糖尿病小鼠血清外泌体在H9C2心肌细胞损伤调控中的作用及机制

Effect and molecular mechanism of serum exosomes of diabetic mice on regulating H9C2 myocardial cell injury

目的探讨糖尿病小鼠(db/db)血清外泌体在体外培养的H9C2心肌细胞损伤中的作用及机制。方法采用db/db小鼠(n=10)及其对照组小鼠(db/+,n=5)制备糖尿病心肌损伤小鼠模型。提取小鼠血清中的外泌体,定量检测其数量并应用PKH26标记为红色荧光;采用Western blotting检测外泌体相关蛋白及外泌体刺激后H9C2细胞炎性因子的表达情况;采用TUNEL检测细胞凋亡情况;采用Rab1a中和抗体进行阻断实验。结果db/db小鼠血清外泌体数量(30.95×109/ml)明显多于db/+小鼠(10.45×109/ml),差异有统计学意义(P<0.01)。体外培养的H9C2细胞能够内吞更多db/db小鼠血清来源的外泌体;应用db/db小鼠血清外泌体干预H9C2细胞可以刺激细胞炎性因子表达明显增加,其中IL-6和IL-1β分别增加6.2倍和2.6倍(P<0.01),且H9C2细胞凋亡明显增多。进一步机制研究发现,Rab1a在db/db小鼠血清外泌体中的表达明显增多,应用Rab1a中和抗体阻断db/db小鼠外泌体中Rab1a表达后可明显抑制H9C2细胞对外泌体的内吞及细胞凋亡。结论db/db小鼠血清来源的外泌体可诱导体外培养的心肌细胞凋亡及发生炎症反应,可能参与了糖尿病心肌损伤的演进。抑制外泌体分泌或干预其调控分子可能成为糖尿病心肌损伤治疗新的研究靶点。

Objective To investigate the effect of exosome in cultured in vitro H9C2 myocardial cells injury of diabetic mice and its mechanism.Methods The mouse model of diabetic myocardial injury was established by using db/db mice(n=10)and their mate mice db/+(n=5).Serum exosomes were isolated and quantitated using the exosome isolation reagent and EXOCET Quantitation kit.The serum exosomes were labeled with PKH26(red fluorescent cell linker)to detect the endocytosis in H9C2 cells.The expressions of exocrine associated protein and inflammatory cytokines in H9C2 cells with or without exosome stimulation were detected by Western blotting.TUNEL was used to detect apoptosis.A neutralizing antibody of Rab1a was used for blocking experiment.Results Db/db mice produced more exosomes than db/+mice(30.95×109/ml vs.10.45×109/ml,P<0.01).Moreover,H9C2 cells cultured in vitro could swallow more serum-exosomes derived from db/db mice.Meanwhile,serum exosome from db/db mice,as used to interfere H9C2 cells,significantly increased the expression of inflammatory cytokines,such as 6.2 folds to IL-6 and 2.6 folds to IL-1β(P<0.01).Furthermore,the apoptosis in H9C2 cells increased compared to those from db/+mice.Mechanism studies announced that the increased expression of Rab1a in exosomes-derived from db/db mice,and blocking the expression of Rab1a in exocrine of db/db mice with Rab1a neutralizing antibody could significantly inhibit the endocytosis and apoptosis of H9C2 cells.Conclusions Serum exosomes isolated from db/db mice may trigger inflammation and apoptosis of cardiomyocytes cultured in vitro,which may be involved in the evolution of diabetic myocardial injury.Inhibition of exosome secretion or intervention of its regulatory molecules may become a new research target for the treatment of diabetic myocardial injury.