重组人骨形态发生蛋白对内毒素致大鼠急性肺损伤的保护作用

Protective effect of recombinant human bone morphogenetic protein on acute lung injury induced by endotoxin in rats

目的:探讨重组人骨形态发生蛋白2在内毒素致大鼠急性肺损伤时的作用及可能的机制。方法:实验于2005-07/2006-03在中国医科大学附属第一医院麻醉实验室完成。①Wistar大鼠60只随机数字法分成3组,每组20只。空白对照组:股静脉1.5h内输注生理盐水5mL;内毒素对照组:经股静脉1h内输注生理盐水3mL,然后再输注内毒素8mg/kg(溶于2mL生理盐水中),30min内输注完毕;重组人骨形态发生蛋白2处理组:骨形态发生蛋白210μg/kg溶于3mL生理盐水中,经股静脉1h内输注,然后输注内毒素(方法如上)。②输液结束后6h,取大鼠肺组织标本,苏木精-伊红染色后观察肺组织形态学的变化;检测肺组织湿/干质量比、肺水含量、肺通透指数等指标反映肺损伤程度;检测丙二醛、一氧化氮、诱导型一氧化氮合成酶、超氧化物歧化酶及髓过氧化物酶的活性来反映氧自由基的代谢;反转录聚合酶链反应、蛋白印迹杂交检测肺组织肿瘤坏死因子、核因子кB、细胞间黏附分子1mRNA与蛋白表达;Kaplan-Meier曲线分析4,6,8,10,12h生存率。结果:①内毒素对照组可见渗出增多,肺泡内充血,肺泡萎陷、断裂融合;重组人骨形态发生蛋白2处理组血管周围炎性细胞浸润,毛细血管扩张肺泡腔内出血减少,炎性细胞浸润较内毒素对照组减少,肺小动脉内膜损伤减轻。②与空白对照组比较,内毒素对照组肺组织湿/干质量比、肺通透指数、丙二醛、一氧化氮含量、诱导型一氧化氮合成酶、髓过氧化物酶活性增加(P<0.01),与内毒素对照组比较,重组人骨形态发生蛋白2处理组各指标值增加(P<0.01)。③与空白对照组、重组人骨形态发生蛋白2处理组比较,内毒素对照组肿瘤坏死因子α、核因子кB、细胞间黏附分子1的蛋白及mRNA表达显著增加(灰度值比为:0.11±0.03,0.26±0.09,0.49±0.13;吸光度值比为:0.17±0.05,0.37±0.09,1.22±0.18;P<0.01)。④在重组人骨形态发生蛋白2预处理组,注射内毒素后4,6,8,10,12h的生存率明显增加(P<0.01)。结论:重组人骨形态发生蛋白2通过抑制核因子кB、肿瘤坏死因子α、细胞间黏附分子1蛋白表达,增强超氧化物歧化酶活性,抑制诱导型一氧化氮合成酶、髓过氧化物酶活性,减少内毒素致急性肺损伤后肺部的炎性反应及氧化应激反应,从而减轻肺损伤程度。

AIM： To investigate the protective role and mechanism of recombinant human bone morphogenetic protein 2 （rhBMP2） on acute lung injury induced by endotoxin in rats. METHODS： The experiment was conducted from July 2005 to March 2006 at the Laboratory of anesthesiology, First Hospital Affiliated To China Medical University. （1） Sixty Wistar rats were randomly divided into 3 groups with 20 animals in each group. Rats in the control group were administrated with 5 mL of normal saline from the femoral vein. Rats in the endotoxin control group were administrated with endotoxin （8 mg/kg, which dissolved in 2 mL of normal saline） within 30 minutes after being administrated with 3 mL of normal saline from the femoral vein in hour. Rats in the rhBMP2 treatment group were infused with rhBMP2 （10 μg/kg） dissolved in 3 mL of normal saline within one hour from the femoral vein, and then was the infusion of endotoxin （in the method mentioned above）. （2） At 6 hours after the transfusion, the left lung removed from the rats was stained by HE to observe the morphological changes. The lung wet/dry weight ratio, and permeation index were determined to show the severity of lung injury. Meanwhile, the activities of malondialdehyde （MDA）, nitrogen monoxidum （NO）, induced nitric oxide synthetase （iNOS）; superoxide dismutase （SOD） and myeloperoxidase （MPO） were determined to show the metabolism of oxygen free radical. While the TNF-α, NF-κB and ICAM-1 gene expressions were measured by western-blot and RT-PCR. The survival ratios at the 4^th, 6^th, 8^th, 10^th and 12^th hours were analyzed by Kaplan-Meier curve. RESULTS： （1） Increased effusion could be seen in the endotoxin control group with congestion in respiratory atrium, which collapsed, broke and confluenced. There was inflammatory cell infiltrate in the periphery of vessels in the rhBMP2 treatment group, and the symptoms were attenuated than those in the endotoxin control group. （2） Compared with the blank control group, the W/D ratio, LPI, MDA, NO, iNOS and MPO were significantly higher than those in the endotoxin control group （P 〈 0.01）. Compared with the endotoxin control group, all indexes increased in the rhBMP2 treatment group （P 〈 0.01）. （3） In comparison with the blank control group and the rhBMP2 treatment group, the protein mRNA expressions of TNF-α, NF-κB and ICAM-1 were significantly increased in the endotoxin control group （gray scale value ratio： 0.11±0.03, 0.26±0.09, 0.49±0.13; the optical density value ratio： 0.17±0.05, 0.37±0.09, 1.22±0.18; P 〈 0.01）. （4） The survival rate dramatically increased at 4, 6, 8, 10 and 12 hours after injection of endotoxin in the rhBMP-2 treatment group （P〈 0.01）. CONCLUSION： The rhBMP2 can reduce the inflammatory reaction and oxidative stress in acute lung injury induced by endotoxin as well as relieve the severity of lung injury by down-regulating the TNF-α, NF-κB and ICAM-1 gene expressions and inhibiting the activity of iNOS and MPO.