目的 探讨解偶联蛋白2(uncoupling protein 2,UCP2)与脂多糖诱导脓毒症大鼠心肌线粒体损伤的关系。方法 通过腹腔注射脂多糖(LPS)建立脓毒症模型。将40只Sprague-Dawley(SD)雄性大鼠随机分为5组,每组8只:对照组(腹腔注射生理盐水)、脓毒症6 h组(LPS-6 h组)、脓毒症12 h组(LPS-12 h组)、脓毒症24 h组(LPS-24 h组)和脓毒症48 h组(LPS-48 h组)。在相应时间点留取大鼠血清和心脏组织,提取心肌线粒体,通过酶标仪检测肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)和活性氧(ROS)水平,流式细胞仪检测线粒体肿胀度和线粒体膜电位(MMP),采用蛋白质免疫印迹试验(Western blot)测定UCP2蛋白表达水平;电镜观察心肌组织线粒体形态学变化。结果 与对照组比较,LPS各组血清CK、CK-MB水平、心肌组织ROS水平和线粒体肿胀度均明显升高(P<0.05),峰值在LPS-24 h组;而LPS各组线粒体膜电位明显下降(P<0.05),LPS-24 h组降至最低。Western blot检测发现LPS各组心肌组织UCP2的表达水平较对照组明显升高(P<0.05),峰值在LPS-24 h组。电镜观察结果发现LPS大鼠心肌线粒体肿胀,线粒体膜部分破碎,有空泡形成,LPS-24 h组病变最严重。LPS大鼠心肌线粒体ROS水平、线粒体肿胀度与UCP2表达量呈正相关(分别r=0.796、0.893,P<0.05);LPS大鼠心肌MMP与UCP2表达量呈负相关(r=-0.903,P<0.05)。结论 在脓毒症大鼠模型中,心肌和心肌线粒体都明显损伤,心肌组织UCP2的表达与线粒体损伤密切相关,推测UCP2可能在脓毒症心肌线粒体损伤中起重要作用。
Abstract
Objective To investigate the correlation between uncoupling protein 2(UCP2) expression and myocardial mitochondria injury in rats with sepsis induced by lipopolysaccharide(LPS).Methods The rat model of sepsis was established through an intraperitoneal injection of LPS.Forty male Sprague-Dawley rats were randomly and equally divided into control group(an intraperitoneal injection of normal saline), sepsis 6 h group(LPS-6 h group), sepsis 12 h group(LPS-12 h group), sepsis 24 h group(LPS-24 h group), and sepsis 48 h group(LPS-48 h group).The serum and heart tissues were harvested at corresponding time points and myocardial mitochondria was extracted.The microplate reader was applied to measure creatine kinase(CK), creatine kinase-MB(CK-MB), and reactive oxygen species(ROS).Flow cytometry was applied to measure the degree of mitochondrial swelling and mitochondrial membrane potential(MMP).Western blot was used to measure the expression level of UCP2.Electron microscopy was applied to observe the morphological changes in heart tissues and myocardial mitochondria.Results Compared with the control group, the LPS groups had significantly increased serum levels of CK, CK-MB, and myocardial ROS, as well as a significantly increased degree of mitochondrial swelling(P<0.05), and these values reached their peaks at 24 hours after LPS injection.The LPS groups had a significant decrease in MMP(P<0.05), which reached the lowest level at 24 hours after LPS injection.Western blot showed that the LPS groups had a significant increase in the expression level of myocardial UCP2 compared with the control group(P<0.05), which reached its peak at 24 hours after LPS injection.The results of electron microscopy showed mitochondrial swelling, partial rupture of the mitochondrial membrane, and cavity formation in rats in the LPS groups.The most severe lesions occurred in the LPS-24 h group.In rats with LPS, the ROS level in the myocardial mitochondria and the degree of mitochondrial swelling were positively correlated with the expression level of UCP2(r=0.796 and 0.893, respectively;P<0.05), while MMP was negatively correlated with the expression level of UCP2(r=-0.903, P<0.05).Conclusions In the rat model of sepsis, the myocardium and myocardial mitochondria have obvious injuries, and the expression level of UCP2 is closely correlated with mitochondrial injury.Therefore, UCP2 might play an important role in myocardial mitochondrial injury in sepsis.
关键词
脓毒症 /
心肌 /
线粒体 /
解偶联蛋白2 /
大鼠
Key words
Sepsis /
Myocardium /
Mitochondria /
Uncoupling protein 2 /
Rats
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基金
国家自然科学基金(81272070);广东省科技计划项目(2014A020212725)。
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