| Objectives: Sufentanil postconditioning attenuates ischemia reperfusion injury, but this effect might be hindered in diabetic animals by inhibition of glycogen synthase kinase-3β(GSK-3β) phosphorylation. Short-term insulin treatment does not restore protection by sevoflurane postconditioning, but whether long-term insulin treatment could restore the cardioprotective effect of sufentanil postconditioning in diabetic rats via phosphorylation of GSK-3β is still unknown. The aims of the current study were to investigate whether sufentanil postconditioning could be maintained by short- or long-term insulin treatment in diabetic rats and to elucidate the role of GSK-3β in this process.Methods:Type 1 diabetes mellitus was induced in male Sprague Dawley rats by a single i.p. injection of 55 mg/kg streptozotocin. The heart was subjected to 30 min of ischemia by occluding the left anterior descending artery, followed by 120 min of reperfusion. 24 healthy rats, weighing 250-280 g, were randomly divided into 3 groups: non-diabetic sham group(NDM-sham, n = 8), non-diabetic ischemia/reperfusion group(NDM-IR, n = 8) and non-diabetic ischemia/reperfusion with sufentanil postconditioning group(NDM-SP, n = 8). 56 diabetic rats were randomly divided into 7 groups: diabetic sham group(DM-sham, n = 8), diabetic ischemia/reperfusion group(DM-IR, n=8), diabetic ischemia/reperfusion with short-term insulin treatment group(DM-IR-Ins-S, n = 8), diabetic ischemia/reperfusion with long-term insulin treatment group(DM-IR-Ins-L, n = 8), diabetic ischemia/reperfusion with sufentanil postconditioning group(DM-SP, n = 8), diabetic ischemia/reperfusion with sufentanil postconditioning and short-term insulin pretreatment group(DM-SP-Ins-S, n = 8), and diabetic ischemia/reperfusion with sufentanil postconditioning and long-term insulin treatment group(DM-SP-Ins-L, n = 8). Groups with the suffix –IR, received 1 ml normal saline at 5 min before the onset of reperfusion; Groups with the suffix –SP, were treated with sufentanil 1ug /kg at 5 min before the onset of reperfusion; Groups with the suffix –Ins-S were treated with, s.c. insulin at 6-8 units per day for 2 days before experiment; Groups with the suffix –Ins-L were treated with, s.c. insulin at 6-8 units per day for 14 days before experiment. Groups with the suffix –sham, received no occlusion or reperfusion. Heart rate and mean arterial pressure were recorded before ischemia, at 30 min after ischemia and at the end of the 120 min reperfusion, respectively. At the end of the 120 min reperfusion, 1 ml blood was collected for c Tn I levels measurement. Hearts were excised for the measurement of AAR, IS and IS/AAR. For analysis of GSK-3β phosphorylation, hearts were collected from all 10 groups(n = 4 per group). At 5 min after reperfusion, heart tissue were collected for themeasurement of p-GSK-3β、GSK-3β by western blotting.Results:Characteristics of rats: One week after receiving STZ(55 mg/kg), diabetic rats showed a 12% decrease in body weight(P < 0.05) and marked increases in fasting plasma glucose(P < 0.05), compared with non-diabetic rats. Both short-and-long-term insulin treatment lowered glucose levels. After long-term insulin treatment, glucose levels were equal to those in the non-diabetic group. In the short-term insulin treatment group, glucose levels were higher than the non-diabetic group. Hemodynamic parameters: No significant differences were seen in all groups at baseline(P > 0.05). All groups showed decreased HR, MAP, and RPP after induction of ischemia/reperfusion, except DM sham and NDM sham groups. Compared with sham groups, HR, MAP, and RPP decreased significantly after 120 min of reperfusion in all ischemia/reperfusion groups(P < 0.05). Other values showed no other statistically significant inter- or intragroup comparisons. Myocardial infarct size: In non-diabetic rats, sufentanil reduced infarct size(P < 0.05). However, sufentanil failed to attenuate infarct size in diabetic rats(P > 0.05). In diabetic rats treated with sufentanil, long-term insulin treatment reduced infarct size( P < 0.05). Short-term insulin treatment did not reduce infarct size(P > 0.05). Neither short- or long-term insulin treatment affected infarct size in diabetic rats that exposed to IR but did not receive sufentanil(P > 0.05 both). c Tn I: c Tn I was increased after ischemia/reperfusion compared to sham groups(P < 0.05). In non-diabetic rats, sufentanil reduced c Tn I levels(P < 0.05). However,sufentanil failed to reduced c Tn I in the diabetic rats(P < 0.05). In diabetic rats treated with sufentanil, long-term insulin treatment reduced c Tn I( P < 0.05). Short-term insulin treatment did not reduce c Tn I(P > 0.05). GSK-3β Western blot analysis: Total GSK-3β was similar across groups(P > 0.05). Sufentanil increased p-GSK-3β in non-diabetic rats(P < 0.05), but not in diabetic rats(P > 0.05). Long-term insulin treatment markedly increased p-GSK-3β levels(P < 0.05), while not in short-term insulin treatment groups(P > 0.05).Conclusions: Cardioprotective effect of sufentanil postconditioning was hindered by diabetes. Sufentanil-induced cardioprotection was restored by long-term insulin treatment. The underlying mechanism may be increased phosphorylation of GSK-3β. |
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