肢体缺血再灌注后肺损伤小鼠肺组织AT1R和Mas受体蛋白表达变化

摘要

目的: 通过观察小鼠肢体缺血再灌注(LIR)后不同时点肺组织血管紧张素Ⅱ 1型受体(AT1R)和Mas受体蛋白表达与肺损伤的变化,探讨局部组织AT1R和Mas受体蛋白表达失衡在LIR急性肺损伤(ALI)中的作用.方法:42只8周龄雄性ICR小鼠随机分为7组,每组6只,其中1组作为对照组,其余6组为再灌注0.5 h、1 h、2 h、4 h、6 h和12 h模型组.模型组小鼠用橡皮圈结扎双后肢根部,缺血2 h后剪断橡皮圈,分别于再灌注后不同时点眼球取血处死小鼠.取肺组织计算脏器系数和湿/干重比;肺泡灌洗液细胞计数和蛋白浓度检测;肺组织病理切片常规HE染色观察肺组织形态变化并进行病理损伤评分;Western blot检测肺组织AT1R和Mas受体蛋白的表达.结果:模型组小鼠肺脏器系数、湿/干重比、肺泡灌洗液细胞计数和蛋白浓度在LIR后显著升高.病理学结果显示,LIR后不同时点小鼠肺组织出现肺泡壁毛细血管扩张和充血、间质和肺泡水肿、血管壁和支气管壁炎症细胞浸润、肺泡间隔增厚、炎症细胞浸润及肺气肿等不同程度的损伤变化,且随着再灌注时间的延长,肺损伤评分逐渐升高.Western blot结果显示,AT1R蛋白在再灌注0.5 h时开始升高,1 h达到最高,之后随再灌注时间的延长,AT1R表达逐渐降低;Mas受体蛋白随再灌注时间延长逐渐升高.结论:LIR引起急性肺损伤,并随再灌注时间的延长损伤逐渐加重;AT1R和Mas受体蛋白表达的变化可能与小鼠LIR后急性肺损伤有关.%AIM: To explore the role of imbalance of local renin-angiotensin system (RAS) in lung injury by observing the changes of angiotensin Ⅱ type 1 receptor (AT1R) and Mas receptor protein expression in the lung and the degree of lung injury subject to limb ischemia-reperfusion (LIR) in the mice.METHODS: Male ICR mice (n=42, 8 weeks old) were randomly assigned into 7 groups (6 in each group), including control group and 6 model groups with LIR of 0.5 h, 1 h, 2 h, 4 h, 6 h and 12 h reperfusion.Tourniquets were used to block the blood flow of the hind limbs of the ICR mice and were released after 2 h ischemia to initiate reperfusion.The mice were sacrificed by eyeball blood withdrawal at different time points after reperfusion.The organ coefficient and wet/dry weight ratio (W/D) of the lung tissue were calculated.Bronchoalveolar lavage fluid (BALF) was taken for cell counting and protein concentration measurement.The histopathological changes of the lung tissues was observed, and the pathological score was calculated.The protein expression of AT1R and Mas receptor in the lung tissues was determined by Western blot.RESULTS: The organ coefficient, W/D of lung tissue, and cell number and protein concentration in BALF of model groups were significantly higher than those in control group after LIR.The pathological changes were found in the lung tissue of model mice, including alveolar capillary dilation and congestion, edema, inflammatory cell infiltration in peripheral vascular, alveolar and bronchial walls, alveolar septal thickening and inflammatory cell infiltration.The lung injury score was elevated gradually along with the extension of reperfusion time.The protein expression of AT1R began to increase at reperfusion time points of 0.5 h and 1 h.With the extension of reperfusion time, the protein expression of AT1R decreased gradually.Conversely, the protein expression of Mas receptor increased gradually with prolonged reperfusion.CONCLUSION: LIR induces acute lung injury gradually.The imbalance of AT1R and Mas receptor expression may be involved in the damage process.