Protective Effects And Mechanism Of N-acetylcysteine On Cardiac Injury Induced By Ischemia/Reperfusion In Diabetic Rats

Part 1 The Establishment of Diabetic Model in S-D RatsObjective To investigate the effect of the dosage of streptozotocin (STZ) on the incidence and the survival rate of diabetic rats. Methods Sixty SD rats were randomly divided into two groups:normal control group (N group,n=12)and experimental group (n=48), and then the rats in the experimental group were randomly reassigned into four groups(A, B, C, D). The rats in A, B, C, D groups were seperately given STZ 50, 55, 60, 65 mg·kg-1 intraperitoneally (ip). The same citric acid buffer was given in N group. The incidence and the survival rate of diabetic rats were compared with each other. Results The incidence and survival rate of rats in B group were significantly higher than those of rats in A group(P<0.05) and there were no significant difference among B, C, D groups as to the above index(P>0.05). Conclusion The dosage of STZ 55 mg·kg-1 was selected to establish experimental diabetic rats.Part 2 Effects of N-acetylcysteine on ischemia/reperfusion-induced ventricular arrhythmia and cardiac dysfunction in diabetic ratsObjective To evaluate the effects of N-acetylcysteine on I/R-induced ventricular arrhythmia and cardiac dysfunction in diabetic rats. Methods 72 rats were randomly divided into six groups: non-diabetic sham operation group(CS, n=12), non-diabetic I/R group(CI/R, n=12), non-diabetic rats treated with NAC group (CN, n=12), diabetic sham operation group(DS, n=12), diabetic I/R group(DI/R, n=12), diabetic rats treated with NAC group (DN, n=12) .Diabetes mellitus was induced by intraperitoneal injection of streptozotocin(STZ) 55mg·kg-1. 1 week after STZ injection the animals in group CN and group DN were treated with NAC 100mg·kg-1 by intragastric administration(two times per day) for 8 weeks. The I/R heart model was made by ligation of the left anterior descending coronary artery (LAD) close to its origin. The LAD was occluded for 30 min followed by removal of ligation to allow reperfusion for 3 h except the sham-operated rats.The severity of arrhythmia was quantified by arrhythmia score(AS). Left ventricular developed pressure(LVDP) and±dp/dtmax were measured and recorded using computerized data acquisition system throughout the experiment. TNF-αand IL-8 determined by enzyme-linked immunosorbent assay (ELISA).Creatine kinase isoenzyme-MB(CK-MB) and myocardial Malondialdehyde (MDA) were measured after the experiment finished. Results (1) After I/R, the values of TNF-α, IL-8, AS, CK-MB and MDA were significantly higher in CN group than those in CS group but lower than those in CI/R group(P<0.05), while LVDP and±dp/dtmax were significantly lower than those in CS but higher than those in CI/R group(P<0.05).(2) LVDP and±dp/dtmax in DN group were significantly higher than those in DI/R group but lower than those in CN group(P<0.05), while the TNF-α, IL-8, AS, CK-MB and MDA were significantly lower in DN group than those in DI/R group but higher than those in CN group(P<0.05). Conclusions: Diabetic rat hearts are more susceptible to I/R-induced arrhythmia and cardiac dysfunction. NAC can improve the recovery of cardiac function and reduce the severity of I/R- induced arrhythmia in both non-diabetic rats and diabetic rats, but the therapeutic effects are less effective in diabetic rats than those in non-diabetic rats. Part 3 Effect of N-acetylcysteine on ischemia/reperfusion-induced cardiocyte apoptosis in diabetic ratsObjective To study the effect of N-acetylcysteine (NAC) on ischemia/ reperfusion (I/R)-induced cardiocyte apoptosis, the content of GSH and GSSG, and the activity of Caspase-3 in diabetic rat hearts. Methods 72 rats were randomly divided into two groups: non-diabetic group and diabetic group with 36 rats in each group. Diabetes mellitus was induced by intraperitoneal injection of streptozotocin(STZ) 55mg·kg-1 and rats serving as controls were given the same volume of sodium citrate. Diabetes mellitus was defined as persistent blood glucose level≥16.7mmol·L-1.The animals in the two groups were randomly reassigned into sham-operated group , I/R group and I/R + treated with NAC group respectively with 12 rats in each group. 1 week after STZ injection, the rats in the two treated with NAC groups were treated with NAC 100mg·kg-1by intragastric administration(two times per day) for 8 weeks. NAC (150mg·kg-1) was injected into peritoneal cavity before operation. The I/R heart model was made by ligation of the left anterior descending coronary artery (LAD) close to its origin. The LAD was occluded for 30 min followed by removal of ligation to allow reperfusion for 3 h except the sham-operated rats. The content of GSH and GSSG, and the activity of Caspase-3 were measured. The apoptosis index (AI) by TUNEL staining was caculated. In addition, the apoptosis of Cardiomyocyte was also confirmed by DNA Ladder. Results (1) After I/R, the activity of Caspase-3, the content of GSSG, the values of AI were higher in diabetic group than those in non-diabetic group, and the content of GSH was lower in diabetic group than that in non-diabetic group(P<0.05). (2) Treatment with NAC decreased the activity of Caspase-3, the content of GSSG, the values of AI but increased the content of GSH in both non-diabetic and diabetic rats(P<0.05), but there were still significant difference about the values of above parameters between diabetic rats and non-diabetic rats(P<0.05). Conclusions:NAC can attenuated cardiomyocyte apoptosis by decreasing the activity of Caspase-3 and increasing the content of GSH, which has protective effect on ischemic/reperfused myocardium injury in both non-diabetic and diabetic rats, but the cardioprotective effect was less effective in diabetic rats than that in non-diabetic rats.Part 4 Effect of N-acetylcysteine on hypoxia-inducible factor-1αand Heme Oxygenase-1 mRNA expression induced by myocardial ischemia-reperfusion in diabetic rat heartsObjective To study the Effect of N-acetylcysteine on hypoxia-inducible factor-1α(HIF-1α) and Heme Oxygenase-1(HO-1) mRNA and protain expression induced by myocardial ischemia-reperfusion in diabetic rat hearts. Methods 72 rats were randomly divided into two groups: non-diabetic group and diabetic group with 36 rats in each group. Diabetes mellitus was induced by intraperitoneal injection of streptozotocin(STZ) 55mg·kg-1 and rats serving as controls were given the same volume of sodium citrate. Diabetes mellitus was defined as persistent blood glucose level≥16.7mmol·L-1.The animals in the two groups were randomly reassigned into sham-operated group , I/R group and I/R + treated with NAC group respectively with 12 rats in each group. 1 week after STZ injection, the rats in the two treated with NAC groups were treated with NAC 100mg·kg-1by intragastric administration(two times per day) for 8 weeks. NAC (150mg·kg-1) was injected into peritoneal cavity before operation. The I/R heart model was made by ligation of the left anterior descending coronary artery (LAD) close to its origin. The LAD was occluded for 30 min followed by removal of ligation to allow reperfusion for 3 h except the sham-operated rats.Myocardial infarct size, the expression of HIF-1α, HO-1 mRNA and protain were detected by RT-PCR and Western blotting in each group. Results (1)The expression of HIF-1αmRNA and protain were not appreciable in group CS, but significantly increased in group CI/R after ischaemia-reperfusion.In group DS, the expression of HIF-1αcould be detected; however, as compared with group CI/R, the expression of HIF-1αwas decreased in group DI/R (P<0.05). The changes in myocardial HO-1 mRNA expression paralleled those of HIF-1α. Myocardial infarct size was greater in diabetic rats than that in non-diabetic rats (P<0.05).(2) The expression of HIF-1αmRNA and protain were higher in DN group than those in DI/R group; The changes in myocardial HO-1 mRNA expression paralleled those of HIF-1α. Conclusions Diabetes increased basal HIF-1αand HO-1 expression, suggesting a state of pseudohypoxia. These findings show that diabetes aggravated myocardial injury induced by ischemia-reperfusion, which is associated with a relatively reduced expression of the HIF-1αand HO-1gene. Treatment with NAC can protective diabetic rat hearts from I/R injury and partly restore The expression of HIF-1αand HO-1.