Research On The Protection And The Mechanism Of The Injure Of Ischemia And Reperfusion In The Cardiocytes Of Rats

Object:To establish myocardial ischemia reperfusion injury model in vitro and rat cardiac myocytes to hypoxia-reoxygenation injury model, use Annexin V-FITC/PI double staining immunohistochemical method by flow cytometry, MTT assay and Western blot were used to detect cell apoptosis, cell proliferation activity, and signal transducer and activator of transcription level of sub-3(STAT3) phosphorylation to observe the role of the protective effect and mechanism of reactive oxygen species (ROS) in myocardial ischemia and reperfusion injury, explore the ROS in the mechanism of ischemic postconditioning activated tyrosine kinase2; signal transduction and transcription factor3(of JAK2-STAT3) pathway.Methods:1. The rats (body weight240～280g) were divided into four groups:(1) blank control group (Sham group):use6/0no damage suture through the left anterior descending artery after thoracotomy, do not block the blood vessels.(2) ischemia-reperfusion injury group (I/R group):6/0no damage suture blocking the left coronary artery, open blood vessels after30min.(3) treated group after ischemia (PostC group):immediately Ischemic postconditioning when the ischemia30min (open10s followed by blocking the10s, repeated3times).(4) MPG group (PostC MPG group):5min intravenous injection of free radical scavenger, the MPG (20mg/kg) in the reperfusion of ischemic postconditioning. Other dealing with the treatment group after ischemia. Intraperitoneal injection of10%chloral hydrate (3mg g) The rat limbs connected ECG monitoring electrodes. Separation of the right common carotid artery placement artery piezometric; Isolated and performed a tracheotomy to connect small animal ventilator, gas source for the indoor air, respiratory rate60beats/min, tidal volume13to15ml. The left side of the4th intercostal thoracotomy, using6/0no damage suture through the left anterior descending artery and block blood vessels. ECG showed ST segment significantly raise that coronary occlusion success. Blocked after30min release the suture revascularization, ECG ST-segment. Eight rats were randomly select the eight experimental rats reperfusion2h take the myocardial specimens, the use of cardiomyocyte apoptosis detection kit reperfusion10min to take myocardial specimens for the detection of JAK2-STAT3pathway activity; cardiomyocyte apoptosis, the detection of myocardial tissue of the immunohistochemical staining of bcl-2and bcl-xL protein levels by Western blot detection of phosphorylated STAT3(P-STAT3) and total of STAT3(t-STAT3) levels.2. Rat myocardial cells (H9C2) by in vitro culture, using the logarithmic growth phase cells as experimental treatment. The experimental part is respectively used at concentrations of0,5,10,20,50,100μmol/L H2O2treated H9C2cells, in order to determine the optimum concentration of H2O2pretreatment. Effect of low concentration H2O2preconditioning on hypoxia/reoxygenation induced cell damage in rats. To determine the optimum pretreatment concentrations after experimental partial cells were divided into four groups:control group:conventional culture. The hypoxia/reoxygenation groups:an advance in5%CO2,95%N2culture box120min,30min reoxygenation. The H2O2pretreated group:a given to determine the concentrations of H2O290min exchange,24h after repetitive hypoxia/reoxygenation processing. The JAK2-STAT3blocker (AG490)+H2O2group:in H2O2pretreatment beforelO min tolOμmol/L AG490processing, more than with the H2O2pretreated group. After completion of treatment using Annexin V-FITC/PI double staining method and FCM method to detect apoptosis; MTT assay for detection of H9C2cell proliferation activity; Western blot method for the detection of signal transducer and activator of transcription3(STAT3) phosphorylation levels.Results:1. Anti-apoptotic effects of ischemic postconditioning:Visible trace of bcl-2and bcl-xL protein expression, no significant cardiac myocyte apoptosis in the myocardial tissue of sham operation group. Ischemia-reperfusion injury group, bcl-2and bcl-xL protein were significantly increased, showing a large number of cardiac myocyte apoptosis. Ischemic postconditioning significantly increase the expression of bcl-2(P<0.05) and bcl-xL (P<0.05) protein levels and inhibition of cardiomyocyte apoptosis (P<0.05) after reperfusion. 2. The MPG (radical scavenger) to deal with the effects of anti-apoptotic effects after ischemia:ischemic postconditioning free radical scavenging agent MPG allows reperfusion in myocardial tissue of bcl-2(P<0.05) and bcl-xL(P<0.05) protein levels were significantly lower, and significantly increased after reperfusion myocardial apoptosis ratio (P<0.05).3. MPG of JAK2-STAT3pathway impact:by Western blot detection found that ischemic postconditioning significantly increase after reperfusion the level of STAT3phosphorylation (P<0.05). The use of MPG before reperfusion significantly reduce the ischemic and post-processing of p-STAT3level (P<0.05), inhibition of JAK2-STAT3pathway activity.4. Different concentrations of H2O2pretreatment in H9C2cells:Compared with other concentrations of20μmol/L group the level of STAT3phosphorylation was significantly increased;20umol/L group compared with50μmol/L and100μmol/L group, myocardial apoptosis rate was significantly reduced;20μmol/L group of myocardial cell viability and cell apoptosis rate and the5and10μmol/L group difference not statistically significant.5.20μmol/L H2O2pretreatment on myocardial cell apoptosis rate, cell viability and the level of STAT3phosphorylation:Hypoxia/reoxygenation group than in the control group apoptosis rate was significantly increased, the difference was statistically significant (P<0.01); H2O2pretreatment group compared with hypoxia/reoxygenation group apoptosis rate were significantly lower, the difference was statistically significant (P<0.01); Between hypoxia/reoxygenation group and AG490groups, apoptosis rate were no significant difference. Hypoxia/reoxygenation group and of AG490+H2O2group of cell vitality than the control group decreased significantly, the difference was statistically significant (P<0.01); H2O2pretreatment group compared with hypoxia/reoxygenation were significantly higher, the difference was statistically significant (P<0.01). The results show that H2O2preconditioning significantly increased the level of STAT3phosphorylation; of AG490+H2O2group STAT3phosphorylation levels were significantly lower than that of H2O2pretreatment group. Conclusions:1. ROS play an important role in the mechanism of anti-cardiomyocyte apoptosis after ischemia treatment. Ischemic post-processing mechanism, ROS possibly through activation of JAK2-STAT3pathway and further raised the level of apoptosis protein bcl-2and bcl-xL, and so on, to play against myocardial cell apoptosis.2.20μmol/L H2O2pretreatment can be a significant activation of JAK2-STAT3pathway after reperfusion, and H9C2cardiomyocytes during hypoxia/reoxygenation injury has adaptive protective effect.