| Background : Myocardial Ischemia/Reperfusion Injury(MIRI)refers to a phenomenon in which myocardial remodeling after a certain period of myocardial ischemia increases the original myocardial injury.The mechanism of MIRI is relatively complex and has not yet been fully elucidated.Existing studies suggest that free radical damage,calcium overload,endothelial cell injury,and myocardial energy metabolism disorder participate in the MIRI process.In addition,studies have shown that the level of inflammatory factors is related to post-ischemic impairment of cardiac function and the number of myocardial necrosis.It is speculated that inflammatory responses also play a very important role in MIRI.P2X7 R can be expressed in cardiomyocytes and up-regulated in myocardial injury.ATP released from damaged cells can activate P2X7 R,induce the NLRP3 inflammasome assembly,promote the secretion of proinflammatory cytokines IL-1β and IL-18,and further expand inflammation reaction.Microvesicles(MV)are membranous vesicles with a diameter of 100 nm to 1 μm released from the activated or apoptosed cells,and can mediate information transmission between cells.Studies in the nervous system have shown that the production of MV is closely related to the activation of P2X7 R.Ranolazine is a clinical drug for the treatment of ventricular arrhythmias and angina pectoris,and its protective mechanism of myocardial ischemia is not yet fully understood.At present,it is still unclear whether MV participate in P2X7R-NLRP3 mediated inflammatory response or ranolazine’s probeb anti-inflammation effect during MIRI.Objective: To investigate whether MVs participate in the P2X7R-NLRP3 medi ated inflammatory response in MIRI and whether ranolazine can play an antiMIRI role through the regulation of immune inflammation.Methods : The experiment was divided into three parts.Firstly,to establish hypoxia/reoxygenation(H/R)model of H9C2 cardiomyocytes and extracting MV from supernatant for next step of identification.Secondly,to investigate the effect of ranolazine on H/R-induced injury of cardiomyocytes.And the last,to study the effect of ranolazine on a rat model of myocardial ischemia/reperfusion.Results:Firstly,H4/R3 was appropriate for induction of H/R model on H9C2 cardiomyocytesn and the viability rate was around 73%.The extracted microvesicles were successfully identified by electron microscope and immunofluorescent tracer.Besides,the pelleted MV protein was quantified at 0.15±0.09 μg/μL.Secondly,we found that ranolazine can effectively down-regulate protein expressions of P2X7 R,NLRP3 and Caspase-1 and reduce MV formation during H/R induced cardiocytes injury(p<0.05).When the P2X7 R was blocked by an inhibitor of A438079,the production of MV was decreased and the intracellular calcium concentration was increased during H/R(p<0.05).Thirdly,we successfully established myocardial ischemia/reperfusion model of rat.The expressions of protein P2X7 R,NLRP3 and Caspase-1 were increased in the I/R rats,high levels of HMGB1 and il-1β in the serum and MV in the plasma were found as well(p< 0.05 vs control).The expressions of protein P2X7 R,NLRP3 and Caspase-1 were significant declined in ranolazine pretreated rat during I/R(p<0.05).And the same downward trends were also founded in the levels of HMGB1,IL-1β and MV(p<0.05).Conclusion:1.Hypoxia 4 h fllowing with reoxygenation 3 h can successfully induce the damage of H9C2 cells to produce MV.2.Ranolazine can reduce the inflammatory response induced by H/R-induced H9C2 cardiomyocyte injury by inhibiting the activation of P2X7R-NLRP3-Caspase-1 and MV.3.The activation of P2X7 R plays an important role in H/R-induced MV production in cardiomyocytes;4.Successfully established rat MIRI model.Ranolazine can reduce IL-1β and MV levels in serum by inhibiting P2X7R-NLRP3-Caspase-1,inhibit inflammatory response during myocardial ischemia/reperfusion,and reduce myocardial injury. |
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