诱导性低温复苏对失血性休克大鼠肠组织Ca^(2+)/钙调蛋白依赖性蛋白激酶Ⅰ表达影响

Effect of induced hypothermia resuscitation on the expression of Ca^(2+)/calmodulin-dependent protein kinaseⅠin intestinal tissues of rats with hemorrhagic shock

目的探讨诱导性低温复苏对失血性休克大鼠肠组织Ca^(2+)/钙调蛋白依赖性蛋白激酶Ⅰ(CaMKⅠ)表达的影响。方法选取成年雄性Wistar大鼠30只,随机分为假手术组(n=6)、常温复苏组(n=12)以及低温复苏组(n=12)。假手术组仅进行外科插管及相关手术操作,不建立失血性休克模型;常温复苏组及低温复苏组每只大鼠放血25 ml/kg,构建失血性休克模型,休克60 min后分别采取38℃和32℃复苏并维持60 min,复苏结束后将大鼠体温恢复至38℃并监测血流动力学3 h。大鼠复苏3 h后,3组均随机选取50%的大鼠处死,取小肠黏膜、肠系膜淋巴结及血液,剩余大鼠进行72 h生存分析。采用定量聚合酶链式反应(PCR)及蛋白质印迹法分别检测3组大鼠肠黏膜中CaMKⅠmRNA及蛋白表达情况,应用酶联免疫吸附试剂盒检测大鼠血清炎性因子表达情况,同时,进行细菌移位检测。结果复苏3 h后,低温复苏组大鼠的心率和平均动脉压(MAP)均低于常温复苏组,而心输出量、每搏量和射血分数均高于常温复苏组,差异有统计学意义(P<0.05)。设定72 h为观察终点,假手术组大鼠于观察终点时全部存活,低温复苏组大鼠的生存时间为(63.50±9.59)h,明显长于常温复苏组的(46.83±19.59)h,差异有统计学意义(P<0.05)。在液体复苏3 h后,低温复苏组CaMKⅠmRNA表达水平、灰度值均明显低于常温复苏组,差异有统计学意义(P<0.05);常温复苏组与低温复苏组CaMKⅠmRNA、蛋白表达水平均明显高于假手术组,差异有统计学意义(P<0.05)。假手术组大鼠血清中CaMKⅠ、肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-1β、IL-6和IL-8水平明显低于常温复苏组和低温复苏组,IL-10水平高于常温复苏组,但低于低温复苏组,差异有统计学意义(P<0.05)。低温复苏组大鼠血清中CaMKⅠ、TNF-α、IL-1β、IL-6和IL-8水平明显低于常温复苏组,IL-10水平明显高于常温复苏组,差异有统计学意义(P<0.05)。复苏3 h后,低温复苏组大鼠血液细菌移位量、肠系膜淋巴结细菌移位量均明显低于常温复苏组,差异有统计学意义(P<0.05)。结论在诱导性低温复苏条件下,失血性休克大鼠肠组织中CaMKⅠmRNA及蛋白水平表达降低,大鼠生存时间延长,CaMKⅠ下调可能是诱导性低温复苏减轻大鼠失血性休克肠缺血再灌注损伤的重要分子机制。

Objective To investigate the effect of induced hypothermia resuscitation on the expression of Ca^(2+)/calmodulin-dependent protein kinase I(CaMKⅠ)in intestinal tissue of rats with hemorrhagic shock.Methods A total of 30 adult male Wistar rats were randomly divided into sham operation group(n=6),normal temperature resuscitation group(n=12)and low temperature resuscitation group(n=12).The sham operation group only performed surgical intubation and related operations,and did not establish hemorrhagic shock model.Each rat in the normal temperature resuscitation group and the low temperature resuscitation group was bloodlet 25 ml/kg,and the hemorrhagic shock model was established.After 60 minutes of shock,the rats were resuscitated at 38℃and 32℃respectively and maintained for 60 minutes.After the resuscitation,the rats’body temperature was restored to 38℃and the hemodynamics were monitored for 3 hours.After 3 hours of resuscitation,50%of the rats in all 3 groups were randomly selected to be killed,and the intestinal mucosa,mesenteric lymph nodes and blood were collected.The survival of the remaining rats was analyzed for 72 hours.Quantitative polymerase chain reaction(PCR)and western blot were used to detect the expression of CaMKⅠmRNA and protein in the intestinal mucosa of the three groups of rats,and the expression of serum inflammatory factors was detected by enzyme-linked immunosorption kit.Meanwhile,bacterial translocation was detected.Results After 3 hours,the heart rate and mean arterial pressure(MAP)of the low-temperature resuscitation group were lower than those of the normal temperature resuscitation group,while the cardiac output,stroke volume and ejection fraction were higher than those of the normal temperature resuscitation group,and the differences were statistically significant(P<0.05).All the rats in the sham operation group survived at 72 h as the end point.The average survival time of rats in the low-temperature resuscitation group was(63.50±9.59)h,which was significantly longer than that in the normal temperature resuscitation group(46.83±19.59)h,and the difference was statistically significant(P<0.05).After 3 hours of liquid resuscitation,the mRNA expression level and gray value of CaMKⅠin the low-temperature resuscitation group were significantly lower than those in the normal temperature resuscitation group,with statistical significance(P<0.05).The mRNA and protein expression levels of CaMKⅠin the normal temperature resuscitation group and the low temperature resuscitation group were significantly higher than those in the sham operation group,with statistical significance(P<0.05).Serum CaMKⅠand tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6 and IL-8 of sham operation group ratswere significantly lower than those of normal temperature resuscitation group and low temperature resuscitation group.The level of IL-10 was higher than that of normal temperature resuscitation group,but lower than that of low temperature resuscitation group,the difference was statistically significant(P<0.05).The levels of CaMKⅠ,TNF-α,IL-1β,IL-6 and IL-8 in serum of rats in low-temperature resuscitation group were significantly lower than those in normal temperature resuscitation group,and the levels of IL-10 were significantly higher than those in normal temperature resuscitation group,with statistical significance(P<0.05).After 3 hours of resuscitation,the blood bacterial displacement and mesenteric lymph node bacterial displacement of rats in the low-temperature resuscitation group were significantly lower than those in the normal temperature resuscitation group,and the difference was statistically significant(P<0.05).Conclusion Under induced hypothermia resuscitation condition,mRNA and protein levels of CaMKⅠin intestinal tissues of hemorrhagic shock rats were decreased,survival time of rats was prolonged.Down-regulation of CaMKⅠmay be an important molecular mechanism of induced hypothermia resuscitation to alleviate intestinal ischemia-reperfusion injury in rats with hemorrhagic shock.