过氧化还原蛋白6对细菌脂多糖诱导的小鼠急性肺损伤氧化应激的保护作用

Roles of peroxiredoxin 6 in the regulation of oxidative stress to lipopolysaccharide-induced acute lung injury

目的探讨过氧化还原蛋白Peroxiredoxin（Prdx）6对细菌脂多糖（LPS）诱导的小鼠急性肺损伤氧化应激的作用及机制。方法应用PCR法对Prdx6基因敲除小鼠行基因型鉴定并应用免疫组织化学法测定Prdx6蛋白在肺脏的表达。将雄性Prdx6基因敲除型小鼠（18只）按随机数字表法分入Prdx6敲除型对照组（9只）、Prdx6敲除型LPS 24 h组（9只）；将雄性野生型C57BL/6J小鼠（18只）按随机数字表法分入野生型对照组（9只）、野生型LPS 24 h组（9只）。各LPS组小鼠气管滴注LPS（5 mg/kg）制备急性肺损伤模型，于给药后24 h分别行肺脏病理检测,BCA法测定BALF内蛋白浓度，比色法测定肺脏总超氧化物歧化酶（T-SOD）活力和过氧化氢、羰基化蛋白和总抗氧化能力（TAOC）,TBA法测定丙二醛的表达。结果Prdx6基因敲除小鼠肺组织免疫组织化学法未检测到Prdx6蛋白表达。两种属小鼠LPS组肺脏病理可见炎症细胞浸润、肺泡间隔增厚及肺泡内出血，Prdx6敲除型较野生型小鼠病理损伤更重。LPS刺激后，野生型LPS 24 h组BALF内的蛋白浓度为（441±54） mg/L，高于野生型对照组的（168±20） mg/L（t=-4.71,P＜0.01）；Prdx6敲除型LPS 24 h组为（770±66）mg/L，高于野生型LPS 24 h组（t=-3.69，P＜0.01）。野生型LPS 24 h组T-SOD为（16.0±1.2） U/mg，低于野生型对照组的（26.5±3.9） U/mg（t=-6.22,P＜0.01）；Prdx6敲除型LPS24 h组为（14.5±5.3）U/mg,与野生型LPS 24 h组差异无统计学意义（t=-0.56,P=0.60）。野生型LPS 24 h组肺脏过氧化氢和丙二醛[分别为（52.3±7.8）nmol/g和（3.3±0.5）nmol/mg]高于野生型对照组[分别为（29.5±3.2）nmok/g和（1.6±0.8）nmol/mg]，差异有统计学意义（t值分别为-4.25和-5.94，均P＜0.01）,Prdx6敲除型LPS 24 h组[分别为（73.5±12.4）nmol/g和（5.9±0.9）nmol/mg],均高于野生型LPS24 h组（t值分别为-3.01和-6.01，均P＜0.05）。野生型LPS24 h组肺脏蛋白羰基为（6.9±1.2）nmol/mg,与野生型对照组的（6.1±0.9）nmol/mg差异无统计学意义（t=-1.62，P=0.15）；Prdx6敲除型LPS 24 h组为（8.9±0.9）nmol/mg，高于野生型LPS 24 h组（t=-2.76,P＜0.05）。野生型LPS 24 h组肺脏TAOC为（4.7±0.6） U/mg,低于野生型对照组的（6.5±0.4） U/mg（t =3.35，P＜0.01）；Prdx6敲除型LPS 24 h组为（3.9 ±0.4） U/mg，低于野生型LPS24 h组（t =2.44，P=0.04）。Prdx6敲除型对照组与其野生型对照组以上参数表达差异无统计学意义。结论在LPS诱导的肺损伤中,Prdx6基因缺失增加了活性氧的产生，加重了氧化应激反应，从而使肺损伤恶化。

ObjectiveTo investigate the roles of peroxiredoxin （Prdx） 6 in the regulation of oxidative stress to lipopolysaccharide （LPS）-induced acute lung injury （ALI） in mice.Methods Prdx6knockout mice were tested for their genotype by PCR and Prdx6 protein expression was measured in lungs by immunohistochemisty.Eighteen male Prdx6 knockout mice were randomised to the Prdx6 knockout control group and the Prdx6 knockout LPS 24 h group, with 9 mice in each group.Eighteen male wild-type C57BL/6J mice were randomised to the wild-type control group and the wild-type LPS 24 h group, with 9 mice in each group. ALI was induced by intratracheal administration of 5 mg/kg LPS. Twenty-four hours after stimulation, lung tissue slides were stained with hematoxylin and eosin for histological evaluation. The concentration of protein in the bronchial alveolar lavage fluid （BALF） was measured by using the Micro BCA Protein Assay Kit.Levels of reactive oxygen species （ROS） in the lungs were quantified by measurement of hydrogen peroxide （H2 O2）. Lipid and protein peroxidation were measured as levels of malondialdehyde （MDA） and protein carbonylation. H2O2, MDA, protein carbonyl, total superoxide dismutase （SOD）activity, and total antioxidative capacity （TAOC） in lungs were measured by using assay kits from the manufacturers.ResultsPrdx6 knockout mice presented their genotype and there was no Prdx6 protein expression in the lung. Increased polymorphonuclear cells in alveoli and bronchial wall thickening were observed in the lungs of LPS groups, which were more severe in Prdx6 knockout LPS 24 h group compared with wild-type LPS 24 h group.LPS instillation induced a significant elevated protein concentration in BALF in wild-type LPS 24 h group （441 ±54） mg/L compared with wild-type control group （168 ± 20）mg/L （t =-4.71, P 〈0.01）.Significantly increased protein level in BALF was observed in Prdx6 knockout LPS 24 h group （770 ±66）mg/L compared with wild-type LPS 24 h group （t =-3.69,P 〈0.01）.LPS instillation induced a significantly decreased SOD activity in wild-type LPS 24 h group （16.0±1.2） U/mg protein compared with wild-type control group （26.5 ± 3.9） U/mg protein （t =6.22, P 〈 0.01）.SOD activity in Prdx6 knockout LPS 24 h group （14.5 ±5.3）U/mg protein was not different from wild-type LPS 24 h group （t =0.56,P =0.60）.LPS instillation induced a significantly increased H2O2 and MDA in wild-type LPS 24h group [H2O2（52.3±7.8） nmol/g protein; MDA （3.3 ±0.5） nmol/mg protein]compared with wild-type control group [H2O2 （29.5 ±3.2） nmol/g protein, （t =-4.25, P 〈 0.01） ; MDA （1.6 ± 0.8） nmol/mg protein, （t =-5.94, P 〈 0.01）].Significantly increased H2 O2 and MDA [H2 O2 （73.5 ± 12.4） nmol/g protein, （t=-3.01, P=0.02）; MDA （5.9±0.9）nmol/mg protein, （t =-6.01, P〈0.01）]were observed in Prdx6 knockout LPS 24 h group compared with wild-type LPS 24 h group. No significant difference of protein carbonylation was observed in wild-type LPS 24 h group （6.9 ± 1.2） nmol/mg protein compared with wild-type control group （6.1±0.9）nmol/mg protein （t =-1.62, P=0.15）.Significantly increased protein earbonylation （8.9 ± 0.9）nmol/g protein was observed in Prdx6 knockout LPS 24 h group compared with wild-type LPS 24 h group （t =-2.76, P = 0.03）.LPS instillation induced a significantly decreased TAOC in wild-type LPS 24 h group （4.7 ± 0.6） U/mg protein compared with wild-type control group （6.5 ＋ 0.4）U/mg protein （t = 3.35, P 〈 0.01）.Significantly decreased TAOC was observed in Prdx6 knockout LPS 24 h group （3.9 ± 0.4） U/mg protein compared with wild-type LPS 24 h group （t =2.44, P = 0.04）.The above parameters were not statistically different between Prdx6 knockout control group and wild-type control group.Conclusion Deletion of peroxiredoxin 6 exaggerated LPS-induced acute lung injury with increased oxidative stress.