烧冲复合伤大鼠心肌损伤及钙蛋白酶的变化规律

Study on changes of calpain and myocardial damage in rats with burn-blast combined injury

目的探讨烧冲复合伤大鼠心肌损伤及钙蛋白酶的变化规律。方法128只雄性sD大鼠按随机数字表法分为对照组、烧伤组、冲击伤组、复合伤组各32只。对照组：37℃温水中12S，烧伤组：94℃的沸水中12S，冲击伤组：5g黑索今距离大鼠左胸壁75cm爆炸，复合伤组：5g黑索今距离大鼠左胸壁75cm爆炸后94℃的沸水中12S。伤后6、24、48、72h4个观察点，采集腹主动脉血和心肌标本；测定左心室射血分数（EF）、左心室短轴缩短指数（FS）；心肌组织HE染色；血清肌钙蛋白T（CTnI）、肌酸激酶同工酶（CK-MB）检测；Tunel染色检测心肌组织细胞凋亡；实时荧光定量聚合酶链反应（RT-PCR）及Western印迹法分析心肌中钙蛋白酶mRNA及蛋白表达变化；荧光光度法检测钙蛋白酶活性。结果复合伤组伤情变化明显，心肌间质水肿明显，可见大面积心肌细胞变性及崩解，中性粒细胞浸润数量增多。伤后24h烧伤组、冲击伤组、复合伤组心功能下降，EF和Fs均显著低于对照组（均P〈0．05）。复合伤组伤后72h时Fs和EF，48h时Fs均显著低于烧伤组、冲击伤组（均P〈0．05）；复合伤组c．rnI上升，各个时间点均显著高于对照组，48h时均屁著高于烧伤组与冲击伤组（均P〈0．05）；复合伤组CK-MB伤后逐渐升高，24h时降低，48h时再升高且均显著高于对照组与烧伤组（均P〈0．05）。伤后烧伤组、冲击伤组、复合伤组心肌凋亡指数均显著高于对照组（均P〈0．05），烧伤组24、48h时分别为（25．3±4．0）、（28．8±5．3），均显著低于同时间点复合伤组的（43．3±9．4）、（53．3±10．4），烧伤组72h时为（31．9±6．7），显著高于冲击伤组的（17．3±6．3）（均P〈0．05）。各时间点复合伤组心肌组织中钙蛋白酶mRNA和蛋白相对表达量均显著高于对照组（均P〈0．05）。复合伤组心肌钙蛋白酶活性伤后24h达到高峰，后逐渐下降，各时间点均显著高于对照组（均P〈0．05）。结论烧冲复合伤后大鼠出现心肌损伤，钙蛋白酶蛋白表达升高、活性增强。

Objective To study myocardial damage and rules of calpain change in rats with burn- blast combined injury. Methods One hundred and twenty-eight male SD rats were randomly divided into control group, burn group, blast group, burn-blast group, with 32 rats in each group. Control group： 37 degrees＇ warm water for 12 s; Burn group： 94 degrees＇ boiling water for 12 s; Blast group： 5 g cyclonite explode in 75 cm distance from left chest wall of rat ; Burn-blast group ： burn group and blast group combined modeling method. At 6, 24, 48, 72 h observation points after injury, abdominal aorta blood samples and myocardial specimen were collected. Left ventricular ejection fraction （EF）, left ventricular fractional shortening index （FS） were measured through color Doppler ultrasound instrument; Myocardial tissue was stained with hematoxylin-eosin （HE） ; serum cardiac troponiu I （CTnl） and creatine kinase isoenzyme （CK- MB） were detected; detection of cell apoptosis in myocardial tissue was performed by terminal deoxynucleotidyl transferase-mediated dUTP notch labeling technique （Tunel）. Expression levels of calpain mRNA level and protein were detected with Real-time fluorescent quantitative polymerase chain reaction （RT-PCR） and Western imprinting method analysis; calpain activity was detected by fluorescence spectrophotometry. Results The injury of burn-blast combined injured rats was obvious, including myocardial interstitial edema, large area of myocardial cell degeneration and disintegration and the number of neutrophil infiltration increased. Cardiac function decreased 24 h after injury in burn group, blast group, burn-blast group ; both EF and FS were significant lower than those of control group （ all P 〈 0. 05 ）. FS at 48, 72 h and EF at 72 h in burn-blast group were significantly lower than those of burn group, blast group at the same time points （ all P 〈 0. 05 ） ; the level of cTnI in burn-blast group rose and was higher than control group at all time points, higher than the burn group, blast group at 48 h （ all P 〈 0. 05 ）. CK-MB in burn- blast group rats increased after injury, lowered at 24 h and rose again at 48 h. The level was significantly higher than control group and burn group （both P 〈 0. 05 ）. Comparing to control group, myocardial apoptosis index in burn group, blast group and burn-blast group were significantly increased （ all P 〈 O. 05 ）. Those of burn group （ 25.3 ± 4. 0） at 24 h and （ 28.8 ± 5.3 ） at 48 h were significantly lowered than burn- blast group （43.3 ±9.4）, （53.3 ±10. 4） at same time points, and bum group （31.9±6. 7） at 72 h was significantly higher than blast group （ 17. 3 ±6. 3 ） （ all P 〈 0. 05 ）. Compared to control group, Calpain mRNA and protein expression in myocardial tissue were significantly increased in bum-blast group at all time points （ all P 〈 0. 05 ）. Calpain activity reached the peak at 24 h after injury, then gradually declined, and was significantly higher than control group （ all P 〈 0. 05 ）. Conclusion Calpain expression and activity increase in burn-blast combined injured rats which leads to myocardial damage.