缺血预处理诱导的外泌体对心肌细胞低氧/复氧损伤及Bcl-2、Bax表达的影响

Effects of ischemic preconditioning induced exosomes on hypoxia/reoxygenation injury and expression of Bcl-2 and Bax in cardiomyocytes

目的探讨缺血预处理(IPC)诱导的外泌体(exosome)对心肌细胞低氧/复氧(H/R)损伤及凋亡蛋白B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关x蛋白(Bax)表达的影响。方法SD大鼠进行IPC或对照处理后,提取血清中的外泌体,标记为IPC-Exo、CON-Exo。采用BCA蛋白定量法测定外泌体浓度。H9c2心肌细胞随机分为Control组、H/R组、H/R+IPC-Exo组和H/R+CON-Exo组。除Control组细胞常规培养外,其余各组细胞均给予5 h低氧和1 h复氧处理,H/R+IPC-Exo组和H/R+CON-Exo组细胞在H/R损伤前分别与相应外泌体共孵育12 h。处理结束后,分别检测各组细胞活力、乳酸脱氢酶活性、细胞凋亡和Bcl-2、Bax蛋白表达情况。结果 IPC明显增加血清外泌体释放。与H/R组比较,IPCExo与H9c2心肌细胞共孵育,可以增加细胞活力、减少乳酸脱氢酶释放、抑制心肌细胞凋亡,减轻心肌细胞H/R损伤;且能上调Bcl-2表达,下调Bax表达。而CON-Exo与心肌细胞共孵育后,对H/R损伤无明显影响。结论缺血预处理诱导的外泌体可以减轻H9c2心肌细胞H/R损伤,其机制可能是通过上调Bcl-2、下调Bax表达,从而发挥心肌保护作用。

Objective To investigate the effects of ischemic preconditioning( IPC) induced exosomes on hypoxia/reoxygenation( H/R) injury and expression of Bcl-2 and Bax in cardiomyocytes. Methods The exosomes were isolated from serum after rats were subjected to IPC or the control treatment and labeled with IPC-Exo,CON-Exo,respectively. The concentration of exosomes was determined by BCA protein assay. In another series of experiment,H9 c2 cardiomyocytes were randomly divided into control,H/R,H/R + IPC-Exo and H/R + CON-Exo groups. The cells in control group were cultured under normal condition. In H/R group,H9 c2 cardiomyocytes were exposed to5 h of hypoxia followed by 1 h of reoxygenation. The cells in H/R + CON-Exo group and H/R + IPC-Exo group were pre-incubated with CON-Exo or IPC-Exo for 12 h before H/R,respectively. Then cell viability,lactate dehydrogenase activity,cell apoptosis and the expression of Bcl-2,Bax protein were detected separately. Results IPC significantly increased the release of serum exosomes. Compared with H/R group,IPC-Exo pre-incubation protected H9 c2 cells from H/R injury by increasing cell viability,inhibiting LDH activity,attenuating cell apoptosisand up-regulating Bcl-2 protein and down-regulating Bax protein. However,CON-Exo failed to prevent H/R injury in H9 c2 cells. Conclusion IPC induced exosomes may exert cardioprotection by reducing H/R injury in H9 c2 cardiomyocytes,the mechanism may be related to up-regulating the expression of Bcl-2 and down-regulating the expression of Bax.