Xiao Jingyuan, Lin Fei, Pan Linghui, Dai Huijun, Jing Ren, Lin Jinyuan, Liang Fangte
Department of Anesthesiology, the Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China. Corresponding author: Lin Fei, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2020 Feb;32(2):188-193. doi: 10.3760/cma.j.cn121430-20190723-00035.
To investigate the protective effect and mechanism of dexamethasone in lung ischemia/reperfusion injury (LIRI) rats.
(1) Part one experiment: 24 Sprague-Dawley (SD) rats were divided into four groups according to the random number method (n = 6): standard ventilation group (N group), normal saline group (NS group), LIRI group, and dexamethasone+LIRI group (DEX group). The rat model of LIRI was established by clamping the left pulmonary hilum for 1 hour and reperfusing it for 2 hours. The DEX group was given dexamethasone 3 mg/kg 5 minutes before reperfusion, and NS group was injected with normal saline. Group N did not receive any treatment. The left lung tissue of the rats in each group were taken alive 2 hours after reperfusion. The lung tissue was harvested for lung wet/dry mass ratio (W/D) measurement. Hematoxylin-eosin (HE) staining and electron microscopy was used to observe the pathological changes of lung tissue and to assess the degree of injury. Ultrastructural changes of lung tissue were observed under electron microscope. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL-1β, IL-6) in lung tissue were detected by enzyme linked immunosorbent assay (ELISA). The expressions of phosphorylated protein kinase B (p-AKT) was detected by Western Blot. (2) Part two experiment: intervention with phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway inhibitor LY294002 to further explore the mechanism of dexamethasone in reducing lung injury induced by LIRI. Twenty-four SD rats were divided into four groups according to the random number method (n = 6): N group, LIRI group, DEX group, and dexamethasone+LY294002+LIRI group (LY group). All the groups except the LY group were treated with membrane and intervention according to part one experiment. The LY group was injected with LY294002 0.3 mg/kg after injection of dexamethasone. The expressions of M1 macrophage polarization markers CD11c, CD16, and M2 macrophage polarization markers CD206, Arg1 were detected by immunohistochemistry.
(1) Part one experiment: compared with N group, the morphological and ultrastructural changes of lung tissue in the LIRI group were significantly changed, lung injury score, lung W/D ratio and TNF-α, IL-1β, IL-6 levels were significantly increased, and p-AKT expression was significantly decreased. Compared with the LIRI group, the morphological and ultrastructural changes of the lung tissue in the DEX group were significantly improved, and the lung injury score was reduced (5.00±0.89 vs. 8.83±0.75), lung W/D ratio and TNF-α, IL-1β, IL-6 levels were significantly decreased [lung W/D ratio: 6.25±0.56 vs. 8.27±0.72, TNF-α (ng/L): 93.28±16.42 vs. 205.90±25.30, IL-1β (ng/L): 130.10±10.81 vs. 209.10±19.20, IL-6 (ng/L): 195.80±21.17 vs. 310.50±20.77], p-AKT expression was significantly increased [p-AKT/AKT: (57.58±8.80)% vs. (36.62±9.25)%], and the differences were statistically significant (all P < 0.05). There was no significant difference in each index between NS group and N group. (2) Part two experiment: compared with the N group, the expression of macrophage polarization markers CD11c, CD16, CD206 and Arg1 in the LIRI group were significantly increased. Compared with the LIRI group, the expressions of CD11c and CD16 in the lung tissue of the DEX group were significantly decreased, and the expressions of CD206 and Arg1 were significantly increased. The intervention of PI3K/AKT signaling pathway inhibitor LY294002 significantly blocked the effect of dexamethasone on LIRI-mediated macrophage polarization (CD11c immunohistochemical score: 7.20±0.36 vs. 5.00±0.34, CD16 immunohistochemical score: 8.20±0.48 vs. 7.40±0.64, CD206 immunohistochemical score: 5.80±0.59 vs. 7.40±0.28, Arg1 immunohistochemical score: 7.20±0.72 vs. 8.80±0.48, all P < 0.05).
Dexamethasone pretreatment can alleviate the intrapulmonary inflammatory response and lung injury caused by LIRI in rats. The mechanism of action is related to the polarization direction of pulmonary macrophagesvia activation of the PI3K/AKT pathway by dexamethasone.
探讨地塞米松对大鼠肺缺血/再灌注损伤（LIRI）的保护作用及机制。
（1）第一部分实验：将24只Sprague-Dawley（SD）大鼠按随机数字法分为四组（n = 6）：标准通气组（N组）、生理盐水组（NS组）、LIRI组和地塞米松+LIRI组（DEX组）。通过夹闭左肺门1小时并再灌注2小时建立LIRI大鼠模型。DEX组在再灌注前5分钟给予地塞米松3 mg/kg，NS组注射生理盐水。N组未接受任何处理。再灌注2小时后处死大鼠，取左肺组织。测定肺组织湿/干质量比（W/D）。采用苏木精-伊红（HE）染色和电子显微镜观察肺组织病理变化并评估损伤程度。在电子显微镜下观察肺组织超微结构变化。采用酶联免疫吸附测定（ELISA）法检测肺组织中肿瘤坏死因子-α（TNF-α）、白细胞介素（IL-1β、IL-6）水平。采用蛋白质免疫印迹法检测磷酸化蛋白激酶B（p-AKT）的表达。（2）第二部分实验：用磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/AKT）通路抑制剂LY294002干预，进一步探讨地塞米松减轻LIRI所致肺损伤的机制。将24只SD大鼠按随机数字法分为四组（n = 6）：N组、LIRI组、DEX组和地塞米松+LY294002+LIRI组（LY组）。除LY组外，其余各组均按第一部分实验进行造模和干预。LY组在注射地塞米松后注射LY294002 0.3 mg/kg。采用免疫组织化学法检测M1巨噬细胞极化标志物CD11c、CD16以及M2巨噬细胞极化标志物CD206、精氨酸酶1（Arg1）的表达。
（1）第一部分实验：与N组比较，LIRI组肺组织形态和超微结构改变明显，肺损伤评分、肺W/D比值及TNF-α、IL-1β、IL-6水平显著升高，p-AKT表达显著降低。与LIRI组比较，DEX组肺组织形态和超微结构改变明显改善，肺损伤评分降低（5.
