目的 探討阿那其根醇提物（EEAP）對(duì)咳嗽變異性哮喘（CVA）大鼠神經(jīng)炎癥和c-Jun N-終端激酶（JNK）/p38絲裂原活化蛋白激酶（p38MAPK）信號(hào)通路的調(diào)控作用。方法 體外實(shí)驗(yàn)中，將大鼠海馬神經(jīng)元H19-7細(xì)胞分為6組，分別為對(duì)照組、模型組、醋酸潑尼松（0.025 mg·mL^-1）陽(yáng)性藥對(duì)照組和EEAP高、中、低質(zhì)量濃度（6.4、3.2、1.6 mg·mL^-1）組，采用脂多糖（LPS）誘導(dǎo)細(xì)胞炎癥模型，采用酶聯(lián)免疫吸附（ELISA）法檢測(cè)各組細(xì)胞上清液中的白細(xì)胞介素-6（IL-6）、IL-8、IL-18的水平，Western blotting法檢測(cè)各組神經(jīng)元細(xì)胞中JNK、p-JNK和p38MAPK的蛋白表達(dá)。動(dòng)物實(shí)驗(yàn)中，將SPF級(jí)雄性SD大鼠隨機(jī)分為對(duì)照組、模型組、醋酸潑尼松（2.5 mg·kg^-1）組和EEAP高、中、低劑量（640、320、160 mg·kg^-1）組，除對(duì)照組外，各組大鼠使用1 mg OVA致敏，大鼠sc新鮮配制的1 mg·mL^-1的OVA溶液，在各大鼠后足跖、腹股溝、腰部、背部和頸部總共取10個(gè)點(diǎn)，每個(gè)點(diǎn)注射0.05 mL，同時(shí)ip 0.5 mL，共計(jì)l mL；造模后第15天開始，大鼠持續(xù)霧化吸入1% OVA 15 d，每天1次，每次20 min以激發(fā)哮喘。對(duì)照組以0.9%氯化鈉溶液代替OVA溶液進(jìn)行sc和霧化吸入。從造模第30天開始，對(duì)照組和模型組ig給予等量0.9%氯化鈉溶液，各給藥組分別ig給予相應(yīng)劑量藥物，每日1次，持續(xù)30 d。采用免疫組織化學(xué)法檢測(cè)海馬區(qū)組織中JNK和p38MAPK的表達(dá)，采用Western blotting法檢測(cè)海馬區(qū)組織中p-JNK的蛋白表達(dá)，采用ELISA法檢測(cè)各組大鼠海馬區(qū)組織中IL-6、IL-8、IL-18的水平。結(jié)果 體外實(shí)驗(yàn)顯示，與對(duì)照組比較，模型組細(xì)胞培養(yǎng)上清液中的IL-6、IL-8、IL-18水平明顯升高（P＜0.05），模型組LPS刺激的神經(jīng)元細(xì)胞中的p-JNK、p38MAPK蛋白表達(dá)水平明顯升高（P＜0.05）；與模型組比較，醋酸潑尼松組及EEAP高、中劑量組細(xì)胞培養(yǎng)上清液中的IL-6、IL-8、IL-18水平明顯降低（P＜0.05），細(xì)胞中p-JNK、p38MAPK蛋白表達(dá)水平明顯降低（P＜0.05）；且EEAP的炎癥因子和蛋白表達(dá)抑制作用呈濃度相關(guān)性。動(dòng)物實(shí)驗(yàn)中，與對(duì)照組比較，模型組大鼠腦組織中IL-6、IL-8、IL-18的水平和JNK、p-JNK和p38MAPK的蛋白表達(dá)水平均升高（P＜0.05）。與模型組比較，醋酸潑尼松組及EEAP各組的IL-6、IL-8、IL-18的水平和JNK、p-JNK和p38MAPK的蛋白表達(dá)水平均降低（P＜0.05）。且EEAP作用具有劑量相關(guān)性。結(jié)論 EEAP抑制CVA誘導(dǎo)的神經(jīng)炎癥，可通過(guò)調(diào)節(jié)炎癥因子IL-6、IL-8、IL-18和JNK、p-JNK/p38MAPK信號(hào)通路發(fā)揮抗炎作用。

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

新疆維吾爾自治區(qū)自然科學(xué)基金面上項(xiàng)目(2021D01C458)