大鼠心肌挫伤后细胞结蛋白破坏与钙超载的实验研究

Experimental study on correlation between desmin degradation and calcium overload in rats with myocardial contusion

目的研究大鼠心肌挫伤(myocardial contusion,MC)后细胞结蛋白(desmin)的破坏与钙超载情况,评价心肌挫伤后结蛋白破坏与钙超载的关系。方法 110只SD大鼠按随机数字表法分为标准对照组(S组,n=10)、单纯致伤组(T组,n=50)和致伤+地尔硫卓组(D组,n=50),BIM-Ⅲ生物撞击机建立胸部撞击致心肌挫伤模型,致伤后D组腹腔注射盐酸地尔硫卓(0.5 mg/kg),分别于伤前,伤后2、4、8、24、48 h检测心肌细胞Ca2+浓度,免疫组化法及Western blot检测心肌细胞结蛋白变化情况,RT-PCR检测结蛋白mRNA表达变化情况。结果①T组伤后2 h心肌细胞Ca2+浓度较S组明显增高[(110.4±17.6)vs(74.0±9.3),P<0.01],伤后4 h达最高值(141.1±17.8)后逐渐下降,48 h恢复至伤前水平(78.6±10.3);②MC后早期即出现严重的结蛋白破坏,T组伤后8 h结蛋白表达量降至最低[与S组比较,(0.11±0.11)vs(1.23±0.27),P<0.01],24 h开始部分恢复,伤后48 h结蛋白表达增高且出现排列紊乱和无规则聚集的现象;③与T组比较,D组显著降低了伤后4、8 h细胞内Ca2+浓度[(84.6±9.7)、(89.3±11.8)],结蛋白表达量在伤后2、4 h两组相较无明显差异,但在伤后8 h,D组(0.55±0.18)高于T组(P<0.05);④T、D组结蛋白mRNA伤后表达增高,24 h达峰值,D组24 h后表达低于T组。结论 MC后早期结蛋白的破坏以细胞的机械损伤为主,随着细胞内Ca2+浓度增高,其介导的心肌结蛋白降解增加。通过伤后应用钙通道阻滞剂降低Ca2+浓度可有效减缓解结蛋白的降解破坏。

Objective To study the correlation between desmin degradation and calcium overload in rats with myocardial contusion（MC）.Methods One-hundred-and-ten SD rats were randomly divided into a trauma（T） group（n=50）,a trauma ＋ diltiazem（D） group（n=50）,and a control（S） group（n=10）.The MC models were established by chest impact with BIM-III Bio-impactor.The animals in the D group were given diltiazem hydrochloride（0.5 mg/kg） through intraperitoneal injection after trauma.The Ca2＋ concentration,protein expression of desmin,and mRNA expression of desmin in myocardial cells were measured by confocal laser scanning microscopy,immunohistochemistry and Western blotting,and RT-PCR,respectively,before and after（2,4,8,24,and 48 h） trauma.Results Compared with that in the S group,the Ca2＋ concentration in myocardial cells in the T group increased significantly 2 h after trauma（110.4±17.6 vs 74.0±9.3,P0.01）,reached the peak 4 h after trauma（141.1±17.8）,and then decreased gradually to the level before trauma 48 h after trauma（78.6±10.3）.Compared with that in the S group,the protein expression of desmin in the T group decreased to the lowest level 8 h after trauma（0.11±0.11 vs 1.23±0.27,P0.01）,began to increase 24 h after trauma（1.06±0.33）,and was higher than the level before trauma 48 h after trauma（1.82±0.41）.Irregular aggregation of desmin was found in the T group at 48 h after trauma.Compared with the T group,the D group had a significantly lower Ca2＋ concentration in 4 and 8 h after trauma（84.6±9.7,89.3±11.8）.The protein expression of desmin showed no significant difference between the T group and D group in 2 and 4 h after trauma,but was higher in the D group than in the T group 8 h after trauma（0.55±0.18 vs 0.11±0.11,P0.05）.Compared with that of the S group（0.62±0.23）,the mRNA expression of desmin in the D group and T group increased after trauma and reached the peak at 24 h after trauma（1.24±0.30 and 1.63±0.31）.The mRNA expression of desmin in the T group was higher than that in the D group in 24 and 48 h after trauma（1.63±0.31 vs 1.24±0.30,P0.05;1.04±0.22 vs 0.78±0.21,P0.05）.Conclusion Desmin disruption is directly caused by mechanical trauma in the early stage of MC,and the increase of intracellular Ca2＋ concentration after trauma can promote desmin degradation.The use of calcium channel blocker diltiazem hydrochloride can decrease intracellular Ca2＋ concentration and thus reduce desmin degradation after trauma.