Objective: To evaluate the effects of penehyclidine hydrochloride on the nuclear factor erythroid 2-related factor 2/antioxidant responsive element (Nrf2/ARE) signaling pathway during endotoxin-induced acute lung injury(ALI) in neonate rats. Methods: Forty 7-day-old Wistar rats weighing 12-18 g were randomly divided into 4 groups (n=10) using a random number table: normal saline group(NS group), acute lung injury(ALI group), penehyclidine hydrochloride group(PHC group) and penehyclidine hydrochloride+ Nrf2 siRNA plasmid group(PNS group). The ALI model was induced with intraperitoneal endotoxin (5.0 mg/kg) in groups ALI, PHC and PNS. In groups PHC and PNS, penehyclidine hydrochloride (2.0 mg/kg) was injected intraperitoneally at 1 h before ALI respectively, while the equal volume of normal saline was administered in groups NS and ALI. The animal of PNS group were inhaled adenovirus packaging of Nrf2-siRNA three times (one time a day) before modeling. At 4 h after endotoxin injection, the rats were sacrificed. The lungs were collected to determine the wet/dry(W/D) lung weight ratio. The expression of Nrf2 and heme oxygen and enzyme 1(HO-1) were determined by Western blotting, contents of tumor necrosis factor-alpha(TNF-α),interleukin10 (IL-10)were determined by enzyme-linked immunosorbent assay(ELISA). The cell apoptosis were determined by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TUNEL),and the apoptotic index was calculated. Results: The W/D ratio in NS, ALI, PHC and PNS groups were (4.2±0.1), (9.6±0.7), (6.5±0.6), (8.3±1.3) respectively. The apoptotic index were (3.7±0.5)%, (31.5±3.2)%, (17.6±4.2)%, (28.1±3.5)%respectively.The contents of TNF-α were (10.3±1.6), (98.5±8.5), (68.5±6.7), (89.9±8.5) pg/ml respectively. The contents of IL-10 were (7.9±0.6), (102.8±9.3), (72.5±5.8), (97.7±9.1) pg/ml respectively.The expression of Nrf2 were (23.2±7.6), (79.8±13.0), (155.5±16.7), (12.0±3.3) respectively. The expression of HO-1 were (31.7±8.6), (90.8±10.3), (147.6±22.5), (61.4±9.7) respectively. There were statistically significant differences among different groups (F=86.013, 154.897, 328.810, 374.198, 333.965, 125.274, all P<0.05). Compared with group NS, the W/D ratio, apoptotic index and the contents of TNF-α, IL-10 increased, the expression of Nrf2 and HO-1 up-regulated in group ALI and group PHC (all P<0.05). Compared with group ALI, the W/D ratio, apoptotic index and the contents of TNF-α, IL-10 decreased, the expression of Nrf2 and HO-1 up-regulated in group PHC (all P<0.05). Compared with group ALI, no significant differences were found in the W/D ratio, apoptotic index and the contents of TNF-α, IL-10 in group PNS(all P>0.05), while the expression of Nrf2 and HO-1 down-regulated in group PNS (all P<0.05). Compared with group PHC, the W/D ratio,apoptotic index and the contents of TNF-α,IL-10 increased, the expression of Nrf2 and HO-1 down-regulated in group PNS (all P<0.05). Conclusion: Nrf2/ARE signaling pathway is involved in the reduction of ALI by penehyclidine hydrochloride in neonate rats.
目的： 评价盐酸戊乙奎醚对新生大鼠内毒素急性肺损伤（ALI）时肺组织核因子E2相关因子-2（Nrf2）/抗氧化反应元件（ARE）信号通路的影响。 方法： 清洁级健康雄性Wistar大鼠40只、体质量12~18 g，7日龄。采用随机数字表法分为4组（n=10）：对照组（NS组）、急性肺损伤组（ALI组）、盐酸戊乙奎醚组（PHC组）和盐酸戊乙奎醚+Nrf2-siRNA质粒组（PNS组）。腹腔注射内毒素5.0 mg/kg制备ALI模型。PHC组和PNS组于内毒素注射前1 h腹腔注射盐酸戊乙奎醚2.0 mg/kg，NS组和ALI组给予等容量生理盐水。PNS组新生大鼠进行模型制备前3 d，吸入采用腺病毒包装的Nrf2-siRNA液3次（1次/d）。于注射内毒素4 h时处死大鼠取肺组织标本，计算肺组织湿/干重比（W/D），采用免疫印迹法（Western blotting）测定Nrf2和血红素氧合酶-1（HO-1）的表达，采用酶联免疫吸附测定法（ELISA）测定肿瘤坏死因子-α（TNF-α）、白细胞介素-10（IL-10）的含量；脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）计数凋亡细胞，计算细胞凋亡指数（AI）。 结果： NS组、ALI组、PHC组、PNS组W/D分别为（4.2±0.1）、（9.6±0.7）、（6.5±0.6）、（8.3±1.3），AI分别为（3.7±0.5）%、（31.5±3.2）%、（17.6±4.2）%、（28.1±3.5）%，TNF-α分别为（10.3±1.6）、（98.5±8.5）、（68.5±6.7）、（89.9±8.5）pg/ml，IL-10分别为（7.9±0.6）、（102.8±9.3）、（72.5±5.8）、（97.7±9.1）pg/ml，Nrf2分别为（23.2±7.6）、（79.8±13.0）、（155.5±16.7）、（12.0±3.3），HO-1分别为（31.7±8.6）、（90.8±10.3）、（147.6±22.5）、（61.4±9.7），差异均有统计学意义（F=86.013、154.897、328.810、374.198、333.965、125.274，均P<0.05）；与NS组相比较，ALI组和PHC组肺组织W/D、AI和TNF-α、IL-10含量升高，Nrf2和HO-1的表达上调（均P<0.05）；与ALI组相比较，PHC组肺组织W/D、AI和TNF-α、IL-10含量下降，Nrf2和HO-1的表达上调（均P<0.05）；与ALI组相比较，PNS组肺组织W/D、AI和TNF-α、IL-10含量差异均无统计学意义（均P>0.05），Nrf2和HO-1的表达下调（均P<0.05）；与PHC组相比较，PNS组肺W/D、AI和TNF-α、IL-10含量升高，Nrf2和HO-1表达下调（均P<0.05）。 结论： Nrf2/ARE信号通路参与了盐酸戊乙奎醚减轻新生大鼠内毒素性ALI。.
Keywords: Acute lung injury; Animals, newborn; Cholinergic antagonists; NF-E2-Related Factor 2; Response elements.