【作者】 马利；

【导师】 姚尚龙；

【作者基本信息】 华中科技大学 ， 麻醉学， 2007， 博士

【摘要】 第一部分糖尿病大鼠模型的建立目的观察不同剂量链脲佐菌素(streptozotocin, STZ)对糖尿病(diabetes mellitus, DM)大鼠成模率及4周存活率的影响。方法SD大鼠60只,随机分出12只作为对照组(N组),其余48只大鼠随机分为4组,分别腹腔注射STZ 50 ( A组)、55 ( B组)、60 ( C组)、65mg·kg-1 ( D组)诱发DM模型,比较各组1周后成模率和4周后存活率。结果以STZ 55mg·kg-1剂量腹腔注射复制DM大鼠模型的成模率高于50 mg·kg-1组(83.33% vs33.33%,P<0.05),随STZ剂量的增加(STZ 60-65mg·kg-1)DM大鼠成模率增高,但存活率降低(83.33% vs 41.67%,P<0.05)。结论综合考虑,STZ-DM大鼠造模宜选取STZ的最佳剂量为55mg·kg-1。第二部分乙酰半胱氨酸对糖尿病大鼠心肌缺血/再灌注后心功能的影响目的观察乙酰半胱氨酸对糖尿病大鼠心肌缺血/再灌注后心律失常和心功能的影响,探讨乙酰半胱氨酸对糖尿病大鼠心肌缺血/再灌注损伤的防治效果。方法72只大鼠先随机分为糖尿病组(36只,链尿佐菌素55mg·kg-1腹腔注射诱导)和非糖尿病对照组(36只)。动物分为正常假手术组(CS组, n=12)、正常缺血/再灌注组(CI/R组,n=12)、正缺血/再灌注+NAC(CN组, n=12)、糖尿病假手术组(DS组, n=12)、糖尿病缺血/再灌注组(DI/R组,n=12)、糖尿病缺血/再灌注+NAC组(DN组, n=12)。建模成功后,NAC处理组灌胃补充NAC(100mg·kg-1),每天2次,手术前1h再给NAC 150mg·kg-1腹腔注射。8周后,采用冠脉结扎及放松的方法复制心肌缺血/再灌注模型。观察标准肢体导联(Ⅱ)心电图、左心室发展压(LVDP)、收缩期和舒张期左心室内压的最大变化速率(±dp/dtmax),采血测肌酸激酶同工酶(CK-MB)活性、肿瘤坏死因子-α(TNF-α)和白介素-8(IL-8)含量,心肌组织匀浆检测丙二醛(MDA)含量。结果(1)基础状态下,糖尿病组与正常组相比,心功能降低(P<0.05);TNF-α和IL-8含量高于正常组(P<0.05)。(2)缺血/再灌注后, CN组和DN组LVDP值和±dp/dtmax高于对应的CI/R组和DI/R组,而上述指标DN组低于CN组(P<0.05);心律失常评分、CK-MB、MDA、TNF-α和IL-8低于对应的CI/R组和DI/R组(P<0.05),而上述指标DN组高于CN组(P<0.05)。DI/R组LVDP值和±dp/dtmax低于CI/R组,而心律失常评分、CK-MB、MDA、TNF-α和IL-8高于CI/R组(P<0.05)。结论8周病程糖尿病大鼠基础心功能降低,炎症反应明显,而且缺血/再灌注后心功能受损程度比非糖尿病组严重;补充NAC既可以改善糖尿病大鼠基础心功能,减轻炎症反应,也可减轻心肌缺血/再灌注损伤的严重程度,改善心功能;但与正常大鼠补充NAC组相比,疗效降低。第三部分乙酰半胱氨酸对糖尿病大鼠心肌缺血/再灌注后细胞凋亡的影响目的本实验拟观察乙酰半胱氨酸对糖尿病大鼠心肌缺血/再灌注后导致的细胞凋亡的影响。方法72只大鼠先随机分为糖尿病组(36只,链尿佐菌素55mg·kg-1腹腔注射诱导)和非糖尿病对照组(36只)。两组大鼠再分别随机分为假手术组、缺血/再灌注组和缺血/再灌注+乙酰半胱氨酸治疗组(乙酰半胱氨酸100mg·kg-1,2次/d,手术前1h再给NAC 150mg·kg-1腹腔注射),每组12只。给予乙酰半胱氨酸8周后进行实验。以穿线结扎及放松左冠脉制备心肌缺血/再灌注模型,组织匀浆检测心肌组织还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)含量和半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的活性;脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)和琼脂糖凝胶电泳检测DNA片段化两种方法检测心肌细胞凋亡并计算凋亡指数。结果(1)正常假手术组未见明显的凋亡,糖尿病假手术组出现少量的凋亡细胞,同时伴有GSH含量降低,GSSG含量和Caspase-3的活性升高(P<0.05);(2)缺血/再灌注后,糖尿病和非糖尿病组均出现明显的心肌细胞凋亡,同时伴有GSH含量降低,GSSG含量和Caspase-3的活性升高,上述变化糖尿病组比非糖尿病组更明显(P<0.05);(3)乙酰半胱氨酸干预的糖尿病和非糖尿病大鼠的心肌细胞凋亡均减轻,同时伴有GSH含量上升,GSSG含量和Caspase-3的活性下降,上述变化非糖尿病组比糖尿病组更明显(P<0.05)。结论乙酰半胱氨酸干预可以通过提高心肌GSH含量、降低Caspase-3的活性减轻糖尿病和非糖尿病大鼠缺血/再灌注引起的心肌细胞凋亡,对缺血/再灌注心肌有保护作用,但糖尿病组的疗效低于非糖尿病组。第四部分乙酰半胱氨酸对糖尿病大鼠心肌缺血/再灌注后低氧诱导因子-1α和血红素加氧酶-1表达的影响目的观察乙酰半胱氨酸对糖尿病大鼠心肌缺血/再灌注后低氧诱导因子-1α(HIF-1α)和血红素加氧酶-1 (HO-1)表达的变化。方法利用链脲佐菌素复制糖尿病大鼠模型,建模成功后,灌胃补充NAC(100mg·kg-1),每天2次,手术前1h再给NAC 150mg·kg-1腹腔注射。动物分为正常假手术组(CS组)、正常缺血/再灌注组(CI/R组)、正常缺血/再灌注+NAC组(CN组)、糖尿病假手术组(DS组)、糖尿病缺血/再灌注组(DI/R组)、糖尿病缺血/再灌注+NAC组(DN组),每组12只,共72只,8周后进行实验。采用冠脉结扎及放松的方法复制心肌缺血/再灌注模型,检测各组心肌梗死范围、HIF-1α和HO-1的mRAN和蛋白表达变化。结果非糖尿病假手术组检测不到HIF-1α和HO-1的mRAN和蛋白表达,糖尿病假手术组检测到少量HIF-1α和HO-1的mRAN和蛋白表达;但是糖尿病组心肌缺血/再灌注后HIF-1α、HO-1mRAN和蛋白表达低于非糖尿病组(P<0.05),心肌梗死范围大于非糖尿病组(P<0.05)。乙酰半胱氨酸干预可以部分恢复糖尿病大鼠心肌缺血/再灌注后HIF-1α和HO-1的mRAN和蛋白的表达,同时减少心肌缺血/再灌注引起的心肌坏死,但是疗效降低(P<0.05)。结论糖尿病加重心肌缺血/再灌注损伤,其机制与糖尿病心肌缺血/再灌注后HIF-1α和HO-1mRAN和蛋白表达相对降低有关。补充NAC可以部分恢复糖尿病大鼠心肌缺血/再灌注后HIF-1α和HO-1mRAN和蛋白表达,有一定的治疗效果。更多还原

【Abstract】 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.更多还原