The Role Of MAPK Pathway In High Glucose-induced Myocardial Apoptosis

Objects:Apoptosis in diabetic cardiomyopathy is one of the mostimportant pathological changes in diabetic cardiomyopathy, Mitogen-activatedprotein kinase (mitogen-activated protein kinases, MAP kinases, MAPK) isone of the important ways of signaling chain network in eukaryotes.It is aclass of serine/threonine protein kinase widely present in eukaryotic cells. Itis a common pathway or focal point that induces gene expression,cellproliferation of nuclear reaction stimulated by a variety of ischemia, hypoxia,hormones, growth factors and cytokines of extracellular. It plays a key role inthe regulation of gene expression and functional activity in thecytoplasm.MAPK pathway plays an important role in apoptosis.The purposeof the study is to investigate high glucose acting on diabetic cardiomyocyteapoptosis and myocardial anti-apoptosis effect of MAPK to explore new targetof drugs prevention in diabetic cardiomyopathy by using primary culturedneonatal rat cardiomyocytes as a model.Methods:1Culture method of cardiomyocytes:Ventricular myocytes were isolatedfrom hearts of the Newborn1-3daysˊneonatal SD rats under sterileconditions.The apicals were obtained and cut into1mm3approximately andthe cyocytes were isolated by mixed digestive enzymes of0.1%trypsin and0.04%collagenase Ⅱ.10%fetal bovine serum was used to terminate digestion.Differential adherent method was used to isolated cardiac cells.Then cellswere cultured at37℃,5%CO2incubator.Brdu was added in the culturemedium to inhibit fibroblast growth. Culture medium was changed every48hours.Inverted phase contrast microscope were used to observemorphology.The purity was identified by immunocytochemical staining.0.4%trypan blue staining was used to measure cardiomyocytes survival. 2Group divided:Neonatal rats’cardiac myocytes were randomly dividedinto seven groups:control group (the glucose was5.6mmol/L); high glucosegroup:(the glucose was25mmol/L);hypertonic control group(addedD-mannitol of19.5mmol/L to control group);DMSO group (high glucosegroup+Pretreated cells for30minutes with1μ l/ml DMSO);SB203580group:(high glucose group+pretreated cells for30minutes with P38inhibitorsSB203580);PD98059group(high glucose group+pretreated cells for30minutes with ERK inhibitors PD98059);SP600125group:(high glucosegroup+pretreated cells for30minutes with JNK inhibitors SP600125).3Detection index：Apoptosis was detected by flow cytometry in neonatalrat cardiomyocytes after cultured for72hours. Expression of cell apoptosisrelated protein caspase-3and expression of p-JNK、 p-P38was detected byWestern blotting assay.4Data analysis: The data was presented as mean±standard deviation (x±s). The statistical analysis was used by SPSS13.0software.The one-wayANOVA and q test were used for pairwise comparisons between groups. P<0.05was considered as statistically significant.P <0.01was considered asstatistically significant difference.Results:1The cardiac myocytes were harvested by extraction of mixed enzymaticdigestion and highly purified by different adhesion time.They showedα-smooth muscle actin positive by cell immunohistochemistry technique.2Flow cytometry results： compared with the control group, thehypertonic group apoptotic ratio had no significant changes (P＞0.05),apoptosis in high glucose group was significantly increased (P<0.01);compared with high glucose group, apoptosis in PD98059group andDMSO groups had no significant changes(P＞0.05), apoptosis in SP600125and SB203580group were both decreased (P <0.01).3The expression of caspase-3: compared with the control group,caspase-3expression in hypertonic group had no significant changes (P＞0.05), and caspase-3expression in high glucose group increased (P <0.01); compared with high glucose group, caspase-3expression in PD98059grouphad no significantly changes(P＞0.05), caspase-3expression in SP600125andSB203580group decreased (P <0.01).4JNK expression of results: compared with the control group, p-JNK inhypertonic group showed no significant changes(P＞0.05),expression ofp-JNK in high glucose was significantly increased(P <0.01);expression ofp-JNK in SP600125group decreased significantly compared with that in highglucose group (P <0.01).5P38expression of results: compared with the control group, p-P38inhypertonic group showed no significant changes(P＞0.05),the expression ofp-P38in high glucose was significantly increased(P <0.01);p-JNK inSP600125group decreased significantly compared with that in high glucosegroup (P <0.01).Conclusions:1High glucose of25mM induced apoptosis of cardiomyocytes incultured SD neonatal rats in vitro.2High glucose may activate caspase-3by activating P38and JNKpathway to induce cardiomyocyte apoptosis.3P38inhibitor and JNK inhibitor had anti-apoptosis in diabeticcardiomyopathy.