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The Study Of P38MAPK Signal Transduction Pathway Of Liraglutide Acting On SD Neonatal Rat Myocardial Cells Under High Glucose Conditions

Posted on:2015-02-03Degree:MasterType:Thesis
Country:ChinaCandidate:Q X ZhuFull Text:PDF
GTID:2254330428974371Subject:Internal Medicine
Abstract/Summary:PDF Full Text Request
Objective: Diabetic cardiomyopathy as an independent cardiovascularcomplications of diabetes has received increasing attention. Myocardialapoptosis plays an important role in the etiology of diabetic cardiomyopathy,and ultimately develop into heart failure. Now,there is no measure can preventthe formation and progression of diabetic cardiomyopathy. Glucagon-likepeptide-1(GLP-1) secreted by intestinal L endocrine cells which belongs toone of an important incretin, GLP-1analogues-liraglutide as antidiabeticdrugs have been widely used clinically,its role out of pancreatic especially onmyocardial cells has attracted wide attention in the field of diabetes. Thisstudy was designed to investigate the effects of liraglutide for SD neonatal ratmyocardial cells under high glucose conditions and its possible mechanism.Methods:1Extract SD neonatal rat cardiomyocytes: Take201-3days old healthySD (Sprague Dawley) rats, kill them and take the apical, application of trypsinand collagenase Ⅱ Digestive and extract the the cells,then through thedifferential adherence method purified cardiomyocytes and when cultivatethem in a cell culture incubator for48hours,we should change culturedmedium and Continue to culture them for24hours. Now,you can observemyocardial cells adhere well and contact with each other and filopodiainterwoven into a network as well as appear synchronous pulse. The cellmorphology observed by inverted phase contrast microscope and fluorescencemethod for identification.2Intervene and divide into groups: divide The myocardial cells culturedfor72hours into7groups,respectively, Normal glucose group (5.5mmol/Lglucose), hypertonic group (5.5mmol/L glucose+19.5mmol/L mannitol), high glucose control group (25.0mmol/L glucose), high glucose+lowconcentration of liraglutide group (25.0mmol/L glucose+50nmol/Lliraglutide), high glucose+medium concentration liraglutide group (25.0mmol/L glucose+100nmol/L liraglutide), high glucose+high concentration ofliraglutide group (25.0mmol/L glucose+200nmol/L liraglutide), Normalglucose group+high concentration liraglutide group (5.5mmol/L glucose+200nmol/L liraglutid). The cells cultured for next72hours (48hours for asolution).3Apoptosis Detection: The seven groups of cells fixed by alcohol andthen Use flow cytometry instrument to detect cell apoptosis rate.4Determination of protein expression: Extract the seven groups of cellsproteins and detecte of apoptosis proteins caspase-3andP-P38MAPK/P38MAPK by Western blot.5Data analysis: The data were presented as mean±standard deviation (x±s) that the statistical analysis using SPSS13.0software. Multiple sampleswere compared by analysis of variance (One-way ANOVA), all pairwisecomparisons between the number of q test. P <0.05as there are significant; P<0.01as there are very significant.Results:1Generally observation: Compared with the cardiomyocytes of normalglucose group, high glucose control group have poorer growth state andweaker pulse, high glucose with liraglutide intervention group betweenlnormal glucose group and high-sugar group, hypertonic control group andlow glucose twith high liraglutide group have no significant difference withthe normal glucose group.2Apoptosis rate: The apoptosis rate of high glucose control group compairedwith normal glucose group increased significantly (19.39±1.37%vs3.33±0.32%, P<0.01), high glucose with liraglutide intervention group islower significantly than high glucose control group (P <0.01), but higher thanlow glucose control group (P <0.01). The apoptosis rate of highconcentrations of liraglutide below the low, medium concentrations of liraglutide group (7.11±1.42%vs12.02±0.88,11.27±1.93,P<0.05), but lowerconcentrations of liraglutide group has no significant difference with mediumconcentrations of liraglutide group (P>0.05), hypertonic control group andnormal glucose with high liraglutide group have no significant difference withthe normal glucose group (3.29±0.48,2.88±0.98vs3.33±0.32, P>0.05).3Western blot:3.1Expression of caspase-3: The expression of caspase-3of high glucosecontrol group compaired with normal glucose group increased significantly (P<0.01), high glucose with liraglutide intervention group is lower than highglucose control group(P <0.01) but higher than lnormal glucose group (P<0.01). The expression of caspase-3of high concentrations of liraglutide(200nmol/L) is lower than the low and medium concentrations of liraglutidegroup (P <0.01), but lower concentrations of liraglutide group has nosignificant difference with medium concentrations of liraglutide group(P>0.05). In addition, hypertonic control group and normal glucose with highliraglutide group have no significant difference with the normal glucose group(P>0.05).3.2Expression of P-p38MAPK: The expression of P-p38MAPK of highglucose control group compaired with normal glucose group increasedsignificantly (P <0.01), high glucose with liraglutide intervention group islower than high glucose control group(P <0.01) but higher than normalglucose group (P <0.01). The expression of P-p38MAPK of highconcentrations of liraglutide is lower than the low and mediumconcentrations of liraglutide group (P <0.01), but lower concentrations ofliraglutide group has no significant difference with medium concentrations ofliraglutide group (P>0.05). In addition, hypertonic control group and normalglucose with high liraglutide group have no significant difference with thenormal glucose group (P>0.05),too.Conclusion:1High glucose can increase the myocardial apoptosis of SD neonatalrat.However liraglutide can fight this myocardial apoptosis. 2The expression of caspase-3of liraglutide intervention group is lowerthan high glucose group, proves that liraglutide can inhibit the expression ofapoptosis protein; high glucose control group increased expression ofphosphorylated P38MAPK than low sugar control groupe and liraglutide canmake the phosphorylate of P38MAPK induced by high glucose weaker,indicating P38MAPK signal transduction pathway is possible one of the wayliraglutide play a protective role on SD neonatal rat cardiac cells.
Keywords/Search Tags:Myocardial cells, Apoptosis, P38MAPK, The liraglutide
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