吗啡预处理诱导的大鼠血清外泌体对H9c2心肌细胞缺氧/复氧损伤的影响

Effects of morphine preconditioning induced serum exosomeon hypoxia/reoxygenation injury in H9c2 cardiomyocytes

目的探讨吗啡预处理(morphine preconditioning,MPC)诱导的大鼠血清外泌体对H9c2心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)损伤的影响。方法大鼠进行MPC或对照处理(CON)后,提取血清中的外泌体,标记为MPC-Exo、CON-Exo。利用透射电镜和Western blot进行外泌体鉴定。H9c2心肌细胞随机分为Control组、H/R组、H/R+CON-Exo组和H/R+MPC-Exo组。除Control组细胞常规培养外,其余各组细胞均给予5 h缺氧和1 h复氧处理,H/R+CON-Exo组和H/R+MPC-Exo组细胞在H/R损伤前,分别与相应外泌体共孵育12 h。分别检测细胞活力、乳酸脱氢酶(lactic acid dehydrogenase, LDH)活性和细胞凋亡。结果外泌体提取物电镜下呈直径30～100 nm的圆形囊泡,并表达外泌体标志性蛋白。MPC-Exo与H9c2心肌细胞共孵育,可以明显减轻心肌细胞H/R损伤,增强细胞活力,减少LDH释放,并抑制心肌细胞凋亡;相反,CON-Exo与心肌细胞共孵育后,对H/R损伤无明显影响。结论 MPC诱导的大鼠血清外泌体可减轻H9c2心肌细胞H/R损伤,发挥心肌保护作用。

Aim To investigate the effect of morphine preconditioning(MPC)induced serum exosomes on hypoxia/reoxygenation(H/R)injury in H9c2 cardiomyocytes.Methods The exosomes were isolated from serum after rats were subjected to MPC or the control treatment(CON)and labeled with MPC-Exo,CON-Exo,respectively.The exosomes were identified by transmission electron microscope and Western blot.In another series of experiment,H9c2 cardiomyocytes were randomly divided into control,H/R,H/R+CON-Exo and H/R+MPC-Exo groups.The cells in control group were cultured under normal condition.In H/R group,H9c2 cardiomyocytes were exposed to 5 h of hypoxia followed by 1 h of reoxygenation.The cells in H/R+CON-Exo group and H/R+MPC-Exo group were pre-incubated with CON-Exo or MPC-Exo for 12 h before H/R,respectively.Then cell viability,lactate dehydrogenase(LDH)activity and cell apoptosis were detected separately.Results Exosomes isolated from rat serum were observed as 30~100 nm diameter round vesicles under transmission electron microscope and expressed the specific markers of exosomes.MPC-Exo pre-incubation protected H9c2 cells from H/R injury by increasing cell viability,inhibiting LDH activity and attenuating cell apoptosis.In contrast,CON-Exo failed to prevent H/R injury in H9c2 cells.Conclusion MPC induced serum exosomes may confer cardioprotection by reducing H/R injury in H9c2 cardiomyocytes.