卡巴胆碱对脂多糖刺激巨噬细胞释放炎症细胞因子的影响及其受体研究

Effects of carbachol on inflammatory cytokine releases from rat peritoneal macrophages induced by lipopolysaccharide and its receptor

目的在体外脂多糖(lipopolysaccharide,LPS)刺激大鼠腹腔巨噬细胞炎症模型中观察拟胆碱药卡巴胆碱对炎症细胞因子释放的影响,并研究其受体途径。方法采集大鼠腹腔巨噬细胞,先给予M样胆碱能受体拮抗剂阿托品或N样胆碱能受体α7亚基特异性拮抗剂α-银环蛇毒素,15min后给予卡巴胆碱或N样胆碱能激动剂烟碱,15min后再给予LPS刺激,4～6h后取细胞培养上清液,ELISA法检测促炎细胞因子TNF-α、IL-6水平和抗炎细胞因子IL-10水平。大鼠腹腔巨噬细胞制作细胞爬片,用高浓度烟碱和卡巴胆碱处理15min后加入异硫氰酸荧光素标记的α-银环蛇毒素(FITC-α-Bgt),在激光扫描共聚焦显微镜下观察结果并摄片。结果卡巴胆碱与烟碱均能抑制LPS刺激后TNF-α、IL-6的释放,对IL-10的释放无影响。阿托品预处理后,卡巴胆碱与烟碱对TNF-α、IL-6释放的抑制作用无明显变化(P>0.05);α-银环蛇毒素预处理后,卡巴胆碱与烟碱对两种促炎细胞因子释放的抑制作用明显减弱(P<0.01)。激光共聚焦显微镜结果显示烟碱和卡巴胆碱处理后,FITC-α-Bgt与细胞的结合被减弱。结论卡巴胆碱在LPS刺激大鼠腹腔巨噬细胞模型中具有抗炎作用,此作用不能被阿托品阻断,可以被α-银环蛇毒素拮抗。表明卡巴胆碱的抗炎作用与烟碱相似,都是通过N样胆碱能受体α7亚基实现的。

Aim To investigate the effects of carbachol on inflammatory cytokine releases from rat peritoneal macrophages induced by lipopolysaccharide and its receptor. Methods Rat peritoneal macrophages were collected and exposed to muscarinic cholinergic receptor antagonist atropine or α-bungarotoxin, an antagonist specifically binds the α7 subunit of nicotinic cholinergic receptor. 15 min later carbachol or nicotine was added into cell culture. After another 15 min, cells were stimulated with LPS. Inflammatory cytokines TNF-α, IL-6, and IL-10 in the cultures media were assayed by ELISA. Rat peritoneal macrophages were exposed to nicotine and carbachol in high concentration for 15 minutes, and then stained with FITC-labeled α-Bungarotoxin and viewed with fluorescent confocal microscopy. Results Increases of TNF-α and IL-6 after LPS stimulation were significantly inhibited in the presence of carbachol or nicotine while the content of IL-10 was not apparently altered. Atropin did not down-regulate the inhibitive effects of both carbachol and nicotine. And α-bungarotoxin significantly down-regulated these effects. Fluorescent confocal microscopy showed that nicotine and carbachol pretreatment markedly reduced the intensity of binding between FITC-labeled α-Bgt and macrophages. Conclusion The results suggested that both carbachol and nicotine play a role in the anti-inflammatory process through the α7 subunit of nAChR.