The Effect Of Erythropoietin On Endoplasmic Reticulum Stress Associated Proteins (GRP78、CHOP) And Myocardial Apoptosis In Neonatal Rats With Asphyxia

Objectives:1. To observe myocardial injury and myocardial apoptosis in neonatal rats afterasphyxia by establishing the model of neonatal rats with normobaric asphyxia.2. To study the existence of the activation of ERS and CHOP apoptosis pathwayin hypoxic-ischemic heart of neonatal rats after asphyxia,and the relationship betweenmyocardial apoptosis and CHOP apoptosis pathway.3. To study the effect of EPO on myocardial injury and the expression ofmyocardial GRP78and CHOP protein in neonatal rats after asphyxia, and to exploreits possible mechanism.Methods:1. Grouping and model building：Totally120newborn Sprague-Dawley(SD) ratsof seven day-old were randomly divided into3groups (n=40in each group):1.sham-operated group,2.asphyxia group,3.EPO-treated asphyxia group, each groupwas further randomly divided into five time points at2h,6h,12h,24h,48h(n=8ateach time point). Establishing the model of neonatal rats with normobaric asphyxia inboth asphyxia group and EPO-treated asphyxia group, but only simulate the processof the model of neonatal rats with normobaric asphyxia in sham-operated group, donot plug stopper and reoxyg enation;10mg/kg(500U/ml) recombinant humanerythropoietin was intraperitoneally injected to the rats of EPO-treated groupimmediately after establishing the model,the rats of other two groups received thesame dose of normal saline(0.9%). Then the blood and myocardial tissue sampleswere obtained in batches at five time points, which were used for the detection ofrelevant index.2. Detect the level of serum myocardial enzymes CK and LDH;the pathologicalchanges of myocardium were observed under optical microscope after HE staining; Myocardial apoptosis were investigated both qualitatively and quantitatively byTUNEL method; the expression of myocardial GRP78and CHOP were measured byimmunohistochemical staining.3. Statistical methods:using the software of SPSS19.0for statistical analysis, allthe datas were expressed as mean±standard error(x±ｓ), and statistical significancebetween groups was evaluated by One-Way ANOVA, the multiple comparison wasevaluated by SNK-q test, P<0.05represented the difference had statistics meaning,P<0.01represented the difference is significant;the relationship between theexpression of myocardial CHOP and the index of myocardial apoptosis was evaluatedby Pearson correlation analysis.Results:1. The levels of serum myocardial enzyme CK: Compared with sham-operatedgroup, the level of CK rised significantly at different time points in asphyxia groupand EPO-treated asphyxia group(P<0.01), it increased at2h, and continued to go upuntil48h. Compared with the asphyxia group, it decreased significantly at each timepoint in EPO-treated asphyxia group(P<0.01).2. The levels of serum myocardial enzyme LDH: Compared with sham-operatedgroup, the level of LDH rised significantly at different time points in asphyxia groupand EPO-treated asphyxia group(P<0.01), it increased at2h, and continued to go upuntil48h. Compared with the asphyxia group, it decreased significantly at each timepoint in EPO-treated asphyxia group(P<0.01).3. The pathological changes of myocardium:Compared with sham-operatedgroup, myocardial cells were arranged disorderly, edema and congestion happened inmyocardial stroma and infiltration of inflammatory cells can be seen in asphyxiagroup at each time points.Compared with the asphyxia group at same time point,edema and congestion of myocardial stroma and infiltration of inflammatory cells canbe alleviated in EPO-treated asphyxia group.4. myocardial apoptosis marked by fluorescence: Sham-operated group only asmall amount of normal apoptotic cell, apoptotic cells significantly increased in asphyxia group, apoptotic cells in EPO-treated asphyxia group was less than that inasphyxia group,but more than that in Sham-operated group.5. Results and analysis of myocardial apoptotic index (AI): Compared withsham-operated group, the AI increased significantly at different time points inasphyxia group and EPO-treated asphyxia group(P<0.01),it rised at2h,and continuedto go up until48h. Compared with the asphyxia group, it decreased significantly ateach time point in EPO-treated asphyxia group(P<0.01).6. The expressions of myocardial GRP78protein: Compared with sham-operatedgroup, the expressions of myocardial GRP78protein increased significantly atdifferent time points in asphyxia group and EPO-treated asphyxia group(P<0.01),itrised at2h,reached the peak at12h, and was still higher than normal at48h.Comparedwith the asphyxia group, it decreased significantly at each time point in EPO-treatedasphyxia group(P<0.01).7. The expressions of myocardial CHOP protein: Compared with sham-operatedgroup, the expressions of myocardial CHOP protein increased significantly atdifferent time points in asphyxia group and EPO-treated asphyxia group(P<0.01),itrised at2h,reached the peak at24h, and was still higher than normal at48h.Compared with the asphyxia group, it decreased significantly at each time point inEPO-treated asphyxia group(P<0.01).8. The correlation analysis between the expression of myocardial CHOP and theindex of myocardial apoptosis:the expression of myocardial CHOP is positivelycorrelated with the myocardial apoptosis index at2h,6h,12h and24h after asphyxia,but there is no correlation between myocardial CHOP and myocardial apoptosis indexat48h.Conclusions:1. myocardial injury and myocardial apoptosis can appear early in neonatal ratsafter asphyxia,and it continue to go up until48h.2. There exist the activation of ERS and CHOP apoptosis pathway in hypoxic-ischemic heart of neonatal rats after asphyxia. 3. The myocardial apoptosis is related with CHOP apoptosis pathway.4. EPO play a protective effect on hypoxic-ischemic heart of neonatal rats afterasphyxia by reducing endoplasmic reticulum stress, interferencing CHOP apoptosispathway and reducing myocardial apoptosis.