| Background and Objective Myocardium is further aggravated by blood flow reperfusion on the basis of ischemia,which is known as myocardial ischemia reperfusion injury and often occurs in cardiac surgery.It not only reduces the clinical efficacy of reperfusion treatment,but also may further aggravate myocardial injury and increase the incidence of heart failure,seriously affecting the patient’s cardiac function.Myocardial hypoxia/reoxygenation(H/R)injury can simulate myocardial ischemia/reperfusion in vitro,which is beneficial to directly observe the changes of morphology and function of damaged cardiomyocytes.It is widely used in the molecular mechanism of IRI.In recent years,we have known that exosomes are vesiclelike bodies with a double-layered membrane structure,containing abundant proteins,lipids,micro RNAs(mi RNA),etc.,and can transmit a variety of signal molecules,which is an important medium for material exchange and information transfer between cells.Mi RNAs are a newly discovered class of endogenous,small molecule,non-coding RNA,which can regulate the apoptosis process of cardiomyocytes through its downstream target genes and signaling pathways,and play an important role in myocardial I/R injury.Our previous study have found that mi R-133b-5p is involved in the myocardial protection of morphine preconditioning(MPC).In view of the previous research basis,the present study intends to further explore whether exosomes are involved in the cardioprotective effects of MPC and the effects of MPC-induced serum exosomes on H/R injury in rat cardiomyocytes and the underlying molecular mechanisms,expecting to provide new ideas and theoretical basis for the study of myocardial protection mechanism of opioids.Methods 1.Isolation and characterization of exosomes The exosomes were isolated from serum after rats were subjected to MPC or the control treatment(CON)by Exo Quick? precipitation solution and labeled with MPC-Exo,CON-Exo,respectively.The exosomes were identified by transmission electron microscope and Western Blot.The concentration of exosomes was determined by the BCA protein assay.2.Labelling and uptake of exosomes The exosomes were labeled with Di I and then the labelled exosomes were added to the culture medium of prepared H9c2 cells for 12 h.After co-culture,the cells were washed with PBS and fixed in 4% paraformaldehyde.The uptake of exosomes by H9c2 cells was observed using fluorescence microscope.3.The role of exosomes in MPC reduce H/R injury in cardiomyocytes H9c2 cardiomyocytes were randomly divided into control,H/R,MPC,MPC+GW4869,H/R+DMSO,H/R+GW4869 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 MPC group were preconditioned with morphine with the final concentration of 1 μmol/L for 10 min and then were cultured for 30 min in morphine-free DMEM liquid culture medium before H/R.The cells in MPC+GW4869 group were pretreated with GW4869 with the final concentration of 5 μmol/L for 12 h and then performed the MPC before H/R.The cells in H/R+DMSO group and H/R+GW4869 group were pretreated with DMSO or GW4869 with the final concentration of 5 μmol/L for 12 h before H/R.After the treatment,cell viability was determined by CCK-8 assay,lactate dehydrogenase(LDH)activity in the culture medium was detected by micro-enzyme labeling and cell apoptosis was assessed by flow cytometry.4.Effects of MPC-Exo on H/R injury in H9c2 cardiomyocytes H9c2 cardiomyocytes were randomly divided into control,H/R,H/R+CON-Exo,H/R+MPCExo.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.After the treatment,cell viability was determined by CCK-8 assay,lactate dehydrogenase(LDH)activity in the culture medium was detected by micro-enzyme labeling and cell apoptosis was assessed by flow cytometry.The expression of mi R-133b-5p in H9c2 cardiomyocytes and exosomes were detected by q RT-PCR,the expression of Fas protein were detected by western blot.Results 1.Isolation and characterization of exosomes Exosomes isolated from rat serum were observed as spheroids with an average size of 30-150 nm using transmission electron microscope.The exosome markers,CD63 and HSP70,was specifically expressed in exosomes.The concentration of serum exosomes in MPC group was significantly higher than those of CON group.The concentration of serum exosomes in MPC group was significantly higher than those of CON group.2.Labelling and uptake of exosomes Fluorescence microscopy showed that a large amount of red particulate matter aggregated in the cytoplasm,suggesting that exosomes can be taken up by H9c2 cardiomyocytes.3.The role of exosomes in MPC reduce H/R injury in cardiomyocytes Compared with H/R group,MPC significantly reduced H/R injury by increased the cell viability,decreased the LDH activity and apoptotic rate(P<0.05).Compared with MPC group,the cell viability was significantly decreased,while the LDH activity,apoptotic rate were dramatically increased in group MPC+GW4869(P<0.05).The cardioprotection of MPC was inhibited by GW4869.4.Effects of MPC-Exo on H/R injury in H9c2 cardiomyocytes MPC-Exo preincubation protected H9c2 cells from H/R injury by increasing cell viability,inhibiting LDH activity and attenuating cell apoptosis and significantly up-regulated the level of mi R-133b-5p in cardiomyocytes(P<0.05).Conversely,CON-Exo had no significant effects on H/R injury and the expression of mi R-133b-5p(P>0.05).Compared with CON-Exo,the expression level of mi R-133b-5p in MPC-Exo is significantly increased(P<0.05).Conclusions MPC alleviates H/R injury in cardiomyocytes depending on exosomes release and exosomes are involved in mediating cardioprotection of MPC;MPC induced serum exosomes may confer cardioprotection by attenuating H/R injury in cardiomyocytes,and the mechanism may be the mediating of mi R-133b-5p transportation. |
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