4R is a tobacco cembranoid that binds to and modulates cholinergic receptors and exhibits neuroprotective and anti-inflammatory activity. Given the established function of the cholinergic system in pain and inflammation, we propose that 4R is also analgesic. Here, we tested the hypothesis that systemic 4R treatment decreases pain-related behaviors and peripheral inflammation via modulation of the alpha 7 nicotinic acetylcholine receptors (α7 nAChRs) in a mouse model of inflammatory pain. We elicited inflammation by injecting Complete Freund’s Adjuvant (CFA) into the hind paw of male and female mice. 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 (2.5 h – 8d) after a single systemic 4R (or vehicle) administration. We evaluated the contribution of α7 nAChRs 4R-mediated analgesia by pre-treating mice with a selective antagonist of α7 nAChRs followed by 4R (or vehicle) administration prior to behavioral tests. We assessed CFA-induced paw edema and inflammation by measuring paw thickness and quantifying 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 α7 nAChR-cre::Ai9 mice to measure the expression of α7 nAChRs 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 α7 nAChR-selective antagonist prevented 4R-mediated analgesia and anti-inflammatory effects, demonstrating that 4R effects are via modulation of α7 nAChRs. We further show that a subset of immune cells in the hind paw expresses α7 nAChRs. However, the number of α7 nAChR-expressing immune cells is unaltered by CFA or 4R treatment, suggesting that 4R effects are independent of α7 nAChR-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.

中文翻译：

---

4R-烟草西松烷醇在炎症性疼痛小鼠模型中的抗痛觉过敏和抗炎作用

4R 是一种烟草西松烷醇，可结合并调节胆碱能受体，并具有神经保护和抗炎活性。鉴于胆碱能系统在疼痛和炎症中的既定功能，我们认为 4R 也具有镇痛作用。在这里，我们测试了这样的假设：全身 4R 治疗通过调节炎症性疼痛小鼠模型中的 α7 烟碱乙酰胆碱受体 (α7 nAChR) 来减少疼痛相关行为和外周炎症。我们通过向雄性和雌性小鼠的后爪注射完全弗氏佐剂（CFA）来引发炎症。然后，我们分别在单次全身 4R（或媒介物）给药之前和之后的不同时间点（2.5 小时 - 8 天）分别使用丙酮、哈格里夫斯和冯弗雷试验评估炎症引起的对冷、热和触觉刺激的超敏反应。我们通过用α7 nAChRs的选择性拮抗剂预处理小鼠，然后在行为测试之前施用4R（或媒介物）来评估α7 nAChRs 4R介导的镇痛作用。我们通过测量爪子厚度并使用苏木精和伊红染色量化注射后爪子中的免疫细胞浸润来评估 CFA 引起的爪子水肿和炎症。最后，我们对 α7 nAChR-cre::Ai9 小鼠的爪子皮肤进行了免疫组织化学和流式细胞术分析，以测量免疫子集上 α7 nAChR 的表达。我们的实验表明，全身给予 4R 可降低雄性和雌性小鼠炎症引起的外周超敏反应，以及雄性小鼠而非雌性小鼠炎症引起的爪子水肿。值得注意的是，在第 1 天单次全身给药后，4R 介导的镇痛和抗炎作用可持续长达 8 天。用 α7 nAChR 选择性拮抗剂预处理可阻止 4R 介导的镇痛和抗炎作用，表明 4R 作用是通过调节实现的α7 nAChR。我们进一步表明后爪中的一部分免疫细胞表达 α7 nAChR。然而，CFA 或 4R 治疗不会改变表达 α7 nAChR 的免疫细胞的数量，这表明 4R 效应与表达 α7 nAChR 的免疫细胞无关。总之，我们的研究结果确定了 4R 烟草西松烷内酯作为雄性和雌性小鼠镇痛剂的新功能，能够以性别依赖性方式减少外周炎症，进一步支持胆碱能系统用于疼痛治疗的药理学靶向。