Liquiritin attenuates IONI-induced trigeminal neuropathic pain via TLR4/MyD88-dependent modulation of microglial M1-like polarization

Trigeminal neuralgia (TN) is a debilitating neuropathic facial pain disorder in which current treatments often provide incomplete or poorly tolerated relief. Microglia-driven neuroinflammation in the trigeminal system, particularly Toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88) signaling, is increasingly recognized as a key driver of neuropathic pain, and network pharmacology suggests that Liquiritin, a major licorice flavonoid with anti-inflammatory actions, may target this pathway. We aimed to determine whether Liquiritin alleviates infraorbital nerve injury (IONI)–induced TN-like neuropathic pain by suppressing microglial M1-like polarization via the TLR4/MyD88 pathway, and to characterize its effects on head-withdrawal thresholds, conditioned place preference, inflammatory and pain mediators and TLR4/MyD88 signaling in vivo and in LPS-stimulated BV2 microglia. Adult female ICR mice (8–10 weeks) underwent infraorbital nerve injury (IONI) or sham surgery and were randomly assigned to Sham + vehicle, IONI + vehicle, IONI + Liquiritin (200 mg/kg, oral) or IONI + Pregabalin (10 mg/kg) groups ( n = 10 per group) treated once daily for 18 days. Mechanical allodynia (head-withdrawal thresholds to von Frey stimulation) and conditioned place preference were assessed, trigeminal tissues were analyzed by Western blotting, immunofluorescence and flow cytometry for microglial markers, inflammatory cytokines, pain mediators and TLR4/MyD88, and TLR4 antagonist/agonist administration, LPS-stimulated BV2 microglia and network pharmacology plus molecular docking were used to interrogate Liquiritin's TLR4/MyD88-dependent actions. In IONI mice, Liquiritin significantly attenuated mechanical allodynia and increased conditioned place preference compared with IONI + vehicle, yielding head-withdrawal threshold and conditioned place preference improvements. Network pharmacology identified 92 Liquiritin-related components, 194 candidate targets, and 41 neuropathic-pain–related overlapping genes enriched in Toll-like receptor signaling, and molecular docking showed favorable binding to IL-1β (−11.2 kcal/mol), TNF-α (−8.35 kcal/mol) and TLR4 (−8.76 kcal/mol). Blocking TLR4/MyD88 signaling with LRU alleviated IONI-induced pain behaviors and reduced trigeminal IL-1β, TNF-α, Iba1, CD32, CGRP and TRPV1 expression, whereas TLR4 agonist TEA partially reversed Liquiritin-induced behavioral and molecular changes, supporting pathway involvement. In trigeminal tissues, Liquiritin decreased TLR4 and MyD88 expression and suppressed microglial M1-like markers together with IL-1β, TNF-α, TRPV1 and CGRP; in LPS-stimulated BV2 microglia, Liquiritin (100 μM) and LRU (10 μg/mL) reduced the production of iNOS, Iba1, CD32 and IL-1β/TNF-α, and serum alanine aminotransferase and aspartate aminotransferase activities were not significantly altered at 200 mg/kg, while serum creatinine increased. Liquiritin alleviated IONI-induced TN-like neuropathic pain in mice, concomitant with reduced microglial M1-like activation, decreased IL-1β/TNF-α and TRPV1/CGRP levels, and down-regulation of TLR4/MyD88 signaling in trigeminal tissues. These in vivo, in vitro and in silico data support Liquiritin as a promising neuroinflammation-modulating candidate targeting TLR4/MyD88 in trigeminal neuropathic pain, warranting further pharmacokinetic, long-term safety and translational studies. • Liquiritin reverses IONI-induced mechanical allodynia and enhances conditioned place preference, with HWT and CPP improvementsversus IONI + vehicle. • Liquiritin suppresses microglial M1-like markers and reduces trigeminal IL-1β and TNF-α levels in IONI mice. • Liquiritin down-regulates TRPV1 and CGRP expression in trigeminal tissues, paralleling behavioral relief. • TLR4 antagonist LRU mimics, and TLR4 agonist TEA partially reverse Liquiritin's analgesic and anti-inflammatory effects via the TLR4/MyD88 pathway. • In LPS-stimulated BV2 microglia, Liquiritin (100 μM) and LRU (10 μg/mL) reduce iNOS, Iba1, CD32 and IL-1β/TNF-α production.