Multi-Omics Insights into the Gut-Spinal Cord Axis: Paeoniflorin Mitigates Diabetic Neuropathic Pain in Rats through Microbial-Metabolic Crosstalk

Painful Diabetic Neuropathy (PDN), a common chronic complication of diabetes, is closely associated with glymphatic system dysfunction and neuroinflammation. This study is the first to explore the mechanism of Paeoniflorin (PF) in treating PDN from the perspective of the gut-spinal cord axis. By establishing a streptozotocin (STZ)/high-fat diet-induced PDN rat model and integrating metagenomics, untargeted metabolomics, and microbial depletion techniques, we demonstrated that PF significantly improved mechanical paw withdrawal threshold (PWT), alleviated insulin resistance, and reduced spinal pro-inflammatory cytokine levels (IL-1β, IL-6, TNF-α) in PDN rats. Dynamic Magnetic resonance imaging (MRI)  revealed that PF restored spinal glymphatic metabolic function, evidenced by enhanced gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) clearance and aquaporin-4 (AQP4) polarization remodeling. Immunofluorescence confirmed that PF suppressed astrocyte activation (total GFAP fluorescence intensity decreased by 61.86% in the PF group vs. PDN group) and restored AQP4 polarity at perivascular endfeet (polarization rate increased from 31.8% to 59.3%). These therapeutic effects were completely reversed by antibiotic intervention (ABX+PF group), highlighting the critical role of gut microbiota. Metagenomic analysis showed that PF enriched short-chain fatty acid (SCFA)-producing genera (Bifidobacterium and Faecalibacterium), activating the tricarboxylic acid (TCA) cycle and arginine metabolism. Metabolomics identified PF-mediated upregulation of anti-inflammatory metabolites (e.g., MGMG 18:2 and N-Acetyl-4-O-acetylneuraminic acid) and suppression of dehydroepiandrosterone sulfate (DHEA-S) synthesis. Our findings demonstrate that PF alleviates PDN by remodeling the gut microbiota-metabolite network, repairing intestinal barrier integrity, and regulating AQP4-dependent waste clearance in the spinal glymphatic system, providing novel therapeutic targets. This study elucidates the regulatory role of the gut-spinal cord axis in PDN and offers a paradigm for investigating "microbiota-host" interactions of low-bioavailability herbal components.