| Background:The treatment of cardiovascular diseases reperfusion,including percutaneous transluminal coronary angioplasty,thrombolytic therapy in patients with myocardial infarction ect.has been widely used in clinical,and also significantly improved the prognosis for patients.However,reperfusion therapy in the recovery of ischemic myocardial blood supply could save the dying myocardium,but simultaneously cause myocardial ischemia and reperfusion injury(MIRI),leading to cardiac dysfunction,such as arrhythmia,myocardial stunning,microvascular blockage,which seriously affect the health of patients.Myocardial ischemic preconditioning(IPC)was an effective mechanism of endogenous myocardial protection,while there were some limitations in clinica.Therefore,exploring the molecular markers that were effective in simulating drugs and combining the corresponding drugs will provide new opportunities for the protection and treatment of clinical myocardial ischemia-reperfusion injury.Previous studies have showed that the effect of fentanyl preconditioning on myocardial protection during myocardial ischemia reperfusion injury was similar to that of IPC.However,the potential molecular mechanism remained to be further studied.In additional,apoptosis acted a vital role in myocardium MIRI.Thus,whether fentanyl preconditioning can reduce reperfusion injury by inhibiting myocardial apoptosis and the specific molecular mechanisms was needed to further study.Long-non-coding RNA(lncRNA)-MALATl has been studied in the regulation of cardiovascular diseases,and its down-regulation can reduce myocardial cell apoptosis,while whether lncRNA-MALAT1 was involved in fentanyl preconditioning myocardial protection was particularly unknown.LncRNA acted as a regulatory molecule,and its expression always changed under varieous pathological states.Moreover,it can inhibit the expression of miRNA by increasing or decreasing the level of miRNA,and also affectinging the expression of miRNA targeting genes,further was involved in the regulation of the development process of pathology.It has been suggested that miR-145 could inhibit cardiomyocytes apoptosis induced by hydrogen peroxide(H2O2)through targeting the expression of apoptosis-related molecule Bnip3 in cardiomyocytes.Objective:The aim of this study was to investigate whether IncRNA-MALAT1 could mediate the progression of ischemia-reperfusion injury of fentanyl preconditioning against cardiomyocytes by regulating miR-145/Bnip3 signaling pathway.Methods:Part 1 Mouse cardiomyocytes HL-1 cells was subjected to hypoxia/reoxygenation treatment and fentanyl pretreatment.The expression of MALAT1,miR-145 and Bnip3 mRNA was detected by real-time PCR and the expression of Bnip3 protein was detected by western blot method,to investigate the effects of fentanyl precondtioning on the expression of MALAT1,miR-145 and Bnip3 in cardiomyocytes under hypoxia/reoxygenation circumstance.Part 2 Over-expressing MALAT1 or inhibiting expression of MALAT1 was performed in mouse HL-1 cardiomyocytes.The expression of miR-145,Bnip3 mRNA and Bnip3,LC-3-I,LC3-II,Beclin-1 proteins were detected by quantitative PCR or western blot method,respectively,to study the effect of MALAT1 on miR-145/Bnip3 signaling.Part 3 1)Mouse cardiomyocytes HL-1 cells was subjected to hypoxia/reoxygenation treatment and fentanyl pretreatment,then IncRNA-MALAT1 over-expression or miR-145 inhibitor modification was performed.Lactate dehydrogenase(LDH)content was detected by ELISA and cell apoptosis was assessed by TUNEL,in order to investigate the effect of MALAT1 overexpression or miR-145 inhibitor on fentanyl cardioprotective.2)IncRNA-MALAT1 silencing was conducted based on hypoxia/reoxygenation model of HL-1 cardiomyocytes.The expression of miR-145,Bnip3 mRNA and Bnip3 protein were detected by quantitative PCR or western blot method,respectively,to study the regulating effect of MALAT1 on miR-145/Bnip3 signaling.Part 4 Male C57BL/6 mice were randomly divided into 5 groups:sham group,I/R group,fentanyl pretreatment + I/R group,fentanyl pretreatment + I/R group +Ad-GFP,fentanyl pretreatment + I/R group + Ad-MALAT1.Serum samples were collected and the content of LDH was detected,to determine the effect of MALAT1 overexpression on the protective effect of fentanyl on myocardium.Results:Part 1 Through real-time PCR and western blot analysis,results showed that the expression of miR-145 was down-regulated and the level of MALAT1 mRNA,Bnip3 mRNA and protein was up-regulated in hypoxia/reoxygenation cardiomyocytes.The effect could be reversed by fentanyl treatment.Part 2 Over-expression MALAT1 could result in up-regulation of Bnip3 expression and down-regulation of miR-145.Silencing MALAT1 induced the up-regulation of miR-145 and down-regulation of Bnip3.Part 3 In hypoxia/reoxygenated HL-1 cardiomyocytes,the up-regulation of miR-145 and the down-regulation of Bnip3 induced by fentanyl were reversed by MALAT1 over-expression.The down-regulation of miR-145 and the up-regulation of Bnip3 in HL-1 cardiomyocytes induced by hypoxia/reoxygenation was strongly reversed by MALAT1 silencing.MALAT1 over-expression can reverse the myocardial protective function of fentanyl and the anti-cardiomyocyte apoptosis effect.Similarly,miR-145 inhibitor can also reverse the myofibrotic function of fentanyl and the anti-cardiomyocyte apoptosis effect.Part 4 In the model of myocardial ischemia and reperfusion in mice,MALAT1 over-expression reversed the myocardium protective effect of fentanyl and promoted the release of serum LDH.Conclusion:1 Cardiomyocyte hypoxia/reoxygenation may cause up-regulation of MALAT1 expression,and this up-regulation of MALAT1 can be inhibited by fentanyl.2 In the model of cardiomyocyte or cardiomyocyte hypoxia/reoxygenation,MALAT1 negatively regulated the expression of miR-145,which was consistent with the expression of Bnip3.3 MALAT1 over-expression or miR-145 inhibitor both could reverse fentanyl precondtioning myocardial function protection and anti-cardiomyocyte apoptosis effect.4 MALAT1/miR-145/Bnip3 signaling pathway was involved in the protective effect of fentanyl precondtioning on myocardial ischemia-reperfusion. |
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