| Objective:Percutaneous coronary intervention(PCI)was a common clinical practice to open stenosis for the treatment of acute myocardial infarction.After blood flow restored in ischemic myocardial tissue,the injury was further aggravated,known as myocardial ischemia/reperfusion(I/R)injury,and treatment of myocardium I/R injury was a major issue.In early stage of our research work,the clinical commonly used calcium channel blockers verapamil,nifedipine and a new type of calcium channel blocker iodide N-n-butyl haloperidol iodide in a does which did not affect cardiac function(call it ’subthreshold dose’),improving the cardiac function of ischemia reperfusion model mice,and reducing the leakage of myocardial enzyme;The expression of macrophage migration inhibitory factor(MIF)could be regulated by verapamil and N-n-butyl haloperidol iodide to reduce the hypoxia/reoxygenation injury of L-VDCC-/-H9c2 myocytes,cardiac endothelial cells and primary myocytes,determining that calcium channel blockers had other ways to antagonize I/R or H/R injury at the overall and cellular level.Our group had further carried out clinical trials to observe the effect of ’subthreshold dose’ verapamil on I/R injury in patients with acute myocardial infarction.This study measured and compared the plasma concentration of calcium channel blockers to provide evidence for observing the ’subthreshold dose’ used by the treatment of myocardial ischemia reperfusion injury.As a secreted protein,MIF played an important role in activating intracellular signaling pathways by binding to cell surface receptor CD74.Therefore,this study investigated whether verapamil and N-n-butyl haloperidol iodide affect MIF concentration in cell culture medium under hypoxic/reoxygenation state.Methods1.C57BL/6JNifdc mice were randomly divided into groups(6 mice in each)and intraperitoneally injected with:Subthreshold dose:verapamil 1.0 mg/kg group;nifedipine 0.3 mg/kg group;diltiazem 0.5 mg/kg group;N-n-butyl haloperidol iodide 1.0 mg/kg group;Minimum effective dose:verapamil 1.5 mg/kg group;nifedipine 0.4 mg/kg group;diltiazem0.75 mg/kg group;N-n-butyl haloperidol iodide 1.5 mg/kg group.Nifedipine groups received blood samples from the posterior orbital venous plexus 5,10,15,and 20 min after intraperitoneal injection,the other groups were 2,5,10 and 15 min after intraperitoneal injection.At each time point,210 μL of blood was collected and centrifuged in an EDTA-treated 1.5 mL centrifuge tube for 10 min(4℃,3000 RPM).The blood samples were frozen at-80℃ until determined by high performance liquid chromatography tandem mass spectrometry.2.Clinical patients were randomly divided into groups.Oral administration before PCI:50 patients in the experimental group were given 20 mL warm water containing verapamil 20 mg orally;30 patients in the placebo group were given 20 mL warm water orally.After administration,1 mL of blood was collected through elbow vein at 1.5 h.The blood was placed in a disposable EDTA-K2 intravenous sample collection container,centrifuged for 10 min(4℃,3000 RPM),and stored frozen at-80℃ until determined by high performance liquid chromatography tandem mass spectrometry.3.Cultured primary cardiomyocytes,cardiac endothelial cells and L-VDCC-/-H9c2 cardiomyocytes were replaced with anoxic fluid(or containing drug)saturated with high purity N2 for 30 min,and placed in the hypoxia workstation(1%O2-5%CO2,37℃),then normal culture medium(or normal culture medium containing drug)was replaced,and placed in the carbon dioxide incubator(37℃,saturated humidity,5%CO2),cell culture medium was collected.The above cell samples were lysed and the cell lysates were collected.Enzyme linked immunosorbent assay(ELISA)was used to determine the concentration of cell lysate and cell culture medium MIF.Result1.In this study,the high performance liquid chromatography tandem mass spectrometry method was established for simultaneous determination of calcium channel blockers.The method was stable and reliable,and could be applied to the determination of calcium channel blockers in human and mice.2.The peak plasma concentrations of the subthreshold dose of verapamil,nifedipine,diltiazem,and N-n-butyl haloperidol iodide in mice were lower than those of the minimum effective dose.3.The plasma samples of 50 patients with clinical acute myocardial infarction were determined,49 of which was lower than the lowest effective concentration 20 ng/mL.4.The MIF concentration of primary cardiomyocytes,cardiac endothelial cells and L-VDCC-/-H9c2 cardiomyocytes in culture medium showed a ’high to low’ aging relationship,which was highest at H:1h/R:1h(endothelial cells:H:1h/R:2h)and lowest at H:4h/R:1h(endothelial cells:H:4h/R:2h).5.The increase of MIF concentration of cardiomyocyte culture medium was earlier than that of intracellular MIF expression under hypoxia state.6.Verapamil and N-n-butyl haloperidol iodide reduced the concentration of MIF in primary myocardial cells,cardiac endothelial cells,and L-VDCC-/-H9c2 myocardial cells at H:1h/R:1h(endothelial cells at H:1h/R:2h).7.Verapamil and N-n-butyl haloperidol iodide increased the concentration of MIF in primary myocardial cells,cardiac endothelial cells,and L-VDCC-/-H9c2 myocardial cells at H:4h/R:1h(endothelial cells at H:4h/R:2h).Conclusion1.Oral administration of verapamil 20 mg in patients with clinical acute myocardial infarction was subthreshold dose.Intravenously injected with verapamil 1.0 mg/kg,nifedipine 0.3 mg/kg,diltiazem 0.5 mg/kg,and N-n-butyl haloperidol iodide 1.0 mg/kg in mice were subthreshold doses by and large,the observed drug effects may be mediated through pathways other than the calcium channel.2.Under hypoxia state,the change of MIF secretion in myocardium was earlier than the change of intracellular expression.3.Combined with the previous results,the reason that verapamil and N-n-butyl haloperidol iodide protected primary myocardial cells,cardiac endothelial cells and L-VDCC-/-H9c2 myocardial cells against H/R injury may be related to the effect on extracellular MIF concentration. |
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