Nociceptor α7nAChR activation blunts neuronal HMGB1 release and attenuates inflammation and nociceptive behavior.

BACKGROUND: High Mobility Group Box 1 (HMGB1) is a nuclear protein that upon extracellular release acts as an alarmin to initiate and amplify inflammation. HMGB1 release from nociceptors contributes to both inflammation and pain; however, the mechanisms for its regulation remain incompletely understood. The cholinergic anti-inflammatory pathway, mediated by α7 nicotinic acetylcholine receptor (α7nAChR) activation, inhibits HMGB1 release from immune cells and reduces inflammation. This study investigates whether α7nAChR signaling similarly inhibits HMGB1 release from nociceptors, thereby affecting pain and inflammation. METHODS: Dorsal root ganglia (DRG) neurons were isolated from C57BL/6 or VGlut2-Cre/ChR2-YFP mice (expressing ChR2 in sensory neurons for optogenetic stimulation at 470 nm). HMGB1 release in vitro was triggered by optogenetic stimulation or exposure to capsaicin (5 µM), in the presence or absence of cholinergic agonists (acetylcholine, GTS-21, PNU-282987), and subsequently measured by ELISA. Immunohistochemistry was used to visualize cellular HMGB1 localization. In vivo models, including optogenetic stimulation and formalin-induced pain-like behavior, were used to evaluate the effects of cholinergic agonists on pain-like behavior, mechanical allodynia and inflammation. α7nAChR knockout (KO) mice served to determine receptor-specific effects. Levels of proinflammatory mediators calcitonin gene-related peptide (CGRP), substance P, HMGB1, and IL-6 were also measured. RESULTS: Optogenetic stimulation of cultured DRG neurons significantly increased HMGB1 release, which was markedly inhibited by cholinergic agonists. Similarly, capsaicin-induced HMGB1 release was suppressed by acetylcholine, GTS-21, and PNU-282987, promoting HMGB1 retention within the nucleus; this effect was abolished in α7nAChR KO neurons. In contrast, the release of CGRP and substance P following optogenetic or capsaicin stimulation of DRG neurons from wild-type mice was not influenced by cholinergic agonists. In vivo, GTS-21 reduced pain-like behaviors and mechanical allodynia in both the formalin-induced and optogenetically-stimulated nociceptive behavior models, as demonstrated by reduced mechanical allodynia and extracellular HMGB1 levels. These effects were absent in α7nAChR KO mice, confirming the critical role of α7nAChR in mediating these responses. CONCLUSION: This study reveals a novel α7nAChR-dependent cholinergic mechanism that reduces nociceptive behavior and inflammation by retaining nuclear HMGB1 in nociceptors. Cholinergic agonists may serve as promising therapeutic agents to mitigate nociceptive behavior and inflammation by targeting α7nAChR in sensory neurons.