严重烧伤大鼠膈肌蛋白氧化应激及抗氧化酶表达的变化

Alteration of oxidative stress and expression of antioxidases in diaphragm of severely burned rats

目的探讨严重烧伤后大鼠膈肌内氧化应激的发生及抗氧化酶表达水平,以期进一步了解严重烧伤后膈肌萎缩和功能障碍的发生机制。方法取80只雄性7~8周龄Wistar大鼠,按随机数字表法分为烧伤组和假伤组,每组40只。将烧伤组大鼠麻醉后背部及腹部分别浸入80℃热水15、8s,制作50%体表总面积Ⅲ度大鼠烫伤(以下称烧伤)模型,伤后立即腹腔注射生理盐水40mL/kg复苏,创面外涂碘伏。假伤组大鼠除浸入37℃温水和不进行液体复苏外,其余处理均与烧伤组相同。分别于伤后2h及1、3、7、14d,每组取8只大鼠麻醉后完整切取膈肌,称取质量,采用酶标仪检测膈肌蛋白羰基化合物含量,采用蛋白质印迹法检测膈肌中过氧化氢酶、超氧化物歧化酶2(SOD2)、谷胱甘肽过氧化物酶1的蛋白表达水平。对数据行析因设计方差分析、t检验、Bonferroni校正。结果(1)2组大鼠伤后2h、1d膈肌质量相近(t=0.453、0.755,P>0.05);烧伤组大鼠膈肌质量于伤后3d开始下降,且明显低于假伤组(t=3.321,P<0.01);烧伤组大鼠膈肌质量于伤后7d开始逐渐增长至伤后14d,但仍明显低于假伤组(t=4.622、4.380,P<0.01)。(2)烧伤组大鼠伤后2h及1、3、7、14d膈肌蛋白羰基化合物含量[(2.7±0.3)、(2.5±0.5)、(2.4±0.4)、(2.5±0.4)、(3.2±0.6)pg/mL]均明显高于假伤组[(1.2±0.4)、(1.6±0.3)、(1.5±0.7)、(1.7±0.3)、(1.8±0.4)pg/mL,t=5.994、3.263、3.666、3.158、5.763,P<0.05或P<0.01]。(3)伤后1、3d,烧伤组大鼠膈肌中过氧化氢酶蛋白表达水平与假伤组相近(t=0.339、0.324,P>0.05);2组大鼠伤后2h及1、3、7、14d膈肌中SOD2蛋白表达水平均相近(t=1.446、1.385、0.757、1.561、0.531,P>0.05);2组大鼠伤后2h及1、3、7d膈肌中谷胱甘肽过氧化物酶1蛋白表达水平均相近(t=0.200、0.729、0.385、1.559,P>0.05)。结论严重烧伤后大鼠膈肌蛋白氧化应激持续发生且抗氧化酶表达相对不足,这可能是烧伤膈肌萎缩的一个重要原因。

Objective To explore the occurrence of oxidative stress and antioxidases expression in diaphragm of severely burned rats,so that the mechanism of respiratory muscle atrophy and dysfunction post-burn injury will be further clarified.Methods Eighty male Wistar rats(aged 7 to 8 weeks)were divided into sham injury group and burn injury group according to the random number table,with 40 rats in each group.Rats in burn injury group were inflicted with 50%total body surface area full-thickness scald(hereinafter referred to as burn)on the back and abdomen by immersing into 80℃water for 15 s and 8 s respectively.Immediately after injury,40 mL/kg normal saline was injected through abdomen for resuscitation,and the wounds were treated with iodine.Except for immersing into 37℃warm water and no resuscitation,the other treatments of rats in sham injury group were the same as those of burn injury group.Whole diaphragms of 8 rats per time point per group were collected after anesthesia at post injury hour(PIH)2 and on post injury day(PID)1,3,7,and 14,and muscle mass was determined.The protein carbonyl content was determined by microplate reader.The protein expressions of catalase,superoxide dismutase 2(SOD2),and glutathione peroxidase 1 were determined by Western blotting.Data were processed with analysis of variance of factorial design,t test,and Bonferroni correction.Results(1)There were no statistically significant differences in the diaphragm mass of rats between the 2 groups at PIH 2 and on PID 1(t=0.453,0.755,P>0.05).The diaphragm mass of rats in burn injury group started to decrease from PID 3,which was significantly lower than that of sham injury group(t=3.321,P<0.01).The diaphragm mass of rats in burn injury group started to increase from PID 7 to PID 14,which was significantly lower than that of sham injury group(t=4.622,4.380,P<0.01).(2)Protein carbonyl content in diaphragm of rats in burn injury group at PIH 2,and on PID 1,3,7,and 14[(2.7±0.3),(2.5±0.5),(2.4±0.4),(2.5±0.4),(3.2±0.6)pg/mL]was significantly higher than that of sham injury group respectively[(1.2±0.4),(1.6±0.3),(1.5±0.7),(1.7±0.3),(1.8±0.4)pg/mL,t=5.994,3.263,3.666,3.158,5.763,P<0.05 or P<0.01].(3)Protein expressions of catalase in diaphragm of rats in burn injury group on PID 1 and 3 were close to those of sham injury group(t=0.339,0.324,P>0.05).There were no statistically significant differences in protein expressions of SOD2 in diaphragm of rats between the 2 groups at PIH 2 and on PID 1,3,7,and 14(t=1.446,1.385,0.757,1.561,0.531,P>0.05).There were no statistically significant differences in protein expressions of glutathione peroxidase 1 in diaphragm of rats in the 2 groups at PIH 2 and on PID 1,3,and 7(t=0.200,0.729,0.385,1.559,P>0.05).Conclusions Continuous oxidative stress and relatively insufficient expression of antioxidases in diaphragm induced by burn injury could be a contributor to diaphragm atrophy.