Monoacylglycerol Lipase Inhibitor Mitigates Severe Acute Pancreatitis in Rats by Modulating the Gut Microbiota-Metabolite-Target Gene Interaction Network

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 signa