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

Role and mechanism of ferroptosis in combined burn-blast injury with acute lung injury in rats

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。对实验组大鼠在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(PaO_(2))和动脉血二氧化碳分压(PaCO_(2)),称重肺组织并计算其湿干重比,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);实验组大鼠肺组织湿干重比、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PaO_(2)明显下降(t=8.85,P<0.05)而PaCO_(2)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,提示铁死亡参与了该疾病的病理生理过程。

ObjectiveTo investigates the role and mechanism of ferroptosis in combined burn-blast injury with acute lung injury in rats.MethodsThis study was an experimental study.Twenty-four 8-week-old male Sprague-Dawley rats were divided into control group and experimental group by random number table method,each containing 12 animals.The rats in experimental group were anesthetized and subjected to explosion treatment to create the model of combined burn-blast injury with acute lung injury,whereas the rats in control group underwent sham injury.At 24 hours post injury,the pathological morphology of lung tissue was observed by hematoxylin-eosin staining and immunohistochemical staining.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(PaO_(2))and arterial partial pressure of carbon dioxide(PaCO_(2))of abdominal aortic blood were measured by automatic animal blood gas analyzer.The lung tissue was weighed and the wet-dry weight ratio was calculated.The total protein concentration in BALF was measured by bicinchoninic acid assay.Lung injury was scored based on hematoxylin-eosin staining.The levels of oxidative stress factors,such as reactive oxygen species,malondialdehyde,superoxide dismutase(SOD),glutathione,and ferrous ion in lung tissue homogenate of rats were detected by related kits.The expression levels of ferroptosis-related molecule glutathione peroxidase 4(GPX4),lipid peroxidation-related molecule 4-hydroxynonenal(4-HNE),and oxidative DNA damage-related molecule 8-hydroxydeoxyguanosine(8-OHdG)in lung tissue were detected by immunofluorescence and immunohistochemistry methods.Mitochondrial morphology in lung tissue cells was observed under transmission electron microscopy.The sample number was all 6.ResultsAt 24 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.At 24 hours post injury,compared with those in control group,the levels of TNF-α,IL-1β,and IL-6 in BALF supernatant of rats in experimental group were significantly increased(with t values of 3.96,9.84,and 10.60,respectively,P<0.05);the wet-dry weight 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),PaO_(2) of abdominal aortic blood decreased significantly(t=8.85,P<0.05)while PaCO_(2) did not change significantly(P>0.05);the levels of SOD and glutathione in the lung tissue homogenate of rats in experimental group were significantly decreased(with t values of 4.36 and 8.56,respectively,P<0.05),while the 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).At 24 hours post injury,immunofluorescence staining and immunohistochemical staining showed that the expression levels 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 levels of 4-HNE in lung tissue of rats in experimental group were 5.93±1.05 and 2.21±0.23,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 levels of 8-OHdG in lung tissue of rats in experimental group were 2.08±0.40 and 1.61±0.29,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).At 24 hours post injury,compared with that in control group,the density of mitochondrial double-layer membrane in the lung tissue cells of rats in experimental group increased,the outer membrane ruptured,and the crista decreased.ConclusionsIn rats with combined burn-blast injury with acute lung injury,there is oxidative DNA damage in lung tissue cells,the imbalance of antioxidant system in lung tissue,and a decrease in the expression of GPX4,the key molecule against ferroptosis,suggesting that ferroptosis is involved in the pathophysiological process of this disease.