Intermedin Protects Against Myocardial Ischemia-reperfusion Injury In Diabetic Rats

BackgroundCardiovascular disease is the leading cause of death inpatients with diabetes,and The mortality rate in diabeticpatients with acute coronary syndromes is higherthan theirnondiabetic counterparts. As diabetic population isexpected to double bythe year2030worldwide, the burdenof cardiovascular disease attributable toepidemic of dia-betes will be increasing. Diabetes mellitus has deleterious effectsnot only oncellular integrity and functions but also on signal trans-ductions.Research showed that diabetic patients were more sensitive to myocardialischemia-reperfusion injury than those non-diabetic patients, and they are morelikely to have serious and fatal myocardial infarction and heart failure. Intermedin(IMD) is a new kind ofcalcitonin gene-related peptide (CGRP) superfamilymembers, and its structure is similar to the CGRP and adrenomedullin, belongingto small molecular polypeptide. They work through the CGRP family receptorssystem-calcitonin receptor (CL)/receptor activity-modifying protein (RAMPs)complexes. Intermedin has obvious protective effects on the cardiovasculardisease, however, the effects of IMD in acute myocardial ischemia reperfusioninjury and itsmechanism, especially in diabetic patients with myocardialreperfusion injury in the rat remain unclear.Therefore, it is of great theoretical andthe actual value to further study the effects and mechanism of IMD in diabetic ratswith myocardial ischemia reperfusion injury.In the present study, we therefore examined the cardioprotective effects ofintermedin against ischemia/reperfusion injury on streptozotocin (STZ)-induceddia-betic rats and explored the underlying mechanism. Part I Experimental study of the protection from IMD in diabeticratmyocardial ischemia reperfusion injuryChapterI Effectsof IMD on oxidativestressinducedbymyocardiumischemia reperfusion indiabetic ratObjectiveTo observe the effects of IMD on myocardial ischemia reperfusion injury indiabetic rats and discuss the mechanism from oxidative stress.Method74healthy male Sprague-Dawley rats were randomly divided into diabeticsgroup(n=50) and non-diabetics group(n=24).Experimental diabetes was inducedin the animals by asingle intraperitoneal administration of STZ dissolved in0.1mol/L citrate buffer (pH4.5) at a dose of55mg/kg.Normal rats received an equalvolume of citrate buffer.Three days post-STZ injection, tail vein bloodglucosesamples were collected and measured with Onetouchglucometer (Johnson&Johnson, USA). The rats wereconsidered diabetic and used for the study only iftheirglucose levels were greater than15mmol/L. Ratswere housed8weeks aftervehicle or STZ injection.Animals were anesthetized intraperitoneally withpento-barbital sodium (50mg/kg) followed by a tracheotomyand an artificialventilation. Blood pressure was recordedfrom the left femoral artery using apressure transducer withheart rate monitored by an electrocardiogram(ECG)throughout the procedure. The left femoral vein was cann-ulated foradministration of drugs. A fourth-intercostal spacethoracotomy was performed,and the pericardium wasexcised to expose the heart. The left anteriordescendingcoronary artery (LAD) was ligated2mm above the leftauricle by a6–0silk suture. A small polypropylene tube wasplaced between the ligature and theLAD. The artery wasoccluded for30min by tightening the ligature. After30minischemia, the ligature was loosened to allow reperfusion for2h. The shamgroup underwent the same surgical proce-dures, apart from tying the6–0silksuture. After2h reper-fusion rats were killed, and parts of the anterior wall of theleft ventricular myocardium near the cardiac apex and bloodsamples wereobtained for further analysisThe rats in non-diabetic group were randomlyreassigned into sham-operated group(NS),I/R group(NIR),the rats in diabeticgroup were randomly reassigned into sham-operated group(DS),I/R group(DIR)and intermedin groups(n=12).Observe the blood glucose, body weight and otherstate changes. Determine the activity of lactatedehydrogenase(LDH) and MBisoenzyme of creatine kinase (CK-MB) in the serum. At the end of experiment,the activities of nitric oxide synthase(NOS), nitric oxide(NO), superoxidedismutase (SOD) and the contents of malondialdehyde(MDA) in myocardiumduring the reperfusion period were measured. Detect the mRNA expression ofp22、p67and gp91in myocardial tissue with PCR.Results1. The blood glucose of rats in diabetic groups was significantly higher thanthat of the respective base value and non diabetic group after3week injectedSTZ(P＜0.05)．And the weight was significantly less than the respective basevalue and non diabetic group (P <0.05or P <0.01). Non diabetic rats eat anddrink normally, their fur was clean and shiny, their reaction was sensitive.Diabetic groups the appetite and the volume of urine were significantly increased,the fur was dirty and dull, unresponsive to external stimulus.2. Compared respectively with those of NS or DS group，the activities ofthe cardiac enzyme LDH and CK-MB as indices of myocardialcellular injurywere measured at the end of reperfusion indifferent groups. Comparing withdiabetic sham group, I/Rcaused a significant increase in LDH and CK-MB india-betic I/R group. The damage evoked by I/R were furtherenhanced in diabeticI/R group comparing with nondiabeticI/R group (both P <0.05). Afterpretreatment with intermedin, the enzyme levels werefound to be attenuated (bothP<0.05vs. Dia I/R group).The activity of SOD, NOS, NO in myocardial weresignificantly lower than that in NS group and DS group (P<0.05); the indexchanges of DIR group was more significant than those of NIR group. Vs those ofDIR group, the activity of CK-MB, LDH in serum, content MDA in myocardial decreased obviously in IMD group, the activity of SOD in myocardial tissue wassignificantly elevated (P <0.05).3. p22、p67and gp91mRAN expression in myocardial tissue of the NIRand DIR was significantly higher than tthat of the NS and DS group, and thedifferences were significant(P<0.05)； DS group vs NS group, there is astatistically significant differences(P<0.05)；Vs the DIR group, p22、p67and gp91mRAN expression levels in myocardial tissue of the IMD group decreasedsignificantly(P<0.05).Chapter II Effects of IMD on ischemia reperfusion-inducedapoptosis in diabetic ratObjectiveObserve the influence of IMD on diabetic rats ischemia-reperfusionmyocardial cell apoptosis and its mechanism.MethodDiabetes model and group are same as the first chapter. Observe themyocardialmorphological changes with light microscopy. Observe the cardiac ultrastructurewith Electron microscope. The apoptotic rate of cardiomyocytes was detected byTUNEL. To detect the protein expression of Caspase-3, Bcl-2, Bax with WesternBlot.Results1. Light microscopy: The part of regional myocardial cells in rats is swellingin NIR and DIR group.Myocardial fiber stripes is unclear or disappeared. Thesechanges were more severe in DIR group. In IMD group, the myocyte is moredegenerate and necrosis than those in DIR group.2. Electron microscopy revealed: In NS group，myocardial cell membrane isintegrity and the particles of matrix is uniform. The myocardial cells of ismild swelling in DS group. Myofibrils arranged neatly. Z line was visible.Mitochondrion is obviously hyperplastic and crest structure is clear. Electrondensity and volume are increased. Myocardial cell structure is not clear in NIRand DIR group. Myofibrils is derangement and sparse. Mitochondrial hyperplasia, edema, and size is different.Part of the mitochondrialcrack, dissolve, formate the dissolved stove. In IMD group, themyocardial ultrastructuremitochondrial damage is significantly reduced than thosein DIR group.3. In NIR and DIR group, myocardial apoptosis rateswere significantly higherthan that in corresponding control group(P<0.05).Vs theNIR group, the apoptotic cells is significantly reduced (P<0.05).4.In NIR and DIR group, Cardiac caspase-3activity was measured by usingcaspase-3activity assay kits (Beyotime institute of biotechnology,)and bax is asignificant increase than that in NS and DS group (P<0.05). Vs the DIR group,the expression of caspase-3and bax in IMD group significantly reduced (P<0.05).Vs NS and DS group, myocardial tissue protein expression of Bcl-2in the NIRand DIR group significantly reduced (P<0.05).In IMD group, theprotein expression of Bcl-2was significantly increased vsDIR group (P<0.05).Chapter IIIEffects of IMD on inflammatory response inducedby myocardium ischemia reperfusion in diabetic ratObjectiveObserve the change of activation of myocardial NF-κB and itsdownstream inflammatoryfactors such as tumor necrosisfactor-alpha(TNF-α),whiteinterleukin-1β (IL-1β) andwhite interleukin-6(IL-6) after myocardium ischemia reperfusion in diabetic rat andits mechanism.MethodDiabetes model and group are same as the first chapter. To determinatethecontent of tumor necrosis factor alpha in (TNF-α), interleukin6(IL-6) andinterleukin1beta (IL-1β)in serum with ELISA method. Determinate mRNAexpression of TNF-α, IL-6and IL-1βinmyocardial tissue withreal time fluorescentquantitative PCR. The translocation of NF-κB in the cardiomyocytes was detectedby immunohistochemistry and the protein expression of NF-κB was determinedwith westernblot.Results 1.In NIR and DIR group, the content of TNF-α, IL-1β and IL-6in serum wassignificantly higher than that in their respective control groups(P<0.05). In DSand DIR group, the above indexes were significantly higher than those in NS andNIR group (P<0.05). The content of TNF-α, IL-6and IL-1β in serum of the ratsin group IMD is lower than that of DIR group (P<0.05).2. After ischemia and reperfusion, the mRNA expression of TNF-α,IL-6,and IL-1β in myocardial tissues in NIR group and DIR were significantlyenhanced than that of their respective control groups, NS group and DSgroup(P<0.05). In IMD group, the expression of mRNA of IL-6, TNF-α andIL-1β decreased obviously than that of DIR group (P<0.05).3. Immunohistochemical results show: NF-κB in myocardial tissues showedlow expression state in NS group and DS group, and the positive expressionappeared in the cytoplasm. In NIR and DIR group,The NF-κB activated, enhancedexpression, positive granules appeared in cytoplasm and nucleus, that is thenuclear translocation.NF-κB activity of IMD group is lower than that of the DI/Rgroup. The nuclei express positive number is significantly less (P<0.05). AfterIschemia reperfusion，the NF-κB cell shift from the iplasma to the nucleus is limit.4. The results Western Blot show: Vs NS and DS group, the NF-κBexpression of the NIR and the DIR group were significantly increased afterischemia and reperfusion (P<0.05).The protein expression of NF-κBin IMDgroup was significantly lower than that in DIR group (P<0.05).Part II Protective effect of intermedin on hypoxia/reoxygenation-inducedinjury of H9c2cells inhigh glucose culture mediumChapterI Building and identification of the eukaryoticexpressionplasmid-intermedin (IMD)Objective:This project aims to build intermedin expression in eukaryotic cells genecarrier pIRES2-EGFP, transfer restructuring plasmid into H9c2myocardial cells,observed the expression of pIRES2-EGFP/IMD plasmid in H9c2cells, andthuslay the foundation for further experiments. Method:First this project, by syntheticing rat IMD CDS region of length,and thenconnectting with the pMD19-TSimple vector, builds pIRES2-EGFP/IMDrecombinant plasmid, whichis tested by restriction enzyme electrophoresis andDNA sequencing. The recombinant plasmid was transfected into the H9c2cellsusing Lipofectamine2000. Then we observed the transfection efficiency withinverted fluorescence microscope and Flow cytometry, selected the optimaltransfection conditions, and detected the expression of IMD mRNA and protein aftertransfection with real time-PCR and western blot.Result:1. Sequencing and restriction analysis showed that this study successfullyconstructed the plasmid of pIRES2-EGFP/IMD.2. The transfection efficiency was the highest when the plasmid:lipofectamine2000transfection complex ratio is3:7.5.3. With real time-PCR and western blot, it finded the expression of IMDmRNA and protein in transfected recombinant plasmid group is higher than in theuntransfected group（P<0.01）.ChapterIIEffectsof IMD on hypoxia/reoxgenation-induced injury ofH9c2cells in high-glucose culture mediumObjective:In thisstudy, we use chemical hypoxiamethod toformatemyocardialischemia-reperfusion mode. H9c2rat cardiaccellsincubated underhighglucose medium. To detect the protective effects and possible molecularmechanism of IMD on hypoxia-reoxygenation injury in H9c2cells under theincubation of the high glucose.Method:H9c2cells were divided into five groups: normal glucose control group(control group); high-glucose control group (HG group); high-glucose hypoxiaand reoxygenation group (the SI/R group); high-glucose IMD plasmid group(IMD group); PD group: the H/R+IMD plasmid+PD98059. The apoptosis rate was observed with flow cytometry. Westernblot were used to detect the proteinsexpression of phosphorylation of ERK1/2.Result:1. SOD activity and myocardialcell viabilitywerestatistically significant in differentgroups(P<0.05). Vs HG group, the SOD activity and myocardialcell viabilityafterhypoxiaand reoxygenationwere significantly lower (P<0.05). After IMD transfected to cells,these indicators significantly increased than the HHR group (P<0.05). After wejoin the ERK1/2inhibitor PD98059into the cell culture medium, the aboveindicators significantly improved vs HHR group (P<0.05).2. LDH activity and MDA level were statistically significant in differentgroups (P<0.05). HG group comparied with the control group: after by highglucose incubated, the LDH and MDA content of myocardial cell significantlyincreased(P<0.05). Vs the HG group above indicators significant increase whenexposed to hypoxia(P<0.01).Vs SI/R group, after IMD transfected cells, itsactivity is significantly reduced(P<0.05). These indicators in PD groupsignificantly improve than the SI/R group (P<0.05). Vs the SI/R group, there is nosignificant difference(P>0.05).3. Cell apoptosis rate was statistically significant in different groups(P<0.05).Vs control group, cell apoptosis rate was significantly increased in HGgroup, and the difference was statistically significant (P<0.05). After hypoxia andreoxygenation, rate of apoptosis increased significantly than in HG group(P<0.05). Rate of apoptosis increased in PD group than in IMD group(P<0.01).Vs the SI/R group, there is no significant difference (P>0.05).4. Western blot showed that:After myocardial cellsincubated by high glucose,pERK1/2protein expressionwas significantlyincreased than in control group(P<0.05).Vs HG group and IMDgroup,the differences were statisticallysignificantin SI/R group (P<0.05). The protein expression of pERK1/2wassignificantly inhibited in PD group (P<0.05). Conclusion:This study confirmed: Diabetes increase myocardialischemia and reperfusioninjury. IMD may bethroughanti-oxidative stress, inhibition of apoptosis,andreduce the inflammatory response, play aprotectiveeffectonmyocardialischemia and reperfusion injuryina high glucose environment.The mechanism may berelatedtotheERK1/2incell signaling pathways.