| Part One:p38MAPK participates in calcium overload and apoptosis during myocardial ischemia-reperfusion injury in rats by down-regulating the expression of SERCA2Objective:To investigate whether p38MAPK-SERCA2 signaling pathway is involved in calcium overload and apoptosis during myocardial ischemia-reperfusion injury in rats.Methods:Sixty male SD rats(body weight 250-300 g)were randomly divided into three groups(n=20 for each group).Sham operation group(Sham group),ischemia/reperfusion group(I/R group),ischemia/reperfusion+SB203580 group(I/R+S group).The p38MAPK inhibitor SB203580(2mg/kg)was intraperitoneally injected into I/R+S group rats 1 hour prior to surgery.The other two groups were injected with an equal volume of vehicle solvent at 1 h before surgery.The animal model of myocardial ischemia-reperfusion injury(MI/RI)was established by ligation of the left anterior descending coronary artery(LAD)for 30 min and then reperfusion for 30 min.The arterial blood gas analysis method was used to monitor the internal environment of rats.The expression of p-p38MAPK,p38MAPK,SERCA2,p-STAT3 and STAT3 protein in myocardial tissue was detected by Western blotting.The mRNA expression of SERCA2,IL-1β,IL-6 was detected by Real-time quantitative PCR(RT-qPCR).The area of ischemia and myocardial infarction determination was detected by Evans Blue and 2,3,5-triphenyl tetrazolium chloride(TTC)double staining method.The concentration of cardiac troponin I(cTnI)in rat serum was detected by ELISA.The calcium concentration in rat cardiomyocytes was detected by calcium detection assay kit.The cardiomyocyte apoptosis was detected by TUNEL staining.Results:There were no significant differences in blood gas analysis results between the groups.Compared with Sham group,myocardial infarct size was significantly increased(P<0.05),serum cTnI concentration was significantly increased(P<0.05),the protein expression of p-p38MAPK,p-STAT3 in myocardial tissues were significantly up-regulated(P<0.05),the protein and mRNA expression of SERCA2 was down-regulated(P<0.05),the mRNA expression of IL-1β and IL-6 and the concentration of intracellular calcium in cardiomyocytes were significantly increased(P<0.05)in I/R group and I/R+S group,the ratio of TUNEL-positive cardiomyocytes in I/R group was increased significantly(P<0.01);Compared with I/R group,myocardial infarct size was significantly decreased(P<0.05),serum cTnI concentration was significantly lowered(P<0.05),and the protein expression of p-p38MAPK and p-STAT3 in myocardial tissues was significant down-regulated(P<0.05),SERCA2 protein and mRNA expression were significantly up-regulated(P<0.05),the mRNA expression of IL-1β and IL-6 were significantly decreased(P<0.05),the concentration of intracellular calcium and the ratio of TUNEL-positive cardiomyocytes were decreased significantly(P<0.01)in I/R+S group.Conclusion:p38MAPK-SERCA2 signaling pathway is involved in myocardial ischemia-reperfusion injury in rats.Inhibition of p38MAPK phosphorylation can protect against MI/RI by reducing calcium overload,myocardial apoptosis,and inflammatory response.Part Two:HSP70 mediates p38MAPK signal to participate in calcium overload and apoptosis during myocardial ischemia-reperfusion injury in ratsObjective:To investigate whether HSP70 mediates p38MAPK signal to participate in calcium overload and apoptosis in myocardial ischemia-reperfusion injury in rats.Methods:80 male SD rats(body weight:250～300g)were randomly divided into 4 groups(n=20 for each group):Sham operation group(Sham group),ischemia/reperfusion group(I/R group),ischemia/reperfusion+Quercetin group(I/R+Q group),ischemia/reperfusion+Quercetin+SB203580 group(I/R+Q+S group).The rats in I/R+Q group was intraperitoneally injected with Quercetin(20mg/kg),the I/R+Q+S group was intraperitoneally injected with Quercetin(20mg/kg)and p38MAPK inhibitor SB203580(2mg/kg),and the other groups were only injected with the same volume of solvent at 1h before surgery.The animal model of myocardial ischemia-reperfusion injury was established by ligating the anterior descending branch of the left coronary artery(LAD)for 30min and then reperfusion for 30min.The arterial blood gas analysis method was used to monitor the internal environment of rats.The expression of HSP70,p-p38MAPK,p38MAPK,SERCA2,p-STAT3 and STAT3 protein in myocardial tissue was detected by Western blotting.The mRNA expression of SERCA2,IL-1β,IL-6 was detected by Real-time quantitative PCR(RT-qPCR).The area of ischemia and myocardial infarction determination was detected by Evans Blue and 2,3,5-triphenyl tetrazolium chloride(TTC)double staining method.The concentration of cardiac troponin I(cTnI)in rat serum was detected by ELISA.The calcium concentration in rat cardiomyocytes was detected by calcium detection assay kit.The cardiomyocyte apoptosis was detected by TUNEL staining.Results:There were no significant differences in arterial blood gas analysis between the four groups(P>0.05).Compared with Sham group,myocardial infarct size was significantly increased(P<0.05),serum cTnI concentration was significantly increased(P<0.05),the protein expressions of HSP70,p-p38MAPK and p-STAT3 in myocardial tissues were significantly up-regulated(P<0.05),and the protein and mRNA expressions of SERCA2 were significantly down-regulated(P<0.05).The mRNA expressions of IL-1βand IL-6 in myocardial tissues were increased significantly(P<0.05),the intracellular calcium concentration and the ratio of TUNEL-positive cardiomyocytes were significantly increased(P<0,05)in I/R group,I/R+Q group and I/R+Q+S group;Compared with I/R group,the myocardial infarct size was significantly increased(P<0.05),the serum cTnI concentration was significantly increased(P<0.05),and the p-p38MAPK and p-STAT3 protein expression levels were significantly up-regulated(P<0.05),HSP70 protein expression,SERCA2 protein and mRNA expression were significantly down-regulated(P<0.05),the mRNA expression of IL-1β and IL-6 in myocardial tissues was significantly increased(P<0.05),the intracellular calcium concentration and the ratio of TUNEL-positive cardiomyocytes were significantly increased(P<0.05)in the I/R+Q group;Compared with I/R+Q group,myocardial infarct size was significantly decreased(P<0.05),serum cTnI concentration was significantly lowered(P<0.05),the protein expressions of p-p38MAPK,p-STAT3 were significantly down-regulated(P<0.05),SERCA2 protein and mRNA expression levels were significantly up-regulated(P<0.05),IL-1β and IL-6 mRNA expressions were significantly decreased(P<0.05),the intracellular calcium concentration and the ratio of TUNEL-positive cardiomyocytes were significantly reduced(P<0.05)in I/R+Q+S group.Conclusion:HSP70 signal is involved in calcium overload and apoptosis during MI/RI,and its mechanism depends on the regulation of p38MAPK phosphorylation level. |
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