucial signaling pathways, exemplified by ACE normalization. Additionally, the elevated levels of arachidonic acid in SAP mice correlated positively with Escherichia-Shigella, an opportunistic pathogen associated with SAP progression and intestinal inflammation [26]. As a precursor to pro-inflammatory eicosanoids, arachidonic acid accumulation can exacerbate chronic inflammation, similar to findings in arthritis [27] and colitis models [28]. This distinctive association with Escherichia-Shigella in SAP presents novel insights, linking microbial-metabolite interactions to disease pathogenesis. Our network analysis connected arachidonic acid to pancreatic HMOX1 expression, a marker of acute pancreatitis [30], and possibly related stress responses in islet cells [31]. We hypothesize that Escherichia-Shigella-driven arachidonic acid metabolism may indirectly modulate HMOX1 via inflammatory intermediates, warranting further mechanistic exploration. These findings suggest a microbial-metabolite axis potentially influencing pancreatic gene regulation in SAP. Our research group has performed a series of preliminary investigations into the therapeutic potential of MAGL suppression. Experimental evidence demonstrated that pharmacological inhibition of MAGL using JZL184 effectively mitigated intestinal barrier dysfunction in rat models of SAP [16]. Transcriptomic analysis further revealed that this therapeutic intervention appears to downregulate critical biological pathways, including cellular adhesion mechanisms and PI3K-Akt signal transduction. Additionally, the treatment was found to regulate post-transcriptional processes through modifying the expression patterns of RNA-binding proteins, subsequently affecting alternative splicing events in target genes. Our study has several limitations. The integration of data from different species (rats and mice) may present challenges in interpretative breadth and generalizability due to potential differences in microbial composition and metabolic pathways. Although there are conserved aspects of microbial and metabolic processes across species, interspecies variability can influence the extent to which findings in one species translate to another, potentially affecting the generalization of our conclusions. Lastly, while we established a microbiota-metabolite-gene regulatory network, the causal mechanisms remain to be validated, necessitating further experimental investigation to confirm these interactions. conclusion: In conclusion, SAP rats demonstrated gut microbial dysbiosis characterized by elevated opportunistic pathogens and depleted probiotics, activating NOD-like receptor and MAPK pathways. JZL184 treatment effectively reshaped the microbiota by suppressing Escherichia–Shigella, enriching Lactobacillus, concurrently inhibiting inflammatory pathways, modulating metabolites (such as ACE-linked aspartic acid), and normalizing pancreatic ACE overexpression as shown through qRT-PCR validation. This integrated mechanism, bridging microbial-metabolite dynamics with ACE regulation, elucidates JZL184's therapeutic efficacy against SAP, providing a foundation for microbiome-targeted therapies and potential clinical applications.
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