目的 探讨阿那其根醇提物（EEAP）对咳嗽变异性哮喘（CVA）大鼠神经炎症和c-Jun N-终端激酶（JNK）/p38丝裂原活化蛋白激酶（p38MAPK）信号通路的调控作用。方法 体外实验中，将大鼠海马神经元H19-7细胞分为6组，分别为对照组、模型组、醋酸泼尼松（0.025 mg·mL^-1）阳性药对照组和EEAP高、中、低质量浓度（6.4、3.2、1.6 mg·mL^-1）组，采用脂多糖（LPS）诱导细胞炎症模型，采用酶联免疫吸附（ELISA）法检测各组细胞上清液中的白细胞介素-6（IL-6）、IL-8、IL-18的水平，Western blotting法检测各组神经元细胞中JNK、p-JNK和p38MAPK的蛋白表达。动物实验中，将SPF级雄性SD大鼠随机分为对照组、模型组、醋酸泼尼松（2.5 mg·kg^-1）组和EEAP高、中、低剂量（640、320、160 mg·kg^-1）组，除对照组外，各组大鼠使用1 mg OVA致敏，大鼠sc新鲜配制的1 mg·mL^-1的OVA溶液，在各大鼠后足跖、腹股沟、腰部、背部和颈部总共取10个点，每个点注射0.05 mL，同时ip 0.5 mL，共计l mL；造模后第15天开始，大鼠持续雾化吸入1% OVA 15 d，每天1次，每次20 min以激发哮喘。对照组以0.9%氯化钠溶液代替OVA溶液进行sc和雾化吸入。从造模第30天开始，对照组和模型组ig给予等量0.9%氯化钠溶液，各给药组分别ig给予相应剂量药物，每日1次，持续30 d。采用免疫组织化学法检测海马区组织中JNK和p38MAPK的表达，采用Western blotting法检测海马区组织中p-JNK的蛋白表达，采用ELISA法检测各组大鼠海马区组织中IL-6、IL-8、IL-18的水平。结果 体外实验显示，与对照组比较，模型组细胞培养上清液中的IL-6、IL-8、IL-18水平明显升高（P＜0.05），模型组LPS刺激的神经元细胞中的p-JNK、p38MAPK蛋白表达水平明显升高（P＜0.05）；与模型组比较，醋酸泼尼松组及EEAP高、中剂量组细胞培养上清液中的IL-6、IL-8、IL-18水平明显降低（P＜0.05），细胞中p-JNK、p38MAPK蛋白表达水平明显降低（P＜0.05）；且EEAP的炎症因子和蛋白表达抑制作用呈浓度相关性。动物实验中，与对照组比较，模型组大鼠脑组织中IL-6、IL-8、IL-18的水平和JNK、p-JNK和p38MAPK的蛋白表达水平均升高（P＜0.05）。与模型组比较，醋酸泼尼松组及EEAP各组的IL-6、IL-8、IL-18的水平和JNK、p-JNK和p38MAPK的蛋白表达水平均降低（P＜0.05）。且EEAP作用具有剂量相关性。结论 EEAP抑制CVA诱导的神经炎症，可通过调节炎症因子IL-6、IL-8、IL-18和JNK、p-JNK/p38MAPK信号通路发挥抗炎作用。

Objective To investigate the regulatory effects of (ethanol extract of the root of Anacyclus pyrethrum, EEAP) on neuroinflammation and c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in cough-variant asthma (CVA) rats. Methods In in vitro experiments, rat hippocampal neuronal cells H19-7 were divided into six groups, namely, the control group, the model group, the prednisone acetate (0.025 mg·mL^-1) positive drug control group, and the EEAP high, medium, and low mass concentration (6.4, 3.2, and 1.6 mg·mL^-1) group, and lipopolysaccharide (LPS) was used to induced cellular inflammation model, and the levels of interleukin-6 (IL-6), IL-8, and IL-18 in the cell supernatants of each group were detected by enzyme-linked immunosorbent assay (ELISA), and the protein expression of JNK, p-JNK, and p38MAPK in the neuronal cells of each group was detected by Western blotting. In the animal experiments, SPF-grade male SD rats were randomly divided into control, model, prednisone acetate (2.5 mg·kg^-1), and EEAP high, medium, and low dose (640, 320, and 160 mg·kg^-1) groups, and rats in each group except the control group were sensitized with 1 mg of OVA, and the rats sc freshly prepared 1 mg ·mL^-1 OVA solution was injected at a total of 10 points in the hindfoot plantar, inguinal, lumbar, dorsal, and cervical regions of each rat, with 0.05 mL injected at each point, while 0.5 mL was injected intraperitoneally, for a total of l mL. Starting on the 15th d after modeling, the rats were continuously nebulized and inhaled with 1% OVA for 15 d, once per day, for 20 min each time in order to stimulate asthma. The control group was sc and nebulized inhalation with 0.9% NaCl solution instead of OVA solution. Starting from the 30th d of modeling, the control group and the model group ig were given equal amounts of 0.9% NaCl solution, and each dosing group ig was given the corresponding dose of drug once a day for 30 d. The expression of JNK and p38MAPK in hippocampal tissues was detected by immunohistochemistry, and the protein expression of p-JNK in the tissues of hippocampal region was detected by Western blotting. ELISA was used to detect the levels of IL-6, IL-8 and IL-18 in the hippocampal area tissues of rats in each group. Results The in vitro experiments showed that the levels of IL-6, IL-8, and IL-18 in cell culture supernatants of the model group were significantly higher compared with those of the control group (P < 0.05), and the protein expression levels of p-JNK and p38MAPK in LPS-stimulated neuronal cells in the model group were significantly higher (P < 0.05). Compared with the model group, the levels of IL-6, IL-8, and IL-18 in cell culture supernatants of the prednisone acetate group and the EEAP high-and medium-dose groups were significantly decreased (P < 0.05). The levels of IL-6, IL-8 and IL-18 in cell culture supernatant were significantly reduced (P < 0.05), and the protein expression levels of p-JNK and p38MAPK in cells were significantly reduced (P < 0.05). And the inhibitory effects of inflammatory factors and protein expression of EEAP showed concentration correlation. In animal experiments, the levels of IL-6, IL-8, IL-18 and the protein expression levels of JNK, p-JNK and p38MAPK in the brain tissues of rats in the model group were elevated compared with those in the control group (P < 0.05). Compared with the model group, the levels of IL-6, IL-8, IL-18 and the protein expression levels of JNK, p-JNK and p38MAPK were decreased in the prednisone acetate group and the EEAP groups (P < 0.05). And the EEAP effect was dose-related. Conclusion EEAP inhibited CVA-induced neuroinflammation and could exert anti-inflammatory effects by regulating inflammatory factors IL-6, IL-8, IL-18 and JNK, p-JNK/p38MAPK signaling pathways.

R285.5

新疆维吾尔自治区自然科学基金面上项目(2021D01C458)