腺苷酸活化蛋白激酶调控线粒体功能在脓毒症中调控心肌细胞损伤的作用和机制研究

doi:10.3969/j.issn.1006-7795.2025.02.018

摘要/Abstract

摘要： 目的探讨腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）调控线粒体功能在脓毒症中调控心肌细胞损伤的作用和机制。方法将40只Balb/c小鼠随机分为假手术组（Sham）（n=10），Sham+阿卡地新（5-aminoimidazole-4-carboxamide ribonucleotide，AICAR）组（n=10），盲肠结扎穿孔（caecal ligation was perforated，CLP）组（n=10）和CLP+AICAR组（n=10）。通过CLP建立脓毒症小鼠模型。采用超声心动图和组织形态学分析用于评估脓毒症诱导的心脏损伤情况。将新生大鼠心肌细胞（neonatal rat cardiomyocytes，NRCMs）与脂多糖（lipoprotein polysaccharide，LPS）（10 μg/mL）孵育24 h以在体外诱导脓毒症模型，并加入AICAR治疗。通过蛋白质免疫印迹（Western Blotting）、酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）和免疫荧光试验测定线粒体功能和动力学。结果与Sham组相比，CLP组小鼠心肌组织中AMPK表达降低，差异有统计学意义（P < 0.05）。与CLP组相比，CLP+AICAR组小鼠心肌组织中AMPK表达增加，差异有统计学意义（P < 0.05）。生存分析显示，CLP导致高病死率（约60%），而AICAR治疗提高了CLP小鼠的存活率（P < 0.05）。与Sham组相比，CLP组心排出量（cardiac output，CO）、搏出量（stroke volume，SV）、左心室舒张压（left ventricular end diastolic volume, LVEDV）降低，差异有统计学意义（P < 0.05），左心室后壁收缩期厚度（left ventricular posterior wall systolic，LVPWs）、左心室舒张末期后壁厚度（left ventricular posterior wall thickness，LVPWd）增加，差异有统计学意义（P < 0.05）。与CLP组相比，CLP+AICAR组小鼠心脏组织中线粒体大小、线粒体嵴数量增加，差异有统计学意义（P＜0.05），并且二氢乙锭（dihydroethidium，DHE）荧光强度和末端脱氧核苷酸转移酶脱氧尿苷三磷酸缺口末端标记（terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling，TUNEL）阳性细胞数降低,差异有统计学意义（P＜0.05）。与LPS组相比，LPS+AICAR组NRCMs的ATP产量、线粒体呼吸速率和复合物Ⅰ、Ⅱ、Ⅲ的活性增加，差异有统计学意义（P＜0.05），线粒体和细胞质活性氧（reactive oxygen species，ROS）水平降低，差异有统计学意义（P＜0.05）。与对照组相比，LPS组NRCMs的线粒体大小降低，差异有统计学意义（P＜0.05），和B淋巴细胞瘤-2相关X蛋白（B-cell lymphoma-2 associated X protein，Bax）、胱天蛋白酶-3（Caspase-3）表达、线粒体分裂指数增加，差异有统计学意义（P＜0.05），并且这种改变可以通过AICAR来减轻（P＜0.05）。结论 AMPK通过其对线粒体结构和功能、能量代谢、氧化应激和细胞凋亡的影响，在维持心脏功能和减轻脓毒性心肌损伤的病理变化中起着至关重要的作用。

关键词: 腺苷酸活化蛋白激酶, 线粒体, 脓毒症, 心肌细胞, 心脏功能

Abstract: Objective To investigate the role and mechanism of AMP-activated protein kinase (AMPK) in regulating mitochondrial function and cardiomyocyte injury in sepsis. Methods Forty Balb/c mice were randomly divided into four groups: Sham group (n=10), Sham+ 5-aminoimidazole-4-carboxamide ribonucleotide(AICAR) group (n=10), CLP group (n=10), and CLP+AICAR group (n=10). A sepsis mouse model was established through cecal ligation and puncture (CLP). Echocardiography and histological analysis were used to assess sepsis-induced cardiac injury. Neonatal rat cardiomyocytes (NRCMs) were incubated with lipopolysaccharide (LPS) (10 μg/mL) for 24 h to induce an in vitro sepsis model, and treated with AICAR. Mitochondrial function and dynamics were assessed by using Western blotting, enzyme-linked immunosorbent assay （ELISA）, and immunofluorescence assays. Results Compared with the Sham group, AMPK expression in the myocardial tissue of CLP mice was significantly reduced (P < 0.05). Compared with the CLP group, AMPK expression in the CLP+AICAR group was significantly increased (P < 0.05). Survival analysis showed that CLP led to a high mortality rate (~60%), while AICAR treatment significantly improved the survival rate of CLP mice (P < 0.05). Compared with the Sham group, cardiac output (CO), stroke volume (SV), and left ventricular end-diastolic volume (LVEDV) were significantly decreased in the CLP group (P < 0.05), while left ventricular posterior wall systolic (LVPWs) and left ventricular posterior wall thickness (LVPWd) were significantly increased (P < 0.05). AICAR treatment alleviated the cardiac dysfunction induced by CLP. Compared with the CLP group, mitochondrial size and the number of mitochondrial cristae in the myocardial tissue of CLP+AICAR group mice were significantly increased (P < 0.05), while DHE fluorescence intensity and the number of TUNEL-positive cells were significantly reduced (P < 0.05). Compared with the LPS group, ATP production, mitochondrial respiration rate, and complex I, II, and III activities in NRCMs of the LPS+AICAR group were significantly increased (P < 0.05), while mitochondrial and cytoplasmic ROS levels were significantly reduced (P < 0.05). Compared with the control group, mitochondrial size in NRCMs of the LPS group was significantly reduced (P < 0.05), while Bax and Caspase-3 expression, as well as mitochondrial fission index, were significantly increased (P < 0.05), and these changes were mitigated by AICAR (P < 0.05). Conclusion AMPK plays a crucial role in maintaining cardiac function and mitigating septic myocardial injury by regulating mitochondrial structure and function, energy metabolism, oxidative stress, and apoptosis.

Key words: AMP-activated protein kinase, mitochondria, sepsis, cardiomyocytes, cardiac function

中图分类号:

R631

黄康, 戴瑶, 伍松柏, 吕建磊, 冯洁. 腺苷酸活化蛋白激酶调控线粒体功能在脓毒症中调控心肌细胞损伤的作用和机制研究[J]. 首都医科大学学报, 2025, 46(2): 314-323.

Huang Kang, Dai Yao, Wu Songbai, Lü Jianlei, Feng Jie. Study on the role and mechanism of AMP-activated protein kinase in regulating mitochondrial function and modulating cardiomyocyte injury in sepsis[J]. Journal of Capital Medical University, 2025, 46(2): 314-323.

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