BAY11－7082对严重烧伤大鼠急性肺损伤及早期肺炎症反应的干预作用

李虎山; 杨薛康; 郝振明; 雷晋

doi:10.3760/cma.j.issn.1009-2587.2018.02.006

BAY11－7082对严重烧伤大鼠急性肺损伤及早期肺炎症反应的干预作用

doi: 10.3760/cma.j.issn.1009-2587.2018.02.006

1. 030009 太原,山西医科大学第六医院烧伤科,山西省烧伤救治中心;
2. 空军军医大学西京医院全军烧伤中心,烧伤与皮肤外科

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出版历程
- 收稿日期: 2017-05-28
- 网络出版日期: 2021-10-28
- 刊出日期: 2018-02-20

Interventional effects of BAY11-7082 on lung inflammatory response at the early stage and acute lung injury of rats with severe burns

1. Burn Care Center of Shanxi Province, Department of Burns, the Sixth Hospital of Shanxi Medical University, Taiyuan 030009, China

摘要

摘要: 目的探讨BAY11－7082对严重烧伤大鼠急性肺损伤及早期肺炎症反应的干预作用。方法 (1)实验1。取12只SD大鼠，按随机数字表法分为正常对照组3只、烧伤组9只。正常对照组大鼠不行特殊处理；烧伤组大鼠背部造成30%体表总面积Ⅲ度烧伤，伤后即刻腹腔注射生理盐水50 mL/kg。烧伤组于伤后12、24、48 h分别取3只大鼠采集腹主动脉血并取肺组织，酶联免疫吸附测定法检测血清中白细胞介素1β(IL－1β)、IL－18含量，实时荧光定量反转录－聚合酶链反应(RT－PCR)法检测肺组织中IL－1β、IL－18 mRNA表达。正常对照组大鼠同前采样检测。(2)实验2。取18只SD大鼠，按随机数字表法分为正常对照组、单纯烧伤组、BAY11－7082干预组，每组6只。正常对照组大鼠不行特殊处理；单纯烧伤组和BAY11－7082干预组大鼠同实验1致伤，伤后即刻分别腹腔注射生理盐水、终质量浓度为8 mg/mL BAY11－7082溶液50 mL/kg。于实验1结果得出的最佳观察时间点收集烧伤各组大鼠肺组织及肺泡灌洗液，苏木素－伊红染色观察肺组织形态并进行肺组织病理损害评分，酶标仪检测肺组织中髓过氧化物酶(MPO)含量及肺泡灌洗液中总蛋白含量，蛋白质印迹法检测肺组织中半胱氨酸天冬氨酸蛋白酶1和核苷酸结合寡聚化结构域样受体3(NLRP3)的蛋白表达，实时荧光定量RT－PCR法检测肺组织中IL－1β、IL－18、NLRP3、半胱氨酸天冬氨酸蛋白酶1 mRNA表达。正常对照组大鼠同前采样检测。对数据行单因素方差分析、LSD－t检验。结果 (1)烧伤组大鼠伤后12、24、48 h血清中IL－1β和IL－18含量显著高于正常对照组(t＝10.55、22.05、12.47、10.60、15.22、11.94，P<0.01)。烧伤组大鼠伤后12、24、48 h肺组织中IL－1β和IL－18 mRNA表达量明显高于正常对照组(t＝3.62、7.19、5.28、3.20、12.62、7.31，P<0.05或P<0.01)。伤后24 h为后续实验最佳观察时间点。(2)伤后24 h，与单纯烧伤组比较，BAY11－7082干预组大鼠炎性细胞浸润及肺泡内红细胞渗出明显减少，肺间质水肿明显消退。单纯烧伤组大鼠肺组织病理损害评分为(9.00±1.00)分，显著高于正常对照组的(1.10±0.26)分(t＝13.23，P<0.01)。BAY11－7082干预组大鼠肺组织病理损害评分为(4.93±0.70)分，虽仍高于正常对照组(t＝8.84，P<0.01)，但较单纯烧伤组明显降低(t＝5.76，P<0.01)。伤后24 h，单纯烧伤组大鼠肺组织中MPO含量[(1.83±0.15)U/mg]及肺泡灌洗液中总蛋白含量[(1.39±0.20)mg/mL]均显著高于正常对照组[(0.51±0.10)U/mg、(0.44±0.05)mg/mL，t＝12.50、7.86，P<0.01]，BAY11－7082干预组大鼠肺组织中MPO含量[(0.91±0.12)U/mg]及肺泡灌洗液中总蛋白含量[(0.60±0.10)mg/mL]显著低于单纯烧伤组(t＝8.36、6.06，P<0.01)。伤后24 h，单纯烧伤组大鼠肺组织中NLRP3和半胱氨酸天冬氨酸蛋白酶1蛋白表达量分别为3.10±0.09、2.99±0.30，显著高于正常对照组的1.00、1.00(t＝9.06、11.28，P<0.01)；BAY11－7082干预组大鼠肺组织中NLRP3和半胱氨酸天冬氨酸蛋白酶1蛋白表达量分别为1.13±0.08、1.81±0.11，显著低于单纯烧伤组(t＝7.24、3.91，P<0.05或P<0.01)。伤后24 h，单纯烧伤组大鼠肺组织中IL－1β、IL－18、NLRP3和半胱氨酸天冬氨酸蛋白酶1的mRNA表达量分别为5.0±0.4、3.32±0.21、3.54±0.42、6.3±1.0，明显高于正常对照组的1.0、1.00、1.00、1.0(t＝13.97、14.14、11.78、7.13，P<0.01)；BAY11－7082干预组大鼠肺组织中IL－1β、IL－18、NLRP3和半胱氨酸天冬氨酸蛋白酶1的mRNA表达量(2.6±0.5、2.00±0.28、1.39±0.21、2.5±0.5)显著低于单纯烧伤组(t＝7.11、5.80、9.99、4.65，P<0.05或P<0.01)。结论严重烧伤大鼠急性肺损伤早期使用BAY11－7082可通过抑制NLRP3，减少半胱氨酸天冬氨酸蛋白酶1的表达，显著降低IL－1β和IL－18的水平，同时减少肺组织中MPO含量及肺泡灌洗液中总蛋白含量，从而减轻肺炎症反应，减轻肺损伤。
- 烧伤 /
- 急性肺损伤 /
- 炎症 /
- 半胱氨酸天冬氨酸蛋白酶1 /
- 白细胞介素1β /
- 白细胞介素18 /
- 核苷酸结合寡聚化结构域样受体3
Abstract: Objective To investigate the interventional effects of BAY11-7082 on lung inflammatory response at the early stage and acute lung injury of rats with severe burns. Methods (1) Experiment 1. Twelve Sprague-Dawley (SD) rats were divided into control (C) group and burn (B) group according to the random number table, with 3 rats in group C and 9 rats in group B. Rats in group C did not receive any special treatment. Rats in group B were inflicted with 30% total body surface area full-thickness burn on the back. Immediately after injury, rats in group B were intraperitoneally injected with normal saline in the dosage of 50 mL/kg. Abdominal aorta blood and lung tissue samples were collected from three rats in group B at post injury hour (PIH) 12, 24, and 48, respectively. The interleukin-1β (IL-1β) and the IL-18 content of serum were determined with enzyme-linked immunosorbent assay. The mRNA expressions of IL-1β and IL-18 in lung tissue were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR). Sample collection and determination in rats of group C were performed as above. (2) Experiment 2. Eighteen SD rats were divided into control (C) group, simple burn (SB) group, and BAY11-7082 intervention (BI) group according to the random number table, with 6 rats in each group. Rats in group C did not receive any special treatment. Rats in groups SB and BI were inflicted with injury as in experiment 1. Immediately after injury, rats in group SB were intraperitoneally injected with normal saline in the dosage of 50 mL/kg, and those in group BI with 8 mg/mL (final mass concentration) BAY11-7082 solution in the dosage of 50 mL/kg. Lung tissue and bronchoalveolar lavage fluid (BALF) of rats with burns were collected at the optimal observation time point concluded from experiment 1. The morphology of lung tissue was observed with hematoxylin-eosin staining, and the pathological damage of lung tissue was graded. The myeloperoxidase (MPO) content of lung tissue and the total protein content of BALF were detected by microplate reader. The protein expressions of nucleotide-binding oligomerization domain-like receptor-3 (NLRP3) and cysteine-aspartic proteases 1 (caspase-1) in lung tissue were determined with Western-blotting. The mRNA expressions of IL-1β, IL-18, NLRP3, and caspase-1 in lung tissue were determined with real-time fluorescence quantitative RT-PCR. Sample collection and determination in rats of group C were performed as above. Data were processed with one-way analysis of variance and LSD-t test. Results (1) The IL-1β and IL-18 content of serum in rats of group B at PIH 12, 24, and 48 were significantly higher than those of group C (t=10.55, 22.05, 12.47, 10.60, 15.22, 11.94, P<0.01). The mRNA expressions of IL-1β and IL-18 in rats of group B at PIH 12, 24, and 48 were significantly higher than those of group C (t=3.62, 7.19, 5.28, 3.20, 12.62, 7.31, P<0.05 or P<0.01). PIH 24 was the optimal observation time point for the following experiment. (2) At PIH 24, compared with those in group SB, the inflammatory cell infiltration and erythrocyte exudates of alveolar in group BI were obviously reduced, and the pulmonary interstitial edema obviously subsided. The pathological damage score of lung tissue in rats of group SB was (9.00±1.00) points, significantly higher than (1.10±0.26) points of group C (t=13.23, P<0.01). The pathological damage score of lung tissue in rats of group BI was (4.93±0.70) points, which was significantly lower than that of group SB (t=5.76, P<0.01) but still significantly higher than that of group C (t=8.84, P<0.01). At PIH 24, the MPO content of lung tissue and the total protein content of BALF in rats of group SB were (1.83±0.15) U/mg and (1.39±0.20) mg/mL, respectively, significantly higher than (0.51±0.10) U/mg and (0.44±0.05) mg/mL of group C (t=12.50, 7.86, P<0.01). The MPO content of lung tissue and the total protein content of BALF in rats of group BI were (0.91±0.12) U/mg and (0.60±0.10) mg/mL, respectively, significantly lower than those of group SB (t=8.36, 6.06, P<0.01). At PIH 24, the protein expressions of NLRP3 and caspase-1 in lung tissue of rats of group SB were 3.10±0.09 and 2.99±0.30, respectively, significantly higher than 1.00 and 1.00 of group C (t=9.06, 11.28, P<0.01). The protein expressions of NLRP3 and caspase-1 in lung tissue of rats of group BI were 1.13±0.08 and 1.81±0.11, respectively, significantly lower than those of group SB (t=7.24, 3.91, P<0.05 or P<0.01). At PIH 24, the mRNA expressions of IL-1β, IL-18, NLRP3, and caspase-1 in lung tissue of rats in group SB were 5.0±0.4, 3.32±0.21, 3.54±0.42, and 6.3±1.0, respectively, significantly higher than 1.0, 1.00, 1.00, and 1.0 of group C (t=13.97, 14.14, 11.78, 7.13, P<0.01). The mRNA expressions of IL-1β, IL-18, NLRP3, and caspase-1 in lung tissue of rats in group BI were 2.6±0.5, 2.00±0.28, 1.39±0.21, and 2.5±0.5, respectively, significantly lower than those of group SB (t=7.11, 5.80, 9.99, 4.65, P<0.05 or P<0.01). Conclusions Applying BAY11-7082 at the early stage of acute lung injury of rats with severe burn can reduce the expression of caspase-1, decrease the levels of IL-1β and IL-18, and decrease the MPO content of lung tissue and the total protein content of BALF through inhibiting NLRP3, thus alleviating the lung inflammatory response and lung injury.
- Burns /
- Acute lung injury /
- Inflammation /
- Caspase 1 /
- Interleukin-1beta /
- Interleukin-18 /
- Nucleotide-binding oligomerization domain-like receptor-3