Inflammatory Regulatory Mechanism Of Mast Cell Involved In The Cardioprotective Effect Of Dexmedetomidine

Part ONE.Cardioprotective effect of dexmedetomidine in patients undergoing cardiac valve replacement with cardiopulmonary bypass:Randomized controlled double-blind studyObjective:To explore the cardioprotective effect of dexmedetomidine in patients undergoing cardiac valve replacement with cardiopulmonary bypass.Methods:Forty adult patients undergoing aortic or mitral valve replacement were randomly divided into two groups(N=20 patients/group):Group DEX,a load dose of dexmedetomidine 1 ?g/kg was given within 10 minutes before induction,and then 0.5?g/kg/h was continuously pumped to the end of operation.Group CON,an equal volume of 0.9%saline was performed.The general data of patients,preoperative LVEF,SBP,HR,and laboratory indicators were noted,as well as the duration of operation,transit,aortic block,extubation,and ICU stay.The changes of SBP and HR before and after the administration of load drug,cut skin,and sternotomy were observed,together with the incidence of hypertension,hypotension,and severe bradycardia after the administration of load dose.The usges of vasoactive drugs and the incidence of PMACE were recorded.The complete blood count was tested before and after operation.The levels of cTnI and TPS in the peripheral blood were detected at preoperation,stop CPB,postoperation,and out of ICU.Multivariate logistic regression was utilized to analyze the accuracy of cTnI,TPS and NLR for predicting PMACE.Results:There was no significant difference in general data and surgical status between the two groups(P>0.05).The change ratio of SBP and HR in Group DEX was higher than those in Group CON after administration of the loading dose,but lower than those in Group CON after cutting skin and sternotomy(P<0.05).Group DEX has an increased incidence of hypertension and severe bradycardia compared with those in Group CON after administration of the loading dose,but the incidence of hypotension was decreased(P<0.05).The usage of phenylephrine and dopamine in Group DEX was lower than that in Group CON,while the usage of atropine was higher in Group DEX than that in Group CON(P<0.05).The incidence of low cardiac output syndrome and malignant arrhythmia was decreased in Group DEX compared with Group CON(P<0.05).Compared with Group CON,the changes of cTnI and TPS were decreased in Group DEX at stop CPB,postoperation,and out of ICU(P<0.05).There was a positive correlation between cTnI and TPS(R2>0.9).The change ratios of neutrophils,monocytes,and leukocytes in Group DEX were higher than those in Group CON,but the change ratios of lymphocytes and NLR were lower(P<0.05).Moreover,the combined use of NLR,TPS,and cTnI to construct a multiple regression model for predicting PMACE can improve the precision of individual predictions,while the AUC of cTnI and NLR,the AUC of TPS and NLR and the AUC of cTnI,TPS and NLR are 0.902,0.892,and 0.895,respectively.Conclusion:The cardioprotective effect of dexmedetomidine in treatment of patients undergoing heart valve replacement with cardiopulmonary bypass can inhibit the stimulation of skin incision and sternotomy,reduce the use of vasoactive drugs and the incidence of PMACE,and decrease the levels of cTnI,TPS,and NLR.In addition,the combination of cTnI,TPS,and NLR has a certain precision in predicting PMACE.Part TWO.Dezmedetomidine pretreatment inhibits degranulation of mast cells against myocardial ischemia/reperfusion injury in ratsObjective:To explore the protective mechanism of dexmedetomidine(DEX)pretreatment against myocardial ischemia/reperfusion(I/R)injury(MIRI)in rats by inhibiting degranulation of mast cells.Methods:A MIRI model was established using rats pretreated with DEX and/or mast cell secretagogue compound 48/80(C48/80),followed by 30 min of ischemia and then 120 min of reperfusion.The preliminary experiment(N=6 rats/group)were assigned to the Sham,MIRI,Group ?(C48/80 0.1 mg/kg.i.v.),Group ?(C48/80 0.5 mg/kg.i.v.)and Group ?(C48/80 1.0 mg/kg.i.v.).Moreover,a total of 72 rats were randomized into 5 groups(N=12 rats/group)as mentioned in the formal experiment:Sham,DEX+I/R(DEX 20 ?g/kg.i.v.),C48/80+I/R(C48/80 0.5 mg/kg.i.v.),and DEX+C48/80+I/R(DEX 20 ?g/kg.i.v.+C48/80 0.5 mg/kg.i.v.).All groups,except Sham group,underwent 30 min of ischemia and then 120 min of reperfusion.Hemodynamics and electrocardiogram were assessed for each heart.TTC staining,hematoxylin-eosin staining,and toluidine blue staining,transmission electron microscope detection,TUNEL staining,ELISA,IHE staining,QRT-PCR,and western blot were detected.Results:The ligation of the left anterior descending coronary artery for 30 minutes of ischemia and 120 minutes of reperfusion was used to establish a rat MIRI model in vivo.The mast cell secretagogue C48/80(0.1,0.5,1.0 mg/kg.i.v.)dose-dependently aggravated these damages induced by I/R injury.Compared with the Sham group,intraoperative hemodynamic disturbances and arrhythmia severity scores,postoperative left ventricular dysfunction and myocardial infarction area ratio,the levels of cTnI and TPS,the myocardial fibers of wavy and edema,the degranulation of mast cells,the ultrastructural damages of left ventricular myocardium and coronary endothelial were significantly increased in the I/R group,while those injury were decreased in the DEX+I/R group and increased in the C48/80+I/R group(P<0.05).In addition,these injuries were partially reversed in the DEX+C48/80+I/R group as compared with C48/80+I/R group(P<0.05).Compared with the Sham group,I/R injury promoted cardiomyocyte apoptosis,increased the positive cell ratio,mRNA and protein expression of HMGB1,TLR4 and NF-?B p65,whereas DEX pretreatment inhibited and C48/80 aggravated these effects(P<0.05).Conclusion:DEX pretreatment may inhibit degranulation of mast cells,repress cascade release of inflammatory factors,and reduce cardiomyocytes apoptosis,thereby preventing I/R injury in vivo.Part THREE.Dexmedetomidine preconditioning alleviates myocardial ischemia/reperfusion injury in isolated rat hearts by inhibiting degranulation of cardiac resident mast cellsObjective:To investigate the cardioprotective mechanism of dexmedetomidine precondition on inhibiting degranulation of cardiac resident mast cells and alleviating MIRI in isolated rat hearts.Methods:Isolated rat hearts on Langendorff perfusion device underwent global ischemia for 30 min and reperfusion for 60 min to induce I/R injury.Pretreatment with C48/80 was conducted using three aproaches to evaluate the effects of C48/80 in pre-experiment as follows(N=6 hearts/group):Control,MIRI,Group ?[(C48/80 1 ?g/mL× 5 min+KHB × 5min)× 4],Group?(C48/80 1?g/mL × 5min+KHB × 5min)and Group ?(a bolus of 20 ?g C48/80).Formal experiments ex vivo consisted of 5 groups(N=12 hearts/group)as follows:Control,I/R,DEX+I/R(10 nM DEX × 30 min),C48/80+I/R(C48/80 1 ?g/mL × 5 min),and DEX+C48/80+I/R(10 nM DEX × 30 min+C48/80 1 ?g/mL × 5 min).All groups,except Control group,underwent global ischemia for 30 min and reperfusion for 60 min to induce I/R injury.Hemodynamics,electrocardiogram and cardiac water content were assessed for each heart.TTC staining,hematoxylin-eosin staining,and toluidine blue staining,transmission electron microscope detection,TUNEL staining,ELISA,IHE staining,QRT-PCR,and western blot were detected.Results:The MIRI Langendorff model of rat isolated heart was successfully constructed by global ischemia for 30 min and reperfusion for 60 min.Repeated intervention of C48/80 induces elution of mast cells in Group ? can improve MIRI,while Group ? and Group ? undergo short-term intervention of C48/80 aggravates I/R damage(P<0.05).I/R injury increased hemodynamic disorders,arrhythmia,myocardial edema and infarction,together with the levels of cTnI and TPS,the myocardial fibers of wavy and edema,the degranulation of mast cells,the ultrastructural damages of left ventricular myocardium and coronary endothelial were increased,whereas DEX pretreatment inhibited and C48/80 aggravated these effects(P<0.05).Compared with the Control group,I/R injury promoted cardiomyocyte apoptosis,increased the positive cell ratio,mRNA and protein expression of HMGB1,TLR4 and NF-?B p65,whereas DEX pretreatment inhibited and C48/80 aggravated these effects(P<0.05).Conclusion:DEX pretreatment inhibits the degranulation of cardiac resident mast cell induced by I/R injury and downregulates the expression of inflammatory factors HMGB1,TLR4,and NF-?B p65.Part FOUR.Dexmedetomidine pretreatment reduces cardiomyocyte damage mediated by mast cell degranulationObjective:To study whether the cardioprotective effect of DEX is associated with the inhibition of mast cell degranulation at the cellular level.Methods:A co-culture system of rat cardiomyocytes H9C2(2-1)and rat mast cells RBL-2H3 was constructed and pretreated with 10 nM DEX and/or 10 ?g/mL C48/80 respectively.The experiment is divided into three groups(N=6 wells/group):Group S(untreated medium cultured for 90 min),Group C(untreated medium cultured for 60 min and treated with 10 ?g/mL C48/80 for 30 min)and Group DC(10 nM DEX pretreated for 60 min,followed by 10 ?g/mL C48/80 treated for 30 min).MTT assay was used for cell viability;flow cytometry and TUNEL assay for cell apoptosis;ELISA for myocardial injury marker cTnI and mast cell marker TPS in supernatant;IHE staining,QRT-PCR and Western blot for the expression of HMGB1,TLR4 and NF-?B p65 of positive cells,mRNA and protein in cardiomyocytes.Results:Compared with Group S,the cell activity of H9C2(2-1)and RBL-2H3 in Group C was significantly decreased,but cell apoptosis and the levels of cTnI and TPS was increased(P<0.05).HMGB1,TLR4 and NF-?B p65 of positive cells,mRNA and protein was significantly increased in Group C compared with that in Group S,but it can be reduced by DEX pretreatment(P<0.05).Conclusion:DEX pretreatment may inhibit the degranulation of mast cells and Inflammation related signaling pathway HMGB1-TLR4-NF-?B p65,thereby reducing the damage of cardiomyocytes mediated by mast cell degranulation.