GV-971 prevents severe acute pancreatitis by remodeling the microbiota-metabolic-immune axis

Severe acute pancreatitis (SAP) is a life-threatening form of acute pancreatitis characterized by pancreatic necrosis, organ failure, and high mortality rates up to 40%. SAP pathogenesis involves acinar cell death, release of DAMPs, and recruitment of innate immune cells (macrophages and neutrophils), leading to a systemic inflammatory response. Intestinal dysfunction and gut microbiota dysbiosis further exacerbate disease severity. Although probiotics and prebiotics can modulate the microbiota and host immunity, clinically approved drugs that beneficially remodel the gut microbiota are limited. GV-971 (sodium oligomannate), approved in China for Alzheimer’s disease, reportedly regulates intestinal microbiota and reduces specific microbial metabolites implicated in neuroinflammation, though its pharmacology remains controversial. Based on the shared roles of microbiota and immune dysfunction in neurodegenerative disease and pancreatitis, the study tests the hypothesis that GV-971 protects against SAP by remodeling the microbiota–metabolic–immune axis, elevating beneficial bacteria and their metabolites (SCFAs) to suppress pro-inflammatory immune polarization.

Prior work shows that gut microbiota dysbiosis worsens SAP and that prebiotics/probiotics can attenuate inflammation and improve gut barrier integrity. Firmicutes-derived butyrate producers (e.g., Faecalibacterium) generate SCFAs with anti-inflammatory effects and barrier support; reduced butyrate has been linked to worse pancreatitis outcomes, and butyrate supplementation can decrease SAP mortality and inflammation. GV-971 has been reported to remodel gut microbiota, lower phenylalanine/isoleucine, and reduce neuroinflammation in Alzheimer’s models, implicating microbiota-mediated immune modulation. However, its efficacy and mechanisms are debated, and its potential in pancreatitis had not been established. The overlap between innate/adaptive immune activation and intestinal barrier impairment in SAP motivates testing microbiota-targeted therapies. The study situates GV-971 within this context, focusing on SCFAs (propionate, butyrate) as candidate mediators.

Animal models: Male C57BL/6 mice (6–8 weeks) were randomly assigned. SAP was induced by intraperitoneal (IP) caerulein (50 µg/kg hourly for 10 or 11 injections depending on figure) followed by a single LPS injection (10 mg/kg). A second SAP model used L-arginine hydrochloride (4 g/kg IP, two hourly injections), with analysis at 72 h. GV-971 treatment: Oral gavage for 7 days at low (100 mg/kg/day), medium (200 mg/kg/day), or high (400 mg/kg/day) doses prior to SAP induction. Readouts 24 h after first caerulein: serum enzymes/cytokines, histopathology, immunohistochemistry, microbiome, metabolomics, and mass cytometry. Safety: Food intake and body weight monitored. Histology and IHC: Pancreas, kidney, lung, ileum fixed, paraffin-embedded, H&E and Sirius Red stained. IHC used anti-MPO, anti-CD68, anti-CD86, anti-CD206, anti-IL-6 antibodies to quantify neutrophils, total/M1/M2 macrophages, and IL-6. Image analysis performed blinded. Serum biochemistry and cytokines: Amylase, lipase, creatinine, ALT measured with commercial kits. Serum IL-6, IL-8, TNF-α quantified by ELISA. Gut microbiota profiling: Cecal contents collected; DNA extracted; V3–V4 16S rRNA amplicons sequenced on Illumina MiSeq. QIIME2 (2019.4) pipeline with cutadapt, DADA2 denoising, MAFFT alignment, FastTree2 phylogeny. Analyses included ASV counts, beta diversity (weighted UniFrac PCoA), and differential taxa at phylum/genus levels. Correlations between genera and pancreatic function indicators were computed (Spearman). Intestinal microbiota transplantation (IMT): Donor cecal contents (from HG+SAP mice) prepared in PBS (100 mg/mL, 20% glycerol) and stored. Recipient mice received an antibiotics (ABX) cocktail in drinking water for 7 days (ampicillin, metronidazole, neomycin, vancomycin), then oral gavage of donor bacterial suspension (10 µL/g) on alternate days for 7 days before SAP induction. Microbiota depletion test: Mice received ABX for 7 days, then GV-971 (high dose) for 7 days, followed by SAP induction to assess dependence on microbiota. Targeted metabolomics: Cecal contents analyzed by UPLC–MS/MS (Acquity UPLC, Xevo TQ-S). A panel of 201 standards was used; internal standards included. Data processed with TMBQ; analyses included PCA/PLS-DA, univariate testing, pathway enrichment, and correlations with pancreatic parameters. Relative class abundances (e.g., SCFAs) were quantified. SCFA supplementation: After ABX, mice received sodium acetate, sodium propionate, or sodium butyrate (400 mg/kg/day, 7 days) prior to SAP induction. Pancreatic and systemic readouts assessed as above. Mass cytometry (CyTOF): Peripheral blood and intestinal cell suspensions prepared, barcoded (20-plex Pd), stained with panels including lineage and activation markers plus Foxp3/RORγt nuclear staining. Acquired on Helios; data normalized, debarcoded, debris/doublets removed; viSNE used to define immune populations and T-cell subsets; frequencies compared between groups. Macrophage depletion: Clodronate liposomes (CL) or PBS liposomes (PL) administered IP on day 6 of GV-971 regimen; SAP induced on day 8; effects on pancreatic injury and enzymes assessed. Cell culture assays: Mouse pancreatic acinar line MPC-83 used to test direct cytoprotection (H2O2 challenge) by propionate/butyrate (no effect). RAW 264.7 macrophages pretreated with sodium propionate (1.6–6.4 mM) or sodium butyrate (0.2–0.8 mM), then stimulated with LPS (1 µg/mL) for 6 h. Outcomes: viability (CCK8), immunofluorescence for CD86/CD206, ELISAs for IL-6/TNF-α/IL-8, RNA-seq, and Western blots. RNA-seq and Western blots: RNA-seq by Novogene; DEGs defined by adjusted p ≤ 0.05 and |log2FC| ≥ 1; KEGG enrichment performed. Western blot targets: p-JNK/JNK, p-ERK1/2/ERK1/2, p-p38/p38, p-p65/p65, GAPDH. Rescue with p38 MAPK agonist U46619 (10 µM) tested against SP/SB effects. Statistics: Normality and variance tested; appropriate parametric/nonparametric tests applied (t-test, ANOVA with Dunnett/Tukey, Kruskal–Wallis with Dunn, two-way ANOVA for curves). p < 0.05 considered significant.

• GV-971 protects in multiple SAP models: In caerulein+LPS SAP, GV-971 (7 days, oral; low/mid/high) reduced serum IL-8 and IL-6 (e.g., IL-8 p < 0.0001; IL-6 p = 0.01 vs SAP), pancreatic IL-6 staining, pathological scores (p < 0.0001), collagen (Sirius) area (p = 0.0001), serum lipase (p < 0.0001) and amylase (p = 0.0002), and improved pancreatic histology (reduced acinar death, leukocyte infiltration, edema). It also reduced kidney and lung injury and improved creatinine and pulmonary pathology. In L-arginine SAP, GV-971 similarly reduced pancreatic injury, inflammatory markers, and serum enzymes. Highest-dose GV-971 showed no adverse effects on food intake or body weight. • Innate immune modulation: SAP increased pancreatic neutrophils (MPO) and macrophages (CD68), with higher M1 (CD86) and lower M2 (CD206). GV-971 reduced neutrophil/macrophage infiltration and shifted macrophage polarization toward M2 (reduced CD86, increased CD206). • Microbiome remodeling: 16S rRNA sequencing of cecal contents (n = 8/group) identified 4157 ASVs. PCoA revealed distinct clustering among control, SAP, and GV-971+SAP. SAP shifted dominant taxa from Firmicutes to Proteobacteria and increased Cyanobacteria; GV-971 pretreatment tended to reverse Firmicutes/Proteobacteria shifts and significantly reduced Cyanobacteria (p < 0.05). At genus level, GV-971 altered Melissococcus, Pseudomonas, Sphingomonas, Ralstonia, Faecalibacterium, and unidentified Streptophyta; Faecalibacterium increased and was negatively correlated with serum IL-6, while other listed genera correlated positively with IL-6. Discussion noted Proteobacteria increased 64.83-fold and Firmicutes decreased to 19.2% of control in SAP; GV-971 showed trends reversing these. • Causality via microbiota: IMT from GV-971-affected donors (after recipient ABX) reduced SAP severity (lower serum IL-8 and pancreatic IL-6; improved histology and Sirius; decreased MPO, CD68, CD86; increased CD206; reduced serum lipase and amylase). Conversely, ABX-mediated microbiota depletion abolished GV-971’s protective effects, indicating microbiota dependence. • Metabolomics identifies SCFAs: Targeted UPLC–MS/MS detected 172 metabolites; GV-971 increased SCFAs, carbohydrates, phenylpropanoic acids, and reduced amino acids and fatty acids. SAP decreased intestinal acetic acid and butyrate vs control; GV-971+SAP increased acetate, propionate, and butyrate vs SAP. Reported p-values: acetate p = 0.007, propionate p = 0.001, butyrate p = 0.003. Pathway enrichment implicated propanoate and butyrate metabolism. Acetate, propionate, and butyrate levels negatively correlated with pancreatic inflammation markers. • SCFA supplementation is protective: After ABX, sodium propionate or sodium butyrate (400 mg/kg/day, 7 days) reduced serum and pancreatic IL-6/IL-8; improved pancreatic histology; decreased MPO and CD68; reduced Sirius collagen; shifted macrophages toward M2 (increased CD206, decreased CD86). They lowered serum lipase (both) and amylase (propionate), and propionate reduced pancreatic weight. They also ameliorated kidney and lung injury and improved ileal villus length and occludin. Sodium acetate did not confer similar benefits. • Immune system rebalancing (CyTOF): SAP increased granulocytes and monocytes and decreased B cells, DCs, and T-cell subsets in peripheral blood. GV-971 reversed granulocyte increases and restored B- and T-cell subsets, including increased Tregs and resting T-cell subsets. In intestinal tissue, SAP increased monocytes/granulocytes and reduced T-cell subsets and DCs; GV-971 increased double-negative T cells and M2 macrophage percentages, and restored barrier integrity (villus length, occludin). • Requirement for macrophages: Clodronate liposome-mediated macrophage depletion prevented GV-971 from reducing serum lipase/amylase and improving pancreatic pathology, indicating macrophage involvement is necessary for protection. • Mechanism in macrophages: In RAW 264.7 cells, sodium propionate and butyrate did not affect viability but reduced LPS-induced M1 polarization (↓CD86+, ↑CD206+) and secretion of IL-6, TNF-α, and IL-8. RNA-seq showed broad transcriptional changes with KEGG enrichment for MAPK signaling. Western blots showed SP/SB inhibited phosphorylation of JNK, ERK1/2, and p38. The p38 agonist U46619 antagonized SP/SB’s anti-inflammatory effects, supporting MAPK pathway mediation. SCFAs did not directly protect pancreatic acinar cells (MPC-83) from H2O2-induced death, suggesting their effects are immune-mediated.

The study demonstrates that GV-971 protects against SAP by reshaping the gut microbiota, elevating beneficial taxa such as Faecalibacterium, and enhancing SCFA production (notably propionate and butyrate). These metabolites, in turn, inhibit macrophage M1 polarization and reduce neutrophil infiltration via suppression of MAPK signaling, thereby dampening pancreatic and systemic inflammation. The protective effects require an intact microbiota and macrophages, as shown by ABX and clodronate experiments, and are transferable by IMT. GV-971 also corrects SAP-induced immune dysregulation in peripheral blood and the intestinal immune microenvironment, while improving intestinal barrier integrity. These results align with and extend literature on SCFAs’ anti-inflammatory roles and butyrate’s benefits in pancreatitis, while newly identifying propionate as protective in SAP and providing a mechanistic link through MAPK inhibition in macrophages. The findings position GV-971—an approved Alzheimer’s therapy—as a candidate adjunct therapy for SAP, leveraging the microbiota–metabolite–immune axis to modulate innate and adaptive immune responses and mitigate organ injury.

Through integrated microbiome, metabolomics, immunophenotyping, and functional experiments, the study establishes GV-971 as an effective protector against SAP in mice. GV-971 remodels gut microbiota (increasing Faecalibacterium), elevates SCFAs (propionate and butyrate), and shifts macrophages toward an anti-inflammatory M2 phenotype by inhibiting MAPK signaling, reducing neutrophil recruitment, and mitigating multi-organ inflammation. The protective effect depends on microbiota and macrophages and is transferable via IMT. These findings support targeting the microbiota–metabolic–immune axis for SAP therapy and suggest repurposing GV-971 or administering SCFAs as potential strategies. Future work should include mechanistic elucidation of how GV-971 modulates specific taxa (e.g., Faecalibacterium), optimization of dosing/timing, validation in additional models and female mice, and clinical translation with trials in SAP patients.

• Preclinical mouse models only; human efficacy and safety in SAP remain untested. • Male mice were used; sex differences were not assessed. • While trends toward reversing Firmicutes/Proteobacteria shifts were observed, some phylum-level changes with GV-971 did not reach statistical significance; causal roles of specific taxa beyond Faecalibacterium were not directly validated. • The mechanism by which GV-971 increases Faecalibacterium is unknown; whether Faecalibacterium directly utilizes GV-971 is unresolved. • SCFAs’ direct effects on other immune populations (e.g., neutrophil chemotaxis) were suggested but not comprehensively dissected in vivo. • The dependence on microbiota suggests potential variability with different baseline microbiomes; generalizability across microbiome contexts is uncertain.