| Part ? Sevoflurane postconditioning attenuated myocardial ischemia/reperfusion injury:the role of TOPK/PTEN/Akt signaling pathway.Objective To investigate the effects of sevoflurane postconditioning (Sevo-PostC) on myocardial ischemia/reperfusion or hypoxia/reoxygenation injury and to determine whether the effects are related to the TOPK/PTEN/Akt signaling pathway.Method Male C57BL/6 mice of 15-16 weeks old were randomly divided into five groups:sham group (Sham); ischemia/reperfusion group (IR); sevoflurane postconditioning group (Sevo-PostC); sevoflurane postconditioning+LY294002 group (Sevo-PostC+LY); sevoflurane postconditioning+HI TOPK-032 group (Sevo-PostC+HI). In vivo myocardial ischemia model was induced by temporarily exteriorizing the heart via a left thoracic incision, and placing an 8-0 silk suture slipknot around the left anterior descending coronary artery. After 45 min ischemia, the slipknot was released and the myocardium was reperfused for 2 h. The arterial blood pressure and heart rate were continuously monitored before and during the entire ischemia/reperfusion procedure. The H9c2 cell were cultured in 1000 mg/dl glucose DMEM supplemented with 10%(v/v) fetal bovine serum for three days and were also randomly divided to five groups:control group (Control); hypoxia/reoxygenation group (HR); sevoflurane postconditioning group (Sevo-PostC); sevoflurane postconditioning+LY294002 group (Sevo-PostC+LY); sevoflurane postconditioning+HI TOPK-032 group (Sevo-PostC+HI). H9c2 cells were subjected to 3 hours hypoxia followed by 6 hours reoxygenation. Myocardial infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining. The cell viability was detected by MTT colorimetric assay. Myocardial CK-MB content was detected by ELISA and LDH content was measured by colorimetric method. Myocardial apoptosis was detected by terminal dexynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL). The protein levels of TOPK, PTEN, Akt and its phosphorylation were measured by Western Blot method.Result In vivo, Sevo-PostC significantly decreased cardiac ischemia/reperfusion injury reflexed by elevated the mean artery pressure at the end of reperfusion period, reduced myocardial infarct size, decreased plasma CK-MB and LDH release as well as myocardial cellular apoptosis when compared with IR group (P<0.05). In vitro, Sevo-PostC remarkably elevated the cellular viability and reduced LDH release after cellular hypoxia/reoxygenation injury (P<0.05). However, the cardioprotective effects induced by Sevo-PostC were abolished by TOPK inhibitor or PI3K inhibitor (P<0.05). The levels of TOPK, PTEN and Akt phosphorylation were significantly increased by Sevo-PostC when compared with IR group (P<0.05). In addition, TOPK inhibitor remarkably blocked the phosphorylation of p-PTEN and p-Akt compared with that in the Sevo-PostC group (P<0.05). However, PI3K inhibitor only inhibited the phosphorylation of p-Akt compared with that in the Sevo-PostC group (P<0.05).Conclusion Sevo-PostC attenuates myocardial ischemia/reperfusion injury via activation of TOPK/PTEN/Akt signaling pathway.Part ? Diabetes blocks the cardioprotective effects of sevoflurane postconditioning by impairing TOPK/PTEN/Akt signaling pathway.Objective To investigate the effects of diabetes on the cardioprotection induced by Sevo-PostC and to explore whether it is related to the impairment of TOPK/PTEN/Akt signaling pathway.Method Diabetic mice were introduced by intraperitoneal injection of 40 mg/kg STZ for five consecutive days. Daily water intake consumption, food consumption, body weight and plasma glucose level were recorded after STZ injection. After eight weeks, all mice were randomly divided into four group:ischemia/reperfusion group (IR); sevoflurane postconditioning group (Sevo-PostC); diabetic+ischemia/reperfusion group (Diabetic+ IR); diabetic+ sevoflurane postconditioning (Diabetic+Sevo-PostC). The H9c2 cell were cultured in 1000 or 4500 mg/dl glucose DMEM supplemented with 10%(v/v) fetal bovine serum for three days and were also randomly divided to eight groups: hypoxia/reoxygenation group (HR); sevoflurane postconditioning group (Sevo-PostC); high glucose+ hypoxia/reoxygenation group (HG+HR); high glucose+sevoflurane postconditioning group (HG+Sevo-PostC); vector adenovirus+hypoxia/reoxygenation group (Ad-vector+HG+HR); vector adenovirus+sevoflurane postconditioning group (Ad-vector+HG+Sevo-PostC); TOPK adenovirus+hypoxia/reoxygenation group (Ad-TOPK+HG+HR); TOPK adenovirus +sevoflurane postconditioning group (Ad-TOPK+HG+Sevo-PostC). Myocardial infarct size was measured using TTC staining. The cell viability was detected by MTT colorimetric assay. Myocardial CK-MB content was detected by ELISA. LDH, and MDA levels were determined by colorimetric method and total SOD was measured by hydroxylamine method. Myocardial apoptosis was detected by TUNEL. Myocardial superoxide anion generation was determined by fluorescent-labeled dihydroethidium staining in in vivo and in vitro models. The protein levels of TOPK, PTEN, Akt and its phosphorylation were measured by Western Blot method.Result In vivo, diabetes significantly increased cardiac ischemia/reperfusion injury reflexed by decreased the mean artery pressure at the end of reperfusion period, enhanced myocardial infarct size, plasma CK-MB and LDH release, myocardial cellular apoptosis and oxidative stress (Diabetic+HR and Diabetic+Sevo-PostC groups vs IR group, P< 0.05), and importantly, it abolished the cardioprotective effects induced by Sevo-PostC (Diabetic+IR group vs Diabetic+Sevo-PostC group, P> 0.05). In vitro, high glucose also increased cellular hypoxia/reoxygenation injury, which decreased the cellular viability, and enhanced LDH release as well as oxidative stress after cellular hypoxia /reoxygenation injury (HG+HR and HG-Sevo-PostC groups vs HR group, P<0.05). High glucose also blocked Sevo-PostC-induced cardioprotection against cellular hypoxia/reoxygenation injury (HG+HR group vs HG-Sevo-PostC group, P>0.05). However, the overexpression of TOPK in H9c2 cells remarkably elevated the cellular viability and decreased LDH release after cellular hypoxia/reoxygenation injury (Ad-TOPK+HG+HR and Ad-TOPK+HG+Sevo-PostC groups vs Ad-vector+HG+HR group, P<0.05). Diabetes or high glucose abolished the elevation of TOPK, PTEN and Akt phosphorylation levels induced by Sevo-PostC (Diabetic+Sevo-PostC group vs Diabetic+IR group, P>0.05; HG+Sevo-PostC group vs HG+IR group, P>0.05). In addition, diabetes or high glucose significantly reduced the levels of TOPK, PTEN and Akt phosphorylation (Diabetic+Sevo-PostC and Diabetic+IR groups vs IR group, P< 0.05; HG+Sevo-PostC and HG+IR groups vs HR group, P<0.05). Importantly, the overexpression of TOPK significantly enhanced the levels of PTEN and Akt phosphorylation in H9c2 cells exposed to high glucose (Ad-TOPK+HG+HR and Ad-TOPK+HG+Sevo-PostC groups vs Ad-vector+HG+HR group, P<0.05).Conclusion Diabetes blocks the cardioprotective effects of sevoflurane postconditioning and exacerbates myocardial ischemia/reperfusion injury due to the impairment of TOPK/PTEN/Akt signaling pathway. |
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