Objective: To observe the effect of electroacupuncture (EA) on vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor receptor-3 (VEGFR-3), proinflammatory factors and apoptosis in myocardial tissue in mice with acute myocardial ischemia (AMI), and to explore the mechanism of EA for AMI.
Methods: Fifty male C57BL/6 mice were randomly divided into a sham operation group, a model group, an EA group, an inhibitor group and an inhibitor+EA group, 10 mice in each group. Except for the sham operation group, the mice in the remaining groups were intervented with ligation at the left anterior descending (LAD) coronary artery to establish AMI model. The mice in the sham operation group were intervented without ligation after thoracotomy. The mice in the EA group were intervented with EA at "Shenmen" (HT 7) and "Tongli" (HT 5), disperse-dense wave, 2 Hz/15 Hz in frequency, 1 mA in current intensity, 30 min each time, once a day, for 3 d. The mice in the inhibitor group were treated with intraperitoneal injection of SAR 131675 (12.5 mg•kg-1•d-1, once a day for 3 d). The mice in the inhibitor+EA group were injected intraperitoneally with SAR 131675 30 min before EA. The ECG before modeling, 30 min after modeling and 3 d after intervention was detected, and the ST segment displacement was recorded; after the intervention, the ELISA method was applied to measure the contents of serum creatine kinase isoenzyme (CK-MB), aspartate aminotransferase (AST) as well as tumor necrosis factor-α (TNF-α) and interleukin-23 (IL-23) in myocardial tissue; the HE staining method was used to observe the morphological changes of myocardial tissue; the immunofluorescence double labeling method was applied to measure the number of co-expression positive cells of VEGF-C/VEGFR-3 in myocardial tissue; the TUNEL method was used to detect the level of cardiomyocyte apoptosis; the Western blot method was applied to measure the protein expressions of VEGF-C, VEGFR-3, b-lymphoma-2 (Bcl-2), activated caspase-3 (Cleaved Caspase-3) and activated poly adenosine diphosphate ribose polymerase-1 (Cleaved PARP-1).
Results: Compared with the sham operation group, in the model group the ST segment displacement was increased (P<0.01); the contents of CK-MB, AST, TNF-α and IL-23 were increased (P<0.01); the arrangement of myocardial fibers was disordered, and interstitial inflammatory cell infiltration was obvious; the number of co-expression positive cells of VEGF-C/VEGFR-3 was decreased (P<0.01); the number of cardiomyocyte apoptosis was increased (P<0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were decreased (P<0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were increased (P<0.01). Compared with the model group, in the EA group the ST segment displacement was decreased (P<0.01); the contents of CK-MB, AST, TNF-α, IL-23 were decreased (P<0.01); the severity of myocardial pathological injury was reduced; the number of co-expression positive cells of VEGF-C/VEGFR-3 was increased (P<0.01); the number of cardiomyocyte apoptosis was reduced (P<0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were increased (P<0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were reduced (P<0.01). There was no significant difference in all the indexes between the model group and the inhibitor group (P>0.05). Compared with the model group, the protein expression of VEGF-C was increased in the inhibitor+EA group (P<0.01). Compared with the inhibitor group, in the EA group the ST segment displacement was decreased (P<0.01); the contents of CK-MB, AST, TNF-α, IL-23 were decreased (P<0.01); the severity of myocardial pathological injury was reduced; the number of co-expression positive cells of VEGF-C/VEGFR-3 was increased (P<0.05); the number of cardiomyocyte apoptosis was reduced (P<0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were increased (P<0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were reduced (P<0.01). Compared with the inhibitor+EA group, all the indexes in the EA group were improved except the protein expression of VEGF-C (P<0.01).
Conclusion: EA could relieve the inflammatory reaction and apoptosis in AMI mice, and its mechanism may be related to activating VEGF-C/VEGFR-3 pathway and promoting lymphangion genesis.
目的：观察电针对急性心肌缺血（AMI）小鼠心肌组织血管内皮生长因子-C（VEGF-C）、血管内皮生长因子受体-3（VEGFR-3）、促炎因子和细胞凋亡的影响，探讨电针治疗AMI的作用机制。方法：将50只雄性C57BL/6小鼠随机分为假手术组、模型组、电针组、抑制剂组和抑制剂+电针组，每组10只。除假手术组外，其余各组小鼠结扎冠状动脉左前降支（LAD）制备急性心肌缺血模型；假手术组开胸后仅穿线不结扎。电针组予电针“神门”“通里”干预，疏密波，频率2 Hz/15 Hz，电流强度1 mA，每次30 min，每天1次，连续3 d；抑制剂组腹腔注射SAR 131675（12.5 mg•kg-1•d-1，每日1次，连续3 d）；抑制剂+电针组于电针前30 min腹腔注射SAR 131675。检测小鼠造模前、造模后30 min、干预3 d后心电图，记录ST段位移值；干预结束后，ELISA法检测血清肌酸激酶同工酶（CK-MB）、天冬氨酸氨基转移酶（AST）及心肌组织肿瘤坏死因子α（TNF-α）、白细胞介素23（IL-23）含量；HE染色法观察心肌组织形态变化；免疫荧光双标法检测心肌组织VEGF-C/VEGFR-3共表达阳性细胞数；TUNEL法检测心肌细胞凋亡水平；Western blot法检测心肌组织VEGF-C、VEGFR-3、B淋巴细胞瘤-2（Bcl-2）、活化的半胱氨酸天冬氨酸蛋白水解酶-3（Cleaved Caspase-3）、活化的多聚腺苷二磷酸核糖聚合酶-1（Cleaved PARP-1）蛋白表达。结果：与假手术组比较，模型组小鼠ST段位移值升高（P<0.01）；CK-MB、AST、TNF-α、IL-23含量升高（P<0.01）；心肌纤维排列紊乱，间质炎性细胞浸润明显；VEGF-C/VEGFR-3共表达阳性细胞数减少（P<0.01）；心肌细胞凋亡数增加（P<0.01）；VEGF-C、VEGFR-3、Bcl-2蛋白表达减少（P<0.01），Cleaved Caspase-3、Cleaved PARP-1蛋白表达增加（P<0.01）。与模型组比较，电针组小鼠ST段位移值降低（P<0.01）；CK-MB、AST、TNF-α、IL-23含量降低（P<0.01）；心肌病理损伤程度减轻；VEGF-C/VEGFR-3共表达阳性细胞数增加（P<0.01）；心肌细胞凋亡数减少（P<0.01）；VEGF-C、VEGFR-3、Bcl-2蛋白表达增加（P<0.01），Cleaved Caspase-3、Cleaved PARP-1蛋白表达减少（P<0.01）。模型组与抑制剂组各指标比较差异无统计学意义（P>0.05）。与模型组比较，抑制剂+电针组小鼠VEGF-C蛋白表达增加（P<0.01）。与抑制剂组比较，电针组小鼠ST段位移值降低（P<0.01）；CK-MB、AST、TNF-α、IL-23含量降低（P<0.01）；心肌病理损伤程度减轻；VEGF-C/VEGFR-3共表达阳性细胞数增加（P<0.05）；心肌细胞凋亡数减少（P<0.01）；VEGF-C、VEGFR-3、Bcl-2蛋白表达增加（P<0.01），Cleaved Caspase-3、Cleaved PARP-1蛋白表达减少（P<0.01）。与抑制剂+电针组比较，除VEGF-C蛋白表达外，电针组各指标均改善（P<0.01）。结论：电针可减轻AMI小鼠炎性反应和细胞凋亡，其机制可能与激活VEGF-C/VEGFR-3通路促进淋巴管生成有关。.
Keywords: VEGF-C/VEGFR-3 pathway; acute myocardial ischemia (AMI); apoptosis; electroacupuncture; inflammatory reaction.