有氧运动通过心肌细胞吞运内皮细胞来源外泌体抑制细胞凋亡
Aerobic Exercise Suppresses Apoptosis by Promoting Endothelial Exosomes from Cardiomyocytes
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摘要: 目的:探讨运动干预对H9C2细胞内吞外泌体及抑制细胞凋亡的影响。方法:C57/BL6小鼠随机分为假手术组(S组)、心梗组(MI组)、心梗有氧运动组(ME组),每组8只,采用左冠状动脉前降支结扎术(left anterior descending of the coronary artery, LAD)制备MI模型,ME组手术结束1周后进行持续6周的有氧运动。训练结束后超声检测心脏LVIDs、LVIDd、FS和EF,Masson染色检测心肌纤维化;使用无外泌体培养基培养HUVEC细胞,并提取外泌体孵育H9C2细胞。使用AMPK激动剂(AICAR,2 mmol/L,24 h)、脂多糖(LPS,10μg/m L,4 h)干预H9C2细胞。NTA和Western Blotting鉴定、检测提取的外泌体;Hoechst 33342染色及TUNEL检测细胞凋亡水平;Western Blotting检测小鼠心肌及H9C2细胞中凋亡相关蛋白Bax、Bcl-2和cleaved-Caspase3。结果:与MI组比较,ME组心功能指标EF和FS显著增加(P<0.05),细胞凋亡蛋白Bax/Bcl-2,cleaved-Caspase3/GAPDH表达显著下降(P<0.05);NTA粒径及Western Blotting鉴定外泌体为阳性结果;正常组细胞无外泌体内吞现象;与单纯LPS诱导的H9C2凋亡组比较,AICAR干预后可显著促进H9C2对外泌体的吞运作用;Hoechst 33342染色及TUNEL检测细胞凋亡阳性数目显著减少(P<0.05),且凋亡相关蛋白Bax/Bcl-2,cleaved-Caspase3/GAPDH表达显著下降(P<0.05)。结论:有氧运动显著抑制心梗心肌细胞凋亡,改善心功能;HUVEC内皮细胞分泌外泌体,且AMPK激动剂AICAR显著促进H9C2心肌细胞对HUVEC细胞来源的外泌体内吞,抑制H9C2心肌细胞凋亡。推测,运动可能通过促进心肌细胞外泌体内吞,抑制心肌细胞凋亡,改善心梗心功能。Abstract: Objective: To investigate the effects of exercise intervention on exosomes and inhibition of apoptosis in H9C2 cells.Methods: Left anterior descending ligature(LAD) was used to prepare MI model in C57/BL6 mice, and they were randomly divided into sham operation group(S), myocardial infarction group(MI), myocardial infarction and aerobic exercise group(ME), 8 mice in each group. One week after the surgery, the ME group performed aerobic exercise training for 6 weeks. After training, the echocardiography was used to detect LVIDs, LVIDd, FS, and EF; the myocardial fibrosis was detected by masson staining.HUVEC cells were cultured in an exosome-free medium, and exosomes were extracted to incubate H9C2 cells. AMPK agonist(AICAR, 2 mmol/L, 24 h) and lipopolysaccharide(LPS, 10 μg/m L, 4 h) were used to interfere H9C2 cells. NTA and Western Blotting were used to identify the extracted exosomes; Hoechst 33342 staining and TUNEL were used to detect apoptosis; Western Blotting was used to detect apoptosis-related proteins of Bax, Bcl-2, and cleaved-Caspase3. Results: Compared with MI group, the EF and FS were significantly increased(P<0.05) and expression of apoptotic protein Bax/Bcl-2 and cleaved Caspase3/GAPDH were significantly decreased(P<0.05) in ME group. NTA particle size and Western Blotting were positive for exosomes; no exocytosis was observed in normal cells. Compared with the H9C2 apoptosis group induced by LPS alone, AICAR intervention can significantly promote H9C2 exosome swallowing function. Hoechst 33342 staining and TUNEL detection significantly reduce the number of positive apoptosis, and the expressions of apoptosis-related protein Bax/Bcl-2 and cleaved-Caspase3/GAPDH were decreased significantly. Conclusion: Aerobic exercise significantly inhibited myocardial cell apoptosis and improved cardiac function in myocardial infarction. HUVEC endothelial cells secreted exosomes, and AMPK agonist AICAR significantly promoted in vivo uptake of HUVEC-derived exosomes by H9C2 cardiomyocytes, and inhibited apoptosis of H9C2 cardiomyocytes. It is speculated that exercise training may improve myocardial function by promoting cardiomyocyte exosomes and inhibiting apoptosis.