| Ischemia heart disease (IHD)is one of the most important of adult mortality in Westerncountries. It had become the second of adult mortality in our country and it is the importantcause of SCD in forensic medicine.Study the progress and mechanism of SCD conduce toevaluation in forensic medicine and clinical medicine to treat and prevent.ObjectiveThe purpose of this study was to investigate the effect of the IMD on myocardial injury inacute myocardial infarction(AMI) and ischemia-reperfusion (I/R)related mechanisms.Methods: There are two parts in this research of animal model and cell culture.1,HealthymaleSprague-Dawley rats weighing 250-300g were divided into 5 groups:control group; ischemia group in which the rats heart were subjected to ischemic for 1.5h ;the intermedin-treated ischemia group which the rats were treated by venous injection(iv) of10-7mol/L intermedin 30min prior to the ischemia treatment; I/R group in which therats heartwere subjected to ischemic for 1h followed by 30 min reperfusion ;the intermedin-treated I/Rgroup which the rats was treated by venous injection(iv) of10-7mol/Lintermedin 30 min prior tothe I/R treatment. MABP,HR,LVSP,LVDP and±LVdp/dtmax were detected.Myocardial injurywas determined by measurement of lactate dehydrogenase(LDH)leakage, MDA creation andsuperoxide dismutase (SOD) activity. Cardiomyocytemorphology observation was detectedthrough HE and eleerton microscope obsevration .The mRNA changes of calcitonin receptor likereceptor (CRLR)and receptor activity modifying protein (RAMP)1/2/3 in left ventricular weremeasured by real-time PCR analysis. Myocardial cAMP and cGMP content was determined byenzyme linked immunosorbent assay(ELISA).Expression of apoptosis related factorsBcl-2 andBax were detected by Immunohistochemistry combining with computer graphiec analysis.NOand NOS were estimated by zymochemistry.2,Cell strain of H9c2 was hypoxia and hypoxia /reoxygenation through chemistry method.The cells were also divided into 5 groups: control group(C); hypoxia group(H); intermedin - treated hypoxia group(H+D);hypoxia / reoxygenation group(H/R); intermedin-treated hypoxia /re-oxygenation group(H/R+D).Cell viability was detected by MTT colorimetry ,LDH activity inthe cell culture fluid and SOD and MDA of cardiomyocytes were detected by colorimetry.Cardiomyocyte morphology observation was detected through elecrton microscope obsevration.Fluo-3-based laser confocal scanning microscopy was used for measuring in intracellularcalcium contentof cultured cardiomyocytes.Flow cytometer based on AnnexinV FITC/PIdoublestaining was used to detect apoptotic rates. The mRNA changes of calcitonin receptor likereceptor (CRLR) and receptor activity modifying protein (RAMP)1/2/3 in cardiomyocytes weremeasured by real-time PCR analysis. Cardiomyocytes cAMP,PKA and cGMP content wasdetermined by enzyme linked immunosorbent assay(ELISA). NO and NOS were estimated byzymochemistry.ResultResults1,①The LVSP of I and I/R group lower than N group,but LVDP higher than N group. TheLVSP increased and LVDP decreasd in two IMD groups campared with two injury group.The±dp/dtmax in left cardiac ventricle of I and I/R group lower than N group, but IMD increasedthem(P<0.05or P<0.01).②LDH leakage and MDA of ischemia groupincreased by 60%and140% compared with the control group, while the SOD activity decrease by 50%,administrationwith IMD attenuated the LDH leakage and MDA by 18% and 48%,SOD activity increase by24%compared with the ischemia group (P<0.05 or P<0.01).LDH leakage and MDA of I/R groupincreased by 87%and 189%compared with the control group, while the SOD activity decrease by84%,administration with IMD attenuated the LDH leakage and MDA both by 41%,and SODactivity increase by 38%(P<0.01)compared with the I/R group.③The results of HE stainingshow no specific alteration. Under transmission electron microscope,obvious nuclei,abundantmitochondria and endoplasmic reticulum were seen in control cells and IMD preconditioningcells; shrinking cell,bubbled mitochondria and endoplasmic reticulum were seen in I cells andthe cells of I/R group .④Immunohistochemistry staining showed the significant decrease ofBCL-2/BAX in ischemia group and I/R group, while an increasement was observed in theIMD-treated groups (P < 0.05 or P<0.01).2,①CRLR,(RAMP)1/2/3 in left ventricular mRNA expression elevated in allexperiment groups (P<0.05 or P<0.01), but in IMD groups higher.②The concentration of cyclic adenosinemonophosphate (cAMP)was increased by 1.30 times and 2.87 times in the ischemia group andIMD-treated group, respectively(both P < 0.05), meanwhile, 1.36times and 2.90 times in the I/Rgroup and IMD-treated group, respectively (both P< 0.05).The concentration of cyclic guanosinemonophosphate (cGMP) was increased by 1.87times and 1.14 times in the I group andIMD-treated group, respectively (both P< 0.05), and 1.94times and 2.27 times in the I/R groupand IMD-treated group, respectively.③The concentration of NO and TNOS in two injurygroups were decreased compare with control group(P<0.01),but IMD-treated groups increasedthe concentration, and IMD-treated I/R groups higher.The activity of iNOS in injury groupsincreased compare with control group, but in IMD-treated groups decreased(P<0.05).3,Cell viability and SOD activity decreased, LDH activity and MDA concentrationincreased in two injury group, but in the IMD preconditioning groups converse. Undertransmission electron microscope,obvious nuclei,abundant mitochondria and endoplasmicreticulum were seen in control cells and IMD preconditioning cells; shrinking cell,bubbledmitochondria and endoplasmic reticulum were seen in hypoxia group cells and the cells ofhypoxia / reoxygenation group .The [Ca2+]i in injury groups increase, and IMD reduce it .Apoptotic rates of two injury groups higher than control group, but the rate of IMDpreconditioning groups lower than homologue injury group.4,CRLR,(RAMP)1/2 in cardiomyocytes mRNA expression decrease in all experimentgroups(P<0.05 or P<0.01), but RAMP3 elevated. CRLR,(RAMP)1/2/3 in cardiomyocytes ofIMD preconditioning groups mRNA expression elevated compare with homologue injurygroup.The concentration of cAMP and PKA has no difference in all experiment groups. Theconcentration of cGMP in injury groups elevated compare with control ,and the concentrationhigher in IMD-treated groups. The concentration of NO and TNOS in two injury groups weredecreased compare with control group,but the concentration of IMD-treated groups increased(P<0.05 or P<0.01). The activity of iNOS has no difference in all experiment groups.ConclusionConclusion:Our results suggest that the protective effect of IMD against myocardium injury is due tothe elevate oxidative stress and inhibition of myocardial apoptosis.The protective effect of IMD through stimulation of its receptor complexes by IMD increases cAMP and NO production incells.Otherwise, IMD has similar protective effect in cardiomyocytes by elevate cell viability andreduce apoptotic rates. |
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