Anti-hyperalgesic and anti-inflammatory effects of 4R-tobacco cembranoid in a mouse model of inflammatory pain

Abstract

Abstract 4R is a tobacco cembranoid that exhibits neuroprotective and anti-inflammatory activity, with previous studies demonstrating that it binds to and modulates cholinergic receptors. Given the established function of the cholinergic system in pain modulation and inflammation, we propose that 4R is also analgesic. In the present study, we tested the hypothesis that systemic 4R treatment decreases pain-related behaviors and peripheral inflammation in a mouse model of inflammatory pain. To do this, we injected Complete Freund’s Adjuvant (CFA) into the hind paw of male and female mice to elicit inflammation. We then assessed inflammation-induced hypersensitivity to cold, heat, and tactile stimulation using the Acetone, Hargreaves, and Von Frey tests, respectively, before and at different time points (2.5h – 8d) after a single systemic 4R (or vehicle) administration. In separate experiments, we pre-treated mice with a selective antagonist of alpha 7 nicotinic acetylcholine receptors (α7nAChRs) followed by 4R (or vehicle) administration prior to behavioral tests. To evaluate CFA-induced paw edema and inflammation, we measured paw thickness and quantified immune cell infiltration in the injected hind paw using hematoxylin and eosin staining. Lastly, we performed immunohistochemical and flow cytometric analyses of paw skin in α7nAChR-cre::Ai9 mice to measure the expression of α7nAChRs on immune subsets. Our experiments show that systemic administration of 4R decreases inflammation-induced peripheral hypersensitivity in male and female mice and inflammation-induced paw edema in male but not female mice. Notably, 4R-mediated analgesia and anti-inflammatory effects lasted up to 8d after a single systemic administration on day 1. Pretreatment with an α7nAChR-selective antagonist prevented 4R-mediated analgesia and anti-inflammatory effects, demonstrating that 4R effects are via modulation of the cholinergic system. We further show that a subset of immune cells in the hid paw expresses α7nAChR. However, the number of α7nAChR-expressing immune cells is unaltered by CFA or 4R treatment, suggesting that 4R effects are independent of α7nAChR-expressing immune cells. Together, our findings identify a novel function of the 4R tobacco cembranoid as an analgesic agent in both male and female mice that reduces peripheral inflammation in a sex-dependent manner, further supporting the pharmacological targeting of the cholinergic system for pain treatment.