【摘要】 目的 探讨银杏叶提取物（EGB761）对小鼠脓毒症急性肺损伤（ALI）的作用及其机制。方法 选择50只雄性昆明小鼠，按随机数字表法分为假手术组（术后腹腔注射等量生理盐水）、模型组（术后腹腔注射等量生理盐水）、低剂量EGB761组（术后立即腹腔注射20 mg/kg EGB761）、高剂量EGB761组（术后立即腹腔注射50 mg/kg EGB761）和高剂量EGB761+核因子E2相关因子2（Nrf2）抑制剂ML385组（于制模前1 h腹腔注射ML385 30 mg/kg，术后立即腹腔注射50 mg/kg EGB761），每组10只。采用盲肠结扎穿孔术（CLP）复制脓毒症ALI模型，假手术组只进行开关腹手术。术后12 h取血清及肺组织，苏木素-伊红（HE）染色后光镜下观察肺组织病理学变化，并进行肺损伤评分；采用酶联免疫吸附试验（ELISA）检测各组小鼠血清中白细胞介素（IL-6、IL-1β）和肿瘤坏死因子-α（TNF-α）水平；采用蛋白质免疫印迹试验（Western blotting）和实时荧光定量反转录-聚合酶链反应（qRT-PCR）检测各组小鼠肺组织中谷胱甘肽过氧化物酶4（GPX4）、Nrf2、溶质载体家族7成员11（SLC7A11）的蛋白表达和Nrf2、GPX4 mRNA表达水平；采用硫代巴比妥酸法检测各组小鼠肺组织中丙二醛（MDA）含量；采用黄嘌呤氧化酶法检测各组小鼠肺组织中超氧化物歧化酶（SOD）活性。结果 与假手术组比较，模型组肺组织中有大量炎症细胞浸润、肺泡结构破坏，肺损伤评分明显升高（分：3.71±0.29比0.33±0.10），血清促炎因子IL-1β、IL-6、TNF-α和肺组织中氧化指标MDA含量均明显升高[IL-1β（ng/L）:0.410±0.002比0.351±0.007,IL-6（ng/L）:0.650±0.010比0.560±0.010,TNF-α（ng/L）:0.320±0.010比0.290±0.002,MDA（nmol/mg）:2.49±0.31比1.77±0.02，均P<0.05]，而肺组织中抗氧化指标SOD活性明显下降（U/mg:20.20±4.61比61.47±8.10,P<0.05）,Nrf2、GPX4、SLC7A11蛋白和GPX4、Nrf2 mRNA表达水平亦均明显下降[Nrf2蛋白表达（Nrf2/β-actin）:0.33±0.11比0.63±0.17,GPX4蛋白表达（GPX4/β-actin）:1.19±0.08比2.50±0.17,SLC7A11蛋白表达（SLC7A11/β-actin）:0.41±0.03比0.67±0.08;GPX4 mRNA(2^-ΔΔCt):0.32±0.00比1.02±0.21,Nrf2 mRNA(2^-ΔΔCt):0.44±0.04比1.01±0.20，均P<0.05]。与模型组比较，低剂量EGB761组和高剂量EGB761组肺损伤程度均有不同程度减轻，高剂量EGB761组肺损伤评分明显降低（分：0.96±0.40比3.71±0.29,P<0.05），肺组织中MDA含量和血清促炎因子IL-1β、IL-6、TNF-α含量均明显降低[MDA（nmol/mg）:1.53±0.09比2.49±0.31,IL-1β（ng/L）:0.350±0.004比0.410±0.002,IL-6（ng/L）:0.570±0.020比0.650±0.010,TNF-α（ng/L）:0.280±0.003比0.320±0.010，均P<0.05]，SOD活性、Nrf2、GPX4、SLC7A11的蛋白表达和GPX4、Nrf2 mRNA表达水平均明显升高[SOD活性（U/mg）:63.78±8.74比20.20±4.61,Nrf2蛋白表达（Nrf2/β-actin）:0.71±0.06比0.33±0.11,GPX4蛋白表达（GPX4/β-actin）:2.03±0.10比1.19±0.08,SLC7A11蛋白表达（SLC7A11/β-actin）:0.65±0.07比0.41±0.03;GPX4 m RNA(2^-ΔΔCt):0.80±0.10比0.32±0.00,Nrf2 mRNA(2^-ΔΔCt):0.84±0.07比0.44±0.04，均P<0.05]。与高剂量EGB761组比较，用Nrf2抑制剂ML385干预后可明显逆转高剂量EGB761的上述作用。结论 银杏叶提取物EGB761可改善CLP诱导的小鼠脓毒症ALI，并可能是通过激活Nrf2上调SLC7A11抑制铁死亡从而发挥保护作用。更多还原

【Abstract】 Objective To investigate the effect and mechanism of Ginkgo biloba extract（EGB761） on acute lung injury（ALI） induced by sepsis in mice. Methods Fifty male Kunming mice were selected, they were randomly divided into sham operation group（intraperitoneal injection of equal amount of normal saline after operation）, model group（intraperitoneal injection of equal amount of normal saline after operation）, low-dose EGB761 group（intraperitoneal injection of 20 mg/kg EGB761 immediately after operation） and high-dose EGB761 group（intraperitoneal injection of 50 mg/kg EGB761 immediately after operation） and high-dose EGB761+nuclear factor E2-related factor 2（Nrf2） inhibitor ML385 group（intraperitoneal injection of ML385 30 mg/kg 1 hour before modeling and 50 mg/kg EGB761 immediately after operation）, with 10 mice in each group. ALI model of sepsis was established by cecal ligation and puncture（CLP）, and the sham operation group only underwent open-closed abdominal surgery. Serum and lung tissue were taken at 12 hours after operation, and the pathological changes of lung tissue were observed under light microscope after hematoxylin-eosin（HE） staining, and the lung injury score was made. Enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of interleukin（IL-6, IL-1β） and tumor necrosis factor-α（TNF-α） in serum of mice in each group. Western blotting test and real-time fluorescence quantitative reverse transcription-polymerase chain reaction（qRT-PCR） were used to detect the protein expression levels of glutathione peroxidase 4（GPX4）, Nrf2, member 11 of solute carrier family 7（SLC7A11） and the mRNA expression levels of Nrf2 and GPX4 mRNA the lung tissues of mice in each group. The content of malondialdehyde（MDA） in the lung tissues of mice in each group was detected by the thiobarbituric acid method. The activity of superoxide dismutase（SOD） in the lung tissues of mice in each group was detected by xanthine oxidase method. Results Compared with the sham operation group, in the model group, a large number of inflammatory cells infiltrated in the lung tissue, the alveolar structure was destroyed, the lung injury score was significantly increased（3.71±0.29 vs. 0.33±0.10）, and serum pro-inflammatory factors IL-1β, IL-6, TNF-α and the content of oxidation index MDA in lung tissue all increased significantly [IL-1β（ng/L）: 0.410±0.002 vs. 0.351±0.007, IL-6（ng/L）: 0.650±0.010 vs. 0.560±0.010, TNF-α（ng/L）: 0.320±0.010 vs. 0.290±0.002, MDA（nmol/mg）: 2.49±0.31 vs. 1.77±0.02, all P < 0.05], while SOD activity in lung tissue decreased significantly（U/mg: 20.20±4.61 vs. 61.47±8.10, P < 0.05）, the expression levels of Nrf2, GPX4, SLC7A11 protein, GPX4, and Nrf2 mRNA all decreased [NRF2 protein expression（Nrf2/β-actin）: 0.33±0.11 vs. 0.63±0.17, GPX4 protein expression（GPX4/β-actin）: 1.19±0.08 vs. 2.50±0.17, SLC7A11 protein expression（SLC7A11/β-actin）: 0.41±0.03 vs. 0.67±0.08; GPX4 mRNA(2^-ΔΔCt): 0.32±0.00 vs. 1.02±0.21, NRF2 mRNA(2^-ΔΔCt): 0.44±0.04 vs. 1.01±0.20, all P < 0.05]. Compared with the model group, the degree of lung injury in the low-dose EGB761 group and the high-dose EGB761 group was reduced to varying degrees, and the lung injury score in the high-dose EGB761 group was significantly reduced（0.96±0.40 vs. 3.71±0.29, P < 0.05）. The contents of MDA in lung tissue and serum proinflammatory factors IL-1β, IL-6 and TNF-α were significantly decreased [MDA（nmol/mg）: 1.53±0.09 vs. 2.49±0.31, IL-1β（ng/L）: 0.350±0.004 vs. 0.410±0.002, TNF-α（ng/L）: 0.280±0.003 vs. 0.320±0.010, all P < 0.05], SOD activity, proteins of Nrf2, GPX4 and SLC7A11, and expression levels of GPX4 and Nrf2 mRNA all increased significantly [SOD activity（U/mg）: 63.78±8.74 vs. 20.20±4.61, the expression of Nrf2 protein（NRF2/β-actin）: 0.71±0.06 vs. 0.33±0.11, the expression of GPX4 protein（GPX4/β-actin）: 2.03±0.10 vs. 1.19±0.08, and the expression of SLC7A11 protein（SLC7A11/β-actin） 0.65±0.07 vs. 0.41±0.03; GPX4 mRNA(2^-ΔΔCt): 0.80±0.10 vs. 0.32±0.00, NRF2 mRNA(2^-ΔΔCt): 0.84±0.07 vs. 0.44±0.04, all P < 0.05]. Compared with high-dose EGB761 group, Nrf2 inhibitor ML385 can obviously reverse the above-mentioned effects of high-dose EGB761 after intervention. Conclusion Ginkgo biloba extract EGB761 can improve the sepsis ALI induced by CLP in mice, and it may play a protective role by activating Nrf2 and up-regulating SLC7A11 to inhibit ferroptosis.更多还原