# Electroacupuncture alleviates pain by activating the MD2/TLR4/NF-κB pathway in the ST36 acupoint

## TL;DR

Electroacupuncture reduces pain by activating the MD2/TLR4/NF-κB pathway at the ST36 acupoint in mice.

## Contribution

Identifies MD2 as a key mediator in the analgesic effects of electroacupuncture at the ST36 acupoint.

## Key findings

- EA increased pain thresholds and activated MD2, TLR4, and NF-κB in the ST36 acupoint.
- MD2 knockdown reduced EA's analgesic effects, while overexpression enhanced them.
- EA modulates the Grem1/BMP4/COX2 axis in the spinal cord via the MD2/TLR4/NF-κB pathway.

## Abstract

The acupuncture acupoint is the critical initial site for the therapeutic efficacy of electroacupuncture (EA). Previous studies have confirmed that the NF-κB pathway within the acupoint region mediates the therapeutic effects of acupuncture. Therefore, this study focuses on an in-depth investigation of the MD2/TLR4/NF-κB axis.

The mouse model of adjuvant-induced arthritis (AIA) was established via intraplantar injection of Complete Freund’s Adjuvant (CFA). EA intervention was applied bilaterally to the Zusanli (ST36) acupoints, and behavioral, molecular, and immunological approaches were integrated to investigate the role of MD2 in EA-mediated analgesia.

EA increased paw thermal withdrawal thresholds (PTWTs) in AIA mice (P < 0.05), accompanied by higher levels of MD2, TLR4, p65, and the phosphorylated form of p65 (p-p65) at the acupoint. Co-immunoprecipitation (Co-IP) confirmed binding between MD2 and TLR4 in ST36 acupoint, while immunofluorescence (IF) revealed co-localization of TLR4 with fibroblasts and mast cells in ST36, suggesting these immune cells are critical targets for signal activation. Lentivirus-mediated knockdown of MD2 in the acupoint partially reversed EA’s analgesic effects and suppressed downstream TLR4/NF-κB pathway activation, whereas MD2 overexpression elicited a partial analgesic effect and promoted pathway activation. Together with spinal cord proteomics data, these findings indicate that modulating MD2 in the acupoint can regulate spinal cord-related signaling pathways. Mechanistically, EA dynamically regulates the equilibrium of the Grem1/BMP4/COX2 axis in the spinal dorsal horn via activation of the MD2/TLR4/NF-κB pathway cascade, achieving systemic analgesia.

This study provides molecular evidence supporting the "acupoint priming" theory in acupuncture and highlights MD2 as a potential therapeutic target for pain management.