735: Flavonoids mitigate pain by interacting with TRPV1: Novel in -silico and in-vivo evidences

Topic: Basic Science
Subtopic: Pain category (ICD-11), Other acute pain category, , , , , , , , , , Pharmacological interventions, Drug development, Behavioral studies

Background and Aims: Transient receptor potential vanilloid 1 (TRPV1) is a key ion channel expressed in nociceptive sensory neurons that mediates pain signaling(1). It is activated by stimuli such as capsaicin, heat, and protons, leading to calcium influx, increased neuronal excitability, and release of pro-inflammatory cytokines and neuropeptides like CGRP and Substance P (2). Although TRPV1 is an important therapeutic target, current modulators (agonists, antagonists, and RNA interference) have limitations including pungency, desensitization, and thermoregulatory side effects (3). Capsazepine, a widely used antagonist, can induce hypothermia and requires high in vivo doses (3). This study aimed to identify safer and cost-effective agents with antinociceptive potential and to elucidate their mechanisms in inhibiting capsaicin-induced TRPV1 activation and its downstream signaling pathways.

Methods: The structure of Capsaicin (2N27) was extracted from 3J5R and the structure of Naringenin, Naringin, and Kaempferol was downloaded from PubChem. The TRPV1 structure was downloaded from RCSB Protein Data Bank (3J5R). The TRPV1 structure was reconstructed by using Modeller. Energy minimization of reconstructed TRPV1 was done using Swiss PDB viewer. Protein-ligand docking was performed using Auto Dock 4.2. Based on docking outcomes, Kaempferol, Naringin, and Naringenin were selected for in-vivo validation. Acute inflammatory pain was induced by intraplantar injection of capsaicin (200 µg/kg) in mice left hind paw (4). Thermal hyperalgesia and mechanical allodynia were assessed using hot plate (5) and von Frey tests (6), respectively. Locomotor activity and anxiety-like behaviors were evaluated before and after 24 h of treatment using open field (7) and elevated plus maze (8) tests. Molecular analyses were conducted using Western blotting, ELISA, and antioxidant assays (9).

Results: Kaempferol (−4.3 kcal/mol) and Naringenin (−5.44 kcal/mol) showed comparatively higher binding affinity near the TRPV1 pore region, whereas Naringin (−2.07 kcal/mol) bind to the capsaicin binding site to TRPV1. Kaempferol, Naringin and Naringenin reduced Capsaicin induced nociceptive responses like paw edema and paw licking. They inhibited thermal hyperalgesia, mechanical allodynia, anxiety-like behaviors(3-3.5 fold) and motor activity(1.25-1.5 fold) induced by Capsaicin. At the cellular level, those three flavonoids decreased expression of TRPV1(1.5-3.3 fold), Calmodulin (8.2-11.4 fold) and CaMKII (2.1-3.2 fold) in the spinal cord. They reduced the level of Substance P (1.2-1.8 fold), CGRP (1.2-2.1) and intracellular reactive oxygen species generation in the paw and spinal cord. Treatment with Kaempferol, Naringin and Naringenin restored SOD (1.5-2.1fold), Catalase (2.3-3.2 fold) and GSH (1.5-3 fold) levels.

Conclusions: The typical flavonoid structures have favored the suitable bindings in TRPV1 channel in rescuing the capsaicin induced cellular events of pain. The comparative analysis using in silico and in vivo systems proved that the Naringenin showed best efficacy among three compounds mitigating acute inflammatory pain conditions. This is a novel evidence based contribution in pain management.

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Presenting Author: Rimi Das
Submitting Author: Rimi Das

Senior Author: Prof. Sanjit Dey

IASP 2026 Global Year theme of Neuropathic Pain: Yes

• Tuhin Bhattacharya: Ph.D - University of Gothenburg
• Sabnur Parvage: M.Sc - University of Calcutta
• Chaitali Mukhopadhyay: Ph.D - University of Calcutta
• Prof. Sanjit Dey: Ph.D - University of Calcutta