Role and mechanism of ferroptosis in acute lung injury associated with combined burn-blast trauma in rats
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摘要:
目的 探讨铁死亡在大鼠烧冲复合伤合并急性肺损伤中的作用及其机制。 方法 该研究为实验研究。取24只8周龄雄性SD大鼠,按照随机数字表法分为对照组和实验组,每组12只,实验组大鼠麻醉后进行爆炸处理以制作烧冲复合伤合并急性肺损伤模型,对照组大鼠行模拟致假伤处理。伤后24 h,采用苏木精-伊红染色和免疫组织化学染色观测肺组织的病理形态,采用酶联免疫吸附测定法检测支气管肺泡灌洗液（BALF）的上清液中肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）及IL-6的水平,采用全自动动物血气分析仪检测腹主动脉血的动脉血氧分压（PaO2）和动脉血二氧化碳分压（PaCO2）,称重肺组织并计算其干湿比重,采用二喹啉甲酸法测定BALF中的总蛋白浓度,基于苏木精-伊红染色对肺损伤情况进行评分,采用相关试剂盒检测大鼠肺组织匀浆液中氧化应激因子活性氧、丙二醛、超氧化物歧化酶（SOD）、谷胱甘肽、亚铁离子水平,采用免疫荧光法和免疫组织化学法检测肺组织中铁死亡相关分子谷胱甘肽过氧化物酶4（GPX4）、脂质过氧化相关分子4-羟基壬烯醛（4-HNE）及DNA氧化损伤相关分子8-羟基脱氧鸟苷（8-OHdG）的表达量,采用透射电子显微镜观察肺组织细胞中线粒体形态。样本数均为6。 结果 伤后24 h,对照组大鼠肺组织结构清晰完整,肺泡壁正常；实验组大鼠的肺组织水肿明显,肺泡壁变厚、结构不清晰。伤后24 h,与对照组相比,实验组大鼠BALF的上清液中TNF-α、IL-1β及IL-6水平均显著升高（t值分别为3.96、9.84、10.60,P<0.05）；实验组大鼠肺组织干湿比重、肺损伤评分以及BALF中总蛋白浓度均明显升高（t值分别为6.91、6.64、10.04,P<0.05）,腹主动脉血的PaO2明显下降（t=8.85,P<0.05）而PaCO2未见明显变化（P>0.05）；实验组大鼠的肺组织匀浆液中SOD及谷胱甘肽水平均明显降低（t值分别为4.36和8.56,P<0.05）,活性氧、丙二醛和亚铁离子水平均明显升高（t值分别为11.55、9.78、14.77,P<0.05）。伤后24 h,免疫荧光染色和免疫组织化学染色显示,实验组大鼠肺组织中GPX4的表达量分别为0.245±0.024、0.786±0.240,明显低于对照组的1.000±0.305、1.000±0.200（t值分别为6.05和2.60,P<0.05）；实验组大鼠大鼠肺组织中4-HNE的表达量分别为5.93±1.05、2.21±0.23,均明显高于对照组的1.00±0.29、1.00±0.23（t值分别为11.13和9.16,P<0.05）；实验组大鼠大鼠肺组织中8-OHdG的表达量分别为2.08±0.40、1.61±0.29,均明显高于对照组的1.00±0.40、1.00±0.26（t值分别为4.72和3.87,P<0.05）。伤后24 h,与对照组相比,实验组大鼠肺组织细胞中线粒体的双层膜密度增加、外膜破裂、嵴减少。 结论 在烧冲复合伤合并急性肺损伤大鼠中,肺组织细胞存在DNA氧化损伤,肺组织中抗氧化体系失衡、抗铁死亡的关键分子GPX4表达下降,提示铁死亡参与了其病理生理过程。 Abstract:Objective To investigates the role and mechanism of ferroptosis in acute lung injury associated with combined burn-blast trauma in rats. Methods This was an experimental study. Twenty-four 8-week-old male Sprague-Dawley rats were randomly divided into control group and experimental group, each containing 12 animals. The rats in experimental group were anesthetized and treated with explosion to make the model of acute lung injury associated with combined burn-blast trauma, whereas the rats in control group underwent sham injury. Twenty-four hours post-injury, the pathological morphology of lung tissue was observed by hematoxylin-eosin staining and immunohistochemical staining methods. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in the supernatant of bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay. The arterial partial pressure of oxygen (PaO2) and arterial partial pressure of carbon dioxide (PaCO2) of abdominal aortic blood were measured by automatic animal blood gas analyzer. The lung tissue was weighed and the dry-wet ratio was calculated. The total protein concentration in BALF was measured by bicinchoninic acid protein concentration determination kit. The levels of oxidative stress factors, such as reactive oxygen species, malondialdehyde, superoxide dismutase (SOD), glutathione, and ferrous ion in lung tissue homogenates of rats were detected by related kits. The expression of ferroptosis related molecule glutathione peroxidase 4 (GPX4), lipid peroxides related molecule 4-hydroxynonenal (4-HNE) and DNA oxidative damage related molecule 8-hydroxydeoxyguanosine (8-OHdG) in lung tissue were detected by immunofluorescence and immunohistochemistry methods. Mitochondrial morphology was observed under transmission electron microscopy. Results Twenty-four hours post-injury, the lung tissue structure of rats in control group was clear and complete, and the alveolar wall was normal; in experimental group, the lung tissue edema of rats was obvious, the alveolar wall became thicker, and the structure was not clear. Twenty-four hours post-injury, compared with those in control group, the levels of TNF-α, IL-1β, and IL-6 in BALF supernatant of experimental group were significantly increased (with t values of 3.96, 9.84, and 10.60, respectively, P<0.05); the dry-wet ratio of lung tissue, lung injury score, and total protein concentration in BALF of rats in experimental group were significantly increased (with t values of 6.91, 6.64, and 10.04, respectively, P<0.05), PaO2 of abdominal aortic blood decreased significantly (t=8.85, P<0.05) while PaCO2 did not change significantly (t=0.20, P>0.05); the levels of SOD and glutathione in the lung homogenate of rats in experimental group were significantly decreased (with t values of 4.36 and 8.56, respectively, P<0.05), while the average levels of reactive oxygen species, malondialdehyde, and ferrous ion were significantly increased (with t values of 11.55, 9.78, and 14.77, respectively, P<0.05). Twenty-four hours post-injury, immunofluorescence staining and immunohistochemical staining showed that the expression of GPX4 in lung tissue of rats in experimental group were 0.245±0.024 and 0.786±0.240, respectively, which were significantly lower than 1.000±0.305 and 1.000±0.200 in control group (with t values of 6.05 and 2.60, respectively, P<0.05). The expression values of 4-HNE in lung tissue of rats in experimental group were 5.93±1.05 and 2.21±0.23 by immunofluorescence and immunohistochemical staining, respectively, which were significantly higher than 1.00±0.29 and 1.00±0.23 in control group (with t values of 11.13 and 9.16, respectively, P<0.05). The expression values of 8-OHdG in lung tissues of rats in experimental group were 2.08±0.40 and 1.61±0.29 by immunofluorescence and immunohistochemical staining, respectively, which were significantly higher than 1.00±0.40 and 1.00±0.26 in control group (with t values of 4.72 and 3.87, respectively, P<0.05). Twenty-four hours post-injury, compared with that in control group, the density of the double membrane of mitochondria in the lung tissue cells of rats in experimental group increased, the outer membrane ruptured, and the crista decreased Conclusions In rats with acute lung injury associated with combined burn-blast trauma in rats, there is oxidative DNA damage in lung tissue cells, the imbalance of antioxidant system in lung tissue, and the expression of GPX4, the key molecule against ferroptosis, is decreased, suggesting that ferroptosis is involved in the pathophysiological process. -
Key words:
- Burn /
- Acute Lung Injury /
- Oxidative Stress /
- Combined Burn and Blast Injury /
- Inhalation Injury /
- Ferroptosis
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参考文献
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图 1 烧冲复合伤合并急性肺损伤大鼠的造模设备及造模后表现。1A.激波管气体爆炸生物杀伤实验系统,由空气瓶、负压真空机、激波管以及多通道数据采集系统等组成；1B.爆炸后,激波管中充满大量烟雾,大鼠全身皮毛烧焦、口鼻处胡须烧断伴肿胀
图 2 2种方法检测的2组大鼠伤后24 h肺组织病理情况。2A、2B.分别为对照组和实验组大鼠肺组织,图2A中肺组织结构完整、肺泡壁无水肿,图2B中肺组织水肿明显,肺泡间隔变厚、结构不清晰、伴出血和炎症细胞浸润 苏木精-伊红×20；2C、2D.分别为对照组和实验组大鼠肺组织中髓过氧化物酶（标记炎症细胞）的表达情况,图2C中肺组织结构清晰,少量炎症细胞（阳性染色为棕色）浸润,图2D中浸润肺组织的炎症细胞较图2C增加 辣根过氧化物酶-二氨基联苯胺×20。
注：对照组大鼠仅进行腹腔注射麻醉处理,实验组大鼠在腹腔注射麻醉后进行爆炸处理以制作烧冲复合伤合并急性肺损伤模型
图 3 2组大鼠伤后24 h肺组织中铁死亡与脂质过氧化及DNA氧化损伤相关分子的表达情况。3A、3B、3C与3D、3E、3F.分别为对照组与实验组GPX4、4-HNE、8-OHdG的表达情况,图3D的绿色荧光强度明显弱于图3A,图3E、3F的绿色荧光强度分别明显强于图3B、3C 异硫氰酸荧光素-4',6-二脒基-2-苯基吲哚；3G、3H、3I与3J、3K、3L.分别为对照组与实验组GPX4、4-HNE、8-OHdG的表达情况,图3J的阳性表达明显低于图3G,图3K、3L的阳性表达分别明显高于图3H、3I 辣根过氧化物酶-二氨基联苯胺
注：对照组大鼠仅进行腹腔注射麻醉处理,实验组大鼠在腹腔注射麻醉后进行爆炸处理以制作烧冲复合伤合并急性肺损伤模型；GPX4为谷胱甘肽过氧化物酶4（反映铁死亡相关情况）,4-HNE为4-羟基壬烯醛（反映脂质过氧化相关情况）,8-OHdG为8-羟基脱氧鸟苷（反映DNA氧化损伤相关情况）；图3A~3F中目标分子阳性染色为绿色,细胞核阳性染色为蓝色;图3G~3L目标分子阳性染色为棕色,细胞核阳性染色为蓝色
图 4 2组大鼠伤后24 h肺组织中细胞线粒体形态 透射电子显微镜×20 000。4A.对照组大鼠肺组织细胞线粒体形态,线粒体的外膜完整、嵴结构清楚；4B.实验组大鼠肺组织线粒体形态,可见线粒体双层膜的密度增加、外膜破裂、嵴减少
注：对照组大鼠仅进行腹腔注射麻醉处理,实验组大鼠在腹腔注射麻醉后进行爆炸处理以制作烧冲复合伤合并急性肺损伤模型；红色箭头指示线粒体
Table 1. 2组大鼠伤后24 h支气管肺泡灌洗液的上清液中炎症因子水平比较（ng/L,
组别 样本数 TNF-α IL-1β IL-6 对照组 6 193±30 86±12 92±10 实验组 6 293±54 151±11 178±17 t值 3.96 9.84 10.60 P值 <0.001 <0.001 <0.001 注：实验组大鼠在腹腔注射麻醉后进行爆炸处理以制作烧冲复合伤合并急性肺损伤模型,对照组大鼠行模拟致假伤处理；TNF-α为肿瘤坏死因子α,IL为白细胞介素 
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Table 2. 2组大鼠伤后24 h肺组织损伤情况比较（
组别 样本数 干湿比重 肺损伤评分（分） BALF中总蛋白浓度（μg/mL） PaO2（mmHg） PaCO2（mmHg） 对照组 6 3.2±1.0 3.2±1.2 245±56 98±8 42±4 实验组 6 9.1±1.9 8.3±1.5 761±112 60±7 43±7 t值 6.91 6.64 10.04 8.85 0.20 P值 <0.001 <0.001 <0.001 <0.001 0.842 注：对照组大鼠仅进行腹腔注射麻醉处理,实验组大鼠在腹腔注射麻醉后进行爆炸处理以制作烧冲复合伤合并急性肺损伤模型；BALF为支气管肺泡灌洗液,PaO2为动脉血氧分压,PaCO2为动脉血二氧化碳分压；1 mmHg=0.133 kPa
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Table 3. 2组大鼠伤后24 h肺组织匀浆液中氧化应激因子水平比较（
组别 样本数 SOD（U/mg） 谷胱甘肽（μmol/g） 丙二醛（mmol/mg） 活性氧（×103 U/L） 亚铁离子（μmol/g） 对照组 6 6.9±1.1 9.8±1.2 3.9±0.8 23±6 7.5±1.4 实验组 6 4.5±0.8 4.2±1.0 9.6±1.2 85±12 19±1.4 t值 4.36 8.56 9.78 11.55 14.77 P值 0.001 <0.001 <0.001 <0.001 <0.001 注：对照组大鼠仅进行腹腔注射麻醉处理,实验组大鼠在腹腔注射麻醉后进行爆炸处理以制作烧冲复合伤合并急性肺损伤模型；SOD为超氧化物歧化酶
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Table 4. 2种方法检测的2组大鼠伤后24 h肺组织中铁死亡与脂质过氧化及DNA氧化损伤相关分子的表达比较（
组别 样本数 免疫荧光法 免疫组织化学法 GPX4 4-HNE 8-OHdG GPX4 4-HNE 8-OHdG 对照组 6 1.000±0.305 1.00±0.29 1.00±0.40 1.000±0.200 1.00±0.23 1.00±0.26 实验组 6 0.245±0.024 5.93±1.05 2.08±0.40 0.786±0.240 2.21±0.23 1.61±0.29 t值 6.05 11.13 4.72 2.60 9.16 3.87 P值 <0.001 <0.001 0.003 0.026 <0.001 0.003 注：对照组大鼠仅进行腹腔注射麻醉处理,实验组大鼠在腹腔注射麻醉后进行爆炸处理以制作烧冲复合伤合并急性肺损伤模型；GPX4为谷胱甘肽过氧化物酶4,4-HNE为4-羟基壬烯醛,8-OHdG为8-羟基脱氧鸟苷
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