Department of Pediatrics, Jinan Maternity and Child Care Hospital, Jinan, China.
Eur Rev Med Pharmacol Sci. 2019 Jan;23(1):361-369. doi: 10.26355/eurrev_201901_16784.
The aim of this study was to detect the oxidative stress response in the rat model of obesity, asthma and obese asthma. Meanwhile, we aimed to investigate the inhibitory effect of neutrophil elastase inhibitor (NEI) on cellular oxidative stress in the body and whether it exerted an effect on the oxidative stress response in obese asthma through the Kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2 (Keap1/Nrf2) pathway.
The obesity and asthma models were established using a total of 70 Sprague-Dawley (SD) rats. All rats were randomly divided into 7 groups. The rats with normal weight were divided into the control (CTR) group (n=10), asthma (ATM) group (n=10) and ATM+NEI group (n=10). Meanwhile, the obese rats were divided into the obesity (OBS) group (n=10), the OBS+NEI group (n=10), the OBS+ATM group (n=10) and the OBS+ATM+NEI group (n=10). After modeling, rats in NEI intervention groups were injected with Sivelestat (5 mg/kg) via the caudal vein twice a day for 1 week. The tests of cough sensitivity to capsaicin and bronchial responsiveness were performed 24 h after the last administration. Lung tissues of rats were collected for hematoxylin-eosin (HE) staining. Meanwhile, the levels of reactive oxygen species (ROS) in heart, lung and kidney tissues were detected via 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The activities of reduced glutathione (GSH), glutathione peroxidase (GSH-Px), H2O2 and total superoxide dismutase (T-SOD) in the heart, lung and kidney tissues were detected using the colorimetric method. The mRNA and protein expressions of Keap1 and Nrf2 messenger ribonucleic acid expressions in the heart, lung and kidney tissues were measured via Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting, respectively.
NEI significantly improved the symptoms and lung pathology in rats with asthma. The level of ROS in the heart, lung and kidney tissues of the OBS group, ATM group and OBS+ATM group was significantly increased. However, NEI markedly inhibited the level of ROS in rats with asthma. The activities of antioxidant stress-related enzymes (reduced GSH, GSH-Px, H2O2 and SOD) in the heart, lung and kidney tissues of the OBS group, ATM group and OBS+ATM group were significantly decreased. However, NEI markedly promoted the activities of the related antioxidant enzymes in oxidative stress response in asthma rats. Besides, the Keap1/Nrf2 signaling pathway in the heart, lung and kidney tissues of the OBS group, ATM group and OBS+ATM group was significantly inhibited, while NEI activated the Keap1/Nrf2 signaling pathway in rats with asthma.
NEI promotes the release of a variety of antioxidant factors, enhances the activity of antioxidant enzymes and improves the symptoms of rats with obese asthma. The possible underlying mechanism may be the activation of the Keap1/Nrf2 signaling pathway.
本研究旨在检测肥胖、哮喘和肥胖型哮喘大鼠模型中的氧化应激反应。同时，我们旨在通过 Kelch 样 ECH 相关蛋白 1/核因子 E2 相关因子 2（Keap1/Nrf2）通路研究中性粒细胞弹性蛋白酶抑制剂（NEI）对体内细胞氧化应激的抑制作用，以及其是否对肥胖型哮喘的氧化应激反应产生影响。
使用总共 70 只 Sprague-Dawley（SD）大鼠建立肥胖和哮喘模型。所有大鼠随机分为 7 组。正常体重大鼠分为对照组（CTR）（n=10）、哮喘（ATM）组（n=10）和 ATM+NEI 组（n=10）。同时，肥胖大鼠分为肥胖（OBS）组（n=10）、OBS+NEI 组（n=10）、OBS+ATM 组（n=10）和 OBS+ATM+NEI 组（n=10）。建模后，NEI 干预组大鼠通过尾静脉每天两次注射 Sivelestat（5mg/kg），共 1 周。最后一次给药后 24 小时进行辣椒素诱导咳嗽敏感性和支气管反应性测试。收集大鼠肺组织进行苏木精-伊红（HE）染色。同时，通过 2'，7'-二氯二氢荧光素二乙酸酯（DCFH-DA）检测心脏、肺和肾脏组织中活性氧（ROS）的水平。采用比色法检测心脏、肺和肾脏组织中还原型谷胱甘肽（GSH）、谷胱甘肽过氧化物酶（GSH-Px）、H2O2 和总超氧化物歧化酶（T-SOD）的活性。采用逆转录-聚合酶链反应（RT-PCR）和 Western blot 分别检测心脏、肺和肾脏组织中 Keap1 和 Nrf2 信使核糖核酸表达的变化。
NEI 显著改善了哮喘大鼠的症状和肺病理学。OBS 组、ATM 组和 OBS+ATM 组大鼠心脏、肺和肾脏组织中的 ROS 水平显著升高。然而，NEI 显著抑制了哮喘大鼠的 ROS 水平。OBS 组、ATM 组和 OBS+ATM 组大鼠心脏、肺和肾脏组织中抗氧化应激相关酶（还原型 GSH、GSH-Px、H2O2 和 SOD）的活性显著降低。然而，NEI 显著促进了哮喘大鼠氧化应激反应中相关抗氧化酶的活性。此外，OBS 组、ATM 组和 OBS+ATM 组大鼠心脏、肺和肾脏组织中的 Keap1/Nrf2 信号通路明显受到抑制，而 NEI 则激活了哮喘大鼠的 Keap1/Nrf2 信号通路。
NEI 促进了多种抗氧化因子的释放，增强了抗氧化酶的活性，改善了肥胖型哮喘大鼠的症状。其可能的潜在机制可能是激活了 Keap1/Nrf2 信号通路。
