烟碱对小鼠中枢神经炎症的改善作用及其机制

Therapeutic effect of nicotine on neuroinflammation in mice and its mechanism

目的 探讨α7亚基N型乙酰胆碱受体(α7nAchRs)激动剂烟碱对小鼠中枢神经炎症和认知行为的改善作用及其机制。方法 取雌性C57BL/6J小鼠60只,采用随机数字表法分为正常组、模型组、烟碱组、甲基牛扁亭柠檬酸盐(MLA)组,每组各15只。模型组、烟碱组和MLA组腹腔注射多聚肌苷酸—多聚胞苷酸[Poly(I∶C)]12 mg/kg制备中枢神经炎症模型,正常组腹腔注射等量生理盐水。烟碱组于造模前30 min腹腔注射烟碱1 mg/kg;MLA组于造模前1 h腹腔注射α7nAchR拮抗剂MLA 5 mg/kg,造模前30 min腹腔注射烟碱1 mg/kg;模型组和正常对照组在同一时间点注射等量生理盐水。造模后3 h,采用水迷宫实验观察小鼠空间学习记忆能力,记录小鼠穿越原平台次数和逃避潜伏期;采用旷场实验观察小鼠自主运动能力,记录小鼠平均运动速度和运动距离;小鼠处死,取脑组织,采用免疫组化法观察海马及前额叶小胶质细胞激活度,评价中枢神经炎症改变程度;采用qPCR法检测脑组织炎症因子IL-6、TNF-α、INF-β、INF-α、IL-1β mRNA,Western blotting法检测脑组织NF-κB p65蛋白磷酸化水平。结果 与正常组比较,模型组水迷宫实验潜伏期延长、穿越平台次数减少,旷场实验平均运动速度和运动距离减少,海马及前额叶小胶质细胞激活度增加,脑组织IL-6、TNF-α、INF-β、INF-α、IL-1β表达增加,脑组织NF-κB p65磷酸化水平增加(P均<0.05);与模型组比较,烟碱组水迷宫检测示潜伏期延长并穿越平台次数增加,旷场实验示平均运动速度以及运动距离均增加,海马及前额叶小胶质细胞激活度降低,脑组织IL-6、TNF-α、INF-β、INF-α、IL-1β表达降低,脑组织NF-κB p65磷酸化水平降低(P均<0.05);与烟碱组比较,MLA组水迷宫检测示潜伏期延长并穿越平台次数减少,旷场实验示平均运动速度以及运动距离均减少,海马及前额叶小胶质细胞激活度增加,脑组织IL-6、TNF-α、INF-β、INF-α、IL-1β表达增加,脑组织NF-κB p65磷酸化水平增加(P均<0.05)。结论 烟碱能够改善Poly(I∶C)所引起的小鼠中枢神经炎症和认知行为改变,其机制与烟碱作用于α7nAchR后减少NF-κB p65磷酸化,进而减少炎症因子释放有关。

nicotine on Poly(I∶C)-induced neuroinflammation and cognitive behavioral changes in mice.Objective To investigate the therapeutic effect ofα7-nicotinic acetylcholine receptor(α7nAchR)agonist Methods Sixty female C57BL/6J mice were randomly divided into the normal control group,Poly(I∶C)group,nicotine group andα7nAchR an-tagonist group(MLA group),with 15 mice in each group.Mice in the model group were given Poly(I∶C)12 mg/kgth-rough intraperitoneal injection to prepare the central nervous inflammation models.Mice in the nicotine group were intraper-itoneally injected with 1 mg/kg nicotine 30 min before injection of Poly(I∶C).Mice in the MLA group were intraperitone-ally injected with 5 mg/kg MLA 1 h before injection of Poly(I∶C)and 1 mg/kg nicotine 30 min before injection of Poly(I∶C).Mice in the model group and the normal control group were injected with the same amount of normal saline at the same time point.Three hours after modeling,6 mice in each group were randomly selected for water maze experiment and open field experiment.The times of crossing the original platform,escape latency,average movement speed and distance of mice were recorded,so as to evaluate the spatial learning and memory ability and autonomous motor ability of mice.Three mice in each group were randomly selected to be killed,and brain tissues were collected.The activation of microglia in hippocampus and prefrontal cortex was observed by immunohistochemical method to evaluate the degree of central ner-vous inflammation in mice.The mRNA levels of IL-6,TNF-α,INF-β,INF-αand IL-1βwere detected by qPCR,and the protein phosphorylation level of NF-κB p65 was detected by Western blotting.Results Compared with the normal con-trol group,water maze test in the Poly(I∶C)group showed prolonged latency and reduced frequency of crossing the plat-form;open field test showed decreased mean motion speed and distance,increased activation of microglia in hippocampus and prefrontal lobe,increased expression levels of IL-6,TNF-α,INF-β,INF-αand IL-1βin the brain tissues,and in-creased level of NF-κB p65 phosphorylation in brain tissues(all P<0.05).Compared with the Poly(I∶C)group,the wa-ter maze test in the nicotine group showed prolonged latency and increased frequency of crossing the platform,the open field test showed increased mean movement speed and distance,decreased activation of microglia in hippocampus and pre-frontal lobe,and decreased expression levels of IL-6,TNF-α,INF-β,INF-αand IL-1βin the brain tissues,and de-creased level of NF-κB p65 phosphorylation in brain(all P<0.05).Compared with the nicotine group,the water maze test in the MLA group showed prolonged latency and decreased frequency of crossing the platform,the open field test showed decreased mean motion speed and distance,increased activation of microglia in hippocampus and prefrontal lobe,and in-creased expression levels of IL-6,TNF-α,INF-β,INF-αand IL-1βin the brain tissues,and increased level of NF-κB p65 phosphorylation in the brain tissues(all P<0.05).Conclusion Nicotine can improve the neuroinflammation and cognitive behavioral changes induced by Poly(I∶C)in mice,and the mechanism is related to the reduction of NF-κB p65 phosphorylation through nicotine acting onα7nAchR,thus reducing the release of inflammatory factors.