中等强度跑台运动阻滞CCL2-CCR2信号通路缓解阿霉素诱导的小鼠心肌损伤作用机制研究

关键词：中等强度跑台运动  阿霉素诱导的心肌损伤  CC基序趋化因子受体 2  单核细胞趋化蛋白1  心肌炎症

Keywords:moderate-intensity treadmill exercise  DOX-induced myocardial injury  CC chemokine receptor-2  chemokine C-C motif ligand 2  myocardial inflammation

摘要：

      摘要 目的：探讨中等强度跑台运动对阿霉素（doxorubicin，DOX）诱导的心肌损伤的保护作用，以及该保护作用是否与阻滞单核细胞趋化蛋白1（chemokine C-C motif ligand 2，CCL2）-CC基序趋化因子受体 2（CC chemokine receptor-2，CCR2）信号通路相关。 方法：8周龄SPF雄性小鼠55只，随机分成空白对照组（control，CON；n=15）；阿霉素注射组（DOX；n=15）、阿霉素+运动组（doxorubicin and aerobic exercise，DOX+EX；n=15）、阿霉素+CCR2拮抗剂组（Doxorubicin and CCR2 antagonist，DOX+CCR2 antagonist；n=10）。DOX腹腔注射用以诱导心肌损伤模型，CCR2拮抗剂腹腔注射用以阻滞CCL2-CCR2信号通路，干预方式为4周中等强度跑台运动。检测阶段，超声心动图检测小鼠心脏功能；HE染色、Masson染色检测各组小鼠心肌损伤情况；生化试剂盒检测各组小鼠血清中心肌损伤标志物肌酸激酶（creatine kinase，CK）、乳酸脱氢酶（lactate dehydrogenase，LDH）的含量；转录组测序分析潜在的作用靶点；免疫蛋白印迹检测各组小鼠心肌组织中核因子κB抑制蛋白（inhibitor of nuclear factor kappa-B，IκB）/核因子κB（nuclear factor-kappa B，NF-κB）信号通路相关蛋白以及B淋巴细胞瘤-2基因（B-cell lymphoma-2，Bcl-2）和Bcl-2相关x蛋白（BCL2 associated X，Bax）凋亡相关蛋白的表达情况；免疫组织化学法检测各组小鼠心肌组织中炎症因子的表达。 结果：成功建立DOX诱导的小鼠心肌损伤模型，4周中等强度跑台运动显著缓解了DOX诱导心肌损伤；转录组结果分析表明CCL2-CCR2信号通路可能参与中等强度跑台运动改善DOX诱导的心肌损伤的过程；4周中等强度跑台运动和CCR2拮抗剂均能显著改善了DOX诱导的心脏泵血功能障碍，改善心肌组织损伤和纤维化，影响IκB/NF-κB信号通路并降低心肌炎症和凋亡。 结论：4周中等强度跑台运动对DOX诱导的小鼠心肌损伤产生保护作用，且这种作用的发挥可能与阻断CCL2-CCR2信号通路密切相关。

Abstract:

      Abstract Objective： To investigate the protective effect of moderate-intensity treadmill exercise on doxorubicin （DOX）-induced myocardial injury and to determine whether this protective effect is associated with blockade of the Chemokine C-C motif ligand 2 （CCL2）- CC chemokine receptor-2 （CCR2） signaling pathway. Method： Fifty-five 8-week-old SPF male mice were randomly divided into four groups: control group （CON， n=15）， doxorubicin group （DOX， n=15）； doxorubicin and aerobic exercise group （DOX+EX， n=15）， and doxorubicin and CCR2 antagonist group （DOX+CCR2 antagonist， n=10）. The myocardial injury model was induced by intraperitoneal injection of DOX and the CCL2-CCR2 signaling pathway was blocked by intraperitoneal injection of CCR2 antagonist.The intervention consisted of four weeks of moderate-intensity treadmill exercise. Echocardiography was used to detect cardiac function. Hematoxylin-eosin （HE） staining and Masson staining were used to detect myocardial injury in each group. Biochemical kits were used to detect the levels of myocardial injury markers creatine kinase （CK） and lactate dehydrogenase （LDH） were measured using biochemical assys. Transcriptome sequencing was used to identify potential targets. Western blot was used to detect the proteins related to the inhibitor of nuclear factor kappa-B （IκB）/ nuclear factor-kappa B （NF-κB） signaling pathway in the cardiac muscle tissues of each group as well as the proteins of B-cell lymphoma-2 （Bcl-2） and BCL2 Associated X （Bax） apoptosis-related proteins. Immunohistochemistry to detect the expression of inflammatory factors in myocardial tissues of mice in each group. Result： A DOX-induced myocardial injury model was successfully established in mice. Four weeks of moderate-intensity treadmill exercise significantly attenuated DOX-induced myocardial injury. Transcriptome analysis showed that the CCL2-CCR2 signaling pathway might be involved in the process of moderate-intensity treadmill exercise to improve DOX-induced myocardial injury. Four weeks of moderate-intensity treadmill exercise and CCR2 antagonist significantly alleviated DOX-induced cardiac pumping dysfunction， ameliorated myocardial tissue damage and fibrosis， influenced the IκB/NF-κB signaling pathway and reduced myocardial inflammation and apoptosis. Conclusion： Four-week moderate-intensity treadmill exercise exerts a protective effect on DOX-induced myocardial injury in mice， and this effect may be related to the blockade of the CCL2-CCR2 signaling pathway.