MicroRNA-874 Mediates The Protection Against Ischemia-reperfusion Injury Of Cardiomyocyte Following Inhalation Anesthesia With Sevoflurane By Targeting STAT3 Through The JAK2/STAT3 Signaling Pathway

Objective Ischemic heart disease(ischemic heart disease,IHD)is the main cause of death in cardiovascular diseases.Myocardial insufficiency caused by decreased coronary blood flow is the reason for IHD.The pathological features of IHD are coronary artery stenosis or blockage.Reperfusion therapy(drug thrombolysis,percutaneous coronary stent implantation,coronary artery bypass grafting and other coronary artery revascularization techniques)is considered to be the most effective therapy for coronary artery ischemia in the recovery of coronary blood supply in the ischemic area of the heart.However,myocardial ischemia-reperfusion(I/R)injury is a major obstacle to the treatment of IHD.It has been found that sevoflurane(SEV)is a volatile anesthetic and inhalation of SEV can reduce I/R injury.SEV has a protective effect on cardiac I/R injury,but the mechanism of regulation of SEV is not clear.More and more studies have shown that microRNA(miRNA)is involved in the occurrence and development of many cardiovascular diseases,especially in IHD.MiRNA can regulate cardiac functions such as signal transduction,myocardial contraction,cardiac growth and morphogenesis.They are also involved in the regulation of cardiomyocyte apoptosis,especially miRNA involved in the pathogenesis of heart diseases such as myocardial infarction(MI)and heart failure(HF).The previous studies found that miRNA-874(miR-874)was highly expressed in I/R injured cells.We find that miR-874 has a regulatory effect on JAK2/STAT3 when we look for the downstream target of miR-874.This study is based on the mechanism of the protective effect of clinical SEV on the heart,which is to explore the application of SEV.Methods 1.Clinical data collection and analysis From January 2012 to December 2015,96 patients(56 males and 40 females,aged 50-75 years,ASA ?-?)undergoing elective mitral valve replacement in Yancheng Hospital affiliated to Southeast University were selected.The patients were randomly divided into two groups:SEV inhalation anesthesia pretreatment group(SEV group,n=48)and propofol(PRO)intravenous anesthesia control group(PRO group,n=48).Using at least one study drug,and at least once a set of cases with post-medication evaluation data.All cases were randomized into groups.Maintenance of anesthesia:the SEV group was injected into SEV via volatile canister after successful anesthesia induced intubation,and SEV inhalation concentration was adjusted by 1.5-2.0%according to the blood pressure of patients;PRO was continuously pumped 0.05-0.2 mg/kg.h in the PRO group after successful anesthesia induced intubation.Both groups were pumped with vecuronium 0.05-0.1 mg/kg.h to maintain muscle relaxation and sufentanil was injected intravenously intermittently to maintain analgesia,the total amount of which was controlled at 1-3 ?g/kg.After aortic occlusion,cardiac infusion of cardioplegic fluid,cardiac pump function,and full-flow CPB replacement,SEV inhalation was stopped immediately in the SEV group,and PRO was injected into the same vein pump in the PRO group to maintain anesthesia,the PRO group continued to maintain the original plan.All intravenous anesthetics were injected into an extracorporeal circulation machine blood pump during CPB(cardiopulmonary bypass,CPB).The Patients' EEG bispectral index was adjusted at 40 to 60 in both groups.The changes of mean arterial pressure(MAP),heart rate(HR)were recorded at the time points of T0(before anesthesia induction),T1(before CPB),T2(at the end of surgery)and T3(4 hours after returning ICU),both the levels of cardiac fatty acid binding protein(H-FABP)and troponin I(cTnl)were recorded at the same times.The time of CPB,the aortic cross-block and operation were recorded,and the usage of sufentanil in the procedure and the conditions of the cardioversion were recorded.2.The role of SEV preconditioning in I/R injury and its molecular mechanism at the level of cells and animals H9C2 cells in logarithmic growth stage were inoculated in 6-well plate with 2×105 cell/holes.After the cells adhered,removed the medium and added SEV.RT-PCR technique was used to detect the relative expression of miR-874,JAK2/STAT3 in normal cardiomyocytes and I/R-induced reinjury cells.A model of myocardial I/R injury was established in 36 randomly selected mice.The mice were anesthetized by intraperitoneal injection of 2%pentobarbital sodium.The mouse's limbs were connected to the electrodes to monitor the ECG.After the successful tracheal intubation,the mouse was mechanically ventilated by a small animal ventilator and connected to a multi-parameter anesthesia gas monitor meanwhile.When the end-tidal carbon dioxide(ETCO2)waveforms appeared,ventilation parameters were set as followed:the tidal volume(VT),5-7 ml/100g;the respiratory rate(RR),60 bpm;the inspiration-expiration ratio,1:2;and the pressure of ETCO2(PETCO2)was adjusted at 13-15 mmHg.Opened the left fourth rib gap,using 6-0 absorbable suture went through the left anterior descending coronary artery and blocked the vessel.The coronary artery occlusion was successful when the ST segments were significantly increased by ECG monitoring.After 30 min,the suture was loosened and the blood followed through the vessel again.The myocardial I/R injury model was successfully established when the ST segments were reduced by the ECG monitor.The mice were randomly divided into 7 groups by random table method.(1)In the control group,the six mice did not receive any treatment.(2)In the sham group,the six mice did not receive the model,but they were sutured with 6-0 non-invasive absorbable suture after thoracotomy.(3)In the I/R group,the six mice were randomly selected without any treatment.(4)In the SEV group,the six mice were randomly selected from the I/R mice.After 30 min of ischemia,the mice were inhaled 2.8%SEV for about 2 min before reperfusion,for 5 times,then reperfused 2 hours.(5)In the SEV+AG490 group,the six mice were randomly selected from the I/R mice and 5 min before reperfusion,AG490 was injected intravenously.The mice were then treated in the same ways as the SEV group.(6)In the SEV+miR-874 inhibitor group,the six mice were randomly selected from the I/R mice.About 24 hours before I/R injury,the miR-874 inhibitor was injected into the myocardium of the mice and then closed the chests.Then the mice were treated in the same ways as the SEV group.(7)In the SEV+miR-874 inhibitor+AG490 group,the six mice were randomly selected from the I/R mice.Intramuscular injection of miR-874 inhibitor was performed 24 hours before I/R injury,and intravenous injection of AG490 was performed 5 min before reperfusion.The mice were treated in the same ways as the SEV group.After the mouse model of myocardial I/R injury was established,SEV was used to pretreat the mouse.When the miR-874 and the JAK2/STAT3 signaling pathways were inhibited,the functional mechanism of miR-874 in I/R injury was explored.Tunel staining was used to detect the apoptosis of cardiomyocytes.The dual-luciferase reporter gene assay was used to not only identify the targeting relationship between miR-874 and STAT3 but also to determine the signal transduction pathway of JAK2/STAT3 and the expression of apoptosis-related genes.Results I.The clinical study found that there were no significant differences between the SEV group and the PRO group in the conditions of the cardioversion,CPB time,aortic block time,operation time and usage of sufentanil.There were no significant differences in the changes of MAP and HR at 4-time points during the operation in both groups.The levels of H-FABP and cTnI in both groups increased gradually from the beginning of the operation to 4 hours after returning to ICU,also the changes were statistically significant.In addition,the levels of H-FABP and cTnI in SEV group were significantly lower than the PRO group at the end of surgery and 4 hours after returning to ICU.It furtherly verified the protective effect of SEV on I/R cardiomyocytes.2.Cell-level studies showed that the relative expression of miR-874 in I/R decreased gradually with the prolongation of I/R time.The activation of miR-874 expression could promote the necrosis of I/R cardiomyocytes,and the inhibition of miR-874 could reduce the necrosis of I/R cardiomyocytes.H9C2 cells were also treated with SEV.RT-PCR was used to detect the expression of JAK2/STAT3 in normal cardiomyocytes and injured cardiomyocytes induced by I/R.It was found that with the prolongation of I/R time,the relative expression of JAK2/STAT3 in I/R cardiomyocytes increased gradually.The inhibited JAK2/STAT3 could promote the apoptosis and necrosis of I/R cardiomyocytes.However,when the JAK2/STAT3 signal transduction pathway was activated,the apoptosis and necrosis of I/R cardiomyocytes were decreased.3.The animal model studies found that the up-regulation of miR-874 was initially observed in the I/R mouse.By inhibiting miR-874,the cardiac function of I/R mouse was improved,cardiomyocyte apoptosis was inhibited(Bax decreased and Bcl-2 increased),and the JAK2/STAT3 signal transduction pathway was activated.STAT3 is the target gene of miR-874,and STAT3 will be up-regulated after inhibiting miR-874.Finally,we found that the role of miR-874 was lost when the JAK2/STAT3 signal transduction pathway was blocked by AG490.Conclusions Through the clinical study,cell level,and the animal model study,it was found that myocardial preconditioning with SEV inhalation anesthesia improved cardiopulmonary function and decreased the levels of cTnI and H-FABP compared with PRO intravenous anesthesia,further verifying the protective effect of SEV preconditioning on myocardial I/R.It revealed that SEV could inhibit the expression of miR-874 in the I/R cardiomyocyte,thus protecting cardiac function.The results show that miR-874 plays an important role in the process of myocardial I/R injury.After pretreatment with SEV,miR-874 can activate the JAK2/STAT3 signal pathway and alleviate the injury of myocardial I/R.In this study,miR-874 inhibitor can target STAT3 through JAK2/STAT3 signal transduction pathway to mediate SEV preconditioning,which can play an important role in myocardial protection,and provides a basis for reducing myocardial I/R injury:immediate or early administration of inhaled anesthetics can exert myocardial protective effect,which also opens up a new treatment for myocardial I/R injury.