SIRT3通过抑制氧化应激保护糖尿病大鼠心肌缺血再灌注损伤的研究
发布时间：2018-10-05 21:48
【摘要】：目的:1.在离体心脏灌流模型上研究心肌缺血/再灌注后SIRT3的表达变化以及心肌乙酰化水平变化。2.构建糖尿病大鼠模型,研究SIRT3在糖尿病大鼠心肌缺血/再灌注模型中对蛋白乙酰化及氧化应激的影响,同时验证SIRT3对糖尿病大鼠心肌缺血/再灌注损伤的保护作用。方法:第一部分:10只雄性SD大鼠,2~3月龄,体重(220-250)g,随机分为对照组(A组,n=5)、实验组(B组,n=5)。两组均采用Langendorff心脏离体灌流模型。A组平行灌注20min心脏功能稳定后再接着平行灌注60min;B组平行灌注20min心脏功能稳定后,全心停止灌注40min再复灌60min制成心肌缺血/再灌注模型。灌注结束后分别用荧光定量PCR(RT-q PCR)和冷冻切片免疫荧光法检测心肌Sirt3的表达情况及心肌整体乙酰化水平。第二部分:24只雄性SD大鼠,2~3月龄,体重(250-300)g,通过腹腔注射链脲佐菌素(Streptozotocin,STZ)诱导建立1型糖尿病(Diabetes mellitus,DM)模型,在注射后的72h、7、14、28天分别取大鼠尾静脉血监测血糖,血糖16.70mmol/L,尿糖+++以上者表示造模成功。造模成功后将DM大鼠随机分为对照组(A组,n=8)、SIRT3抑制剂组(B组,n=8)和SIRT3激活剂组(C组,n=8)。饲养5周后,A组行生理盐水灌胃4周;B组行尼克酰胺灌胃4周;C组行白藜芦醇灌胃4周。三组DM大鼠均采用Langendorff心脏离体灌流建模方法,平行灌注20min、全心停止灌注40min、复灌60min,制成心肌缺血/再灌注模型。灌注过程中,心肌缺血前后记录不同实验组的心功能变化包括心率(HR)、左心室发展压(LVDP)、左室收缩压最大上升/下降速率(±dp/dtmax)等。分别在平行灌注20 min时和再灌注1h时收集冠脉流出液,运用ELISA方法检测肌钙蛋白T(Tn-T)含量。灌注结束后,通过TTC染色测定灌注后心肌梗死面积,分别用冰冻切片免疫荧光和western bolt测定caspase3的表达水平以反映心房细胞凋亡,并测定心肌组织乙酰化水平,以及SOD活性及丙二醛(MDA)的含量。结果:第一部分:各组灌注结束后心肌Sirt3 m RNA表达量比较,A组是B组的1.7倍,差异有统计学意义(p0.05)。各组免疫荧光检测心肌组织乙酰化水平相比较,B组比A组明显升高。第二部分:各组心肌缺血/再灌注后的心率、左心室发展压、左心室收缩压最大上升速率及左心室收缩压最大下降速率比较,差异均有统计学意义(p0.05),且结果显示C组上述各指标高于A组、B组,B组各指标最低,差异有统计学意义(p0.05)。各组缺血/再灌注后的心肌梗死面积比较,差异有统计学意义,B组明显大于A组、C组而C组最低,差异有统计学意义(p0.05)。各组心肌缺血/再灌注后用冰冻切片免疫荧光检测心肌caspase 3蛋白表达量,B组明显高于A、C组,C组最低,差异有统计学意义(p0.05)。各组心肌缺血/再灌注后用western blot检测心肌乙酰化水平,结果表明B组明显高于A、C组,C组最低,差异有统计学意义(p0.05)。各组缺血/再灌注后心肌组织SOD活性相比较,C组高于A组、B组而B组最低,差异有统计学意义(p0.05)。各组缺血/再灌注后心肌MDA水平相比较,B组明显高于A、C组,C组最低,差异有统计学意义(p0.05)。结论:1.正常成年大鼠心脏经离体缺血/再灌注后SIRT3的表达量有明显下调,而心肌整体乙酰化水平显著升高。2.SIRT3活性增强可以降低糖尿病大鼠心肌缺血/再灌注损伤,其保护糖尿病心肌的机制可能与抑制心肌氧化应激有关。
[Abstract]:Purpose: 1. The changes of the expression of SIRT3 after myocardial ischemia/ reperfusion and the changes of myocardial calcium level were studied in the model of heart perfusion. To study the effect of SIRT3 in myocardial ischemia/ reperfusion model of diabetic rats and to verify the protective effect of SIRT3 on myocardial ischemia/ reperfusion injury in diabetic rats. Methods: The first part: 10 male SD rats, 2 ~ 3 months old and 220-250 g, randomly divided into control group (group A, n = 5), experimental group (group B, n = 5). Langendorff perfusion model was used in both groups. In group A, the heart function was stable after 20min, and then perfused with 60min in parallel. After the cardiac function of 20min in group B was stable, the whole heart stopped perfusion for 40min and then poured for 60min to prepare the model of myocardial ischemia/ reperfusion. At the end of perfusion, the expression of Srt3 and the level of myocardial ischemia were detected by fluorescence quantitative PCR (RT-q PCR) and frozen section immunofluorescence assay, respectively. Part 2: 24 male SD rats, 2-3 months old and 250-300 g body weight (250-300) g were induced by Streptozoocin (STZ), and blood glucose was monitored at 72h, 7, 14 and 28 days after injection. More than + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + DM rats were randomly divided into control group (group A, n = 8), SIRT3 inhibitor group (group B, n = 8) and SIRT3 activator group (group C, n = 8). After 5 weeks of feeding, group A was given normal saline for 4 weeks, and the group B was given intragastric intragastric administration for 4 weeks, and the group C was given intragastric intragastric intragastric administration for 4 weeks. Three groups of DM rats were perfused with Langendorff's heart-to-body perfusion model, perfused in parallel for 20min, the whole heart was stopped for 40min, and the reperfusion was 60min to prepare the model of myocardial ischemia/ reperfusion. The cardiac function changes of different experimental groups were recorded before and after myocardial ischemia, including heart rate (HR), left ventricular development pressure (LVDP), maximum systolic blood pressure rising/ descending rate (Vdp/ dtmax), etc. The levels of troponin T (Tn-T) were measured by ELISA in 20 min parallel perfusion and 1h reperfusion respectively. At the end of perfusion, the area of myocardial infarction was determined by TCTC staining, and the expression level of caspase3 was measured by using frozen section immunofluorescence and western boldt, respectively, to reflect the apoptosis of atrial cells, and to determine the level of myocardial infarction and the levels of SOD and MDA. Results: In the first part, the expression of Sirt3 mRNA in myocardium was compared with that of group B, and group A was 1. 7 times of group B. The difference was statistically significant (P0.05). Compared with group A, group B was significantly higher than group A in group B. In the second part, the heart rate, left ventricular development pressure, maximum left ventricular systolic blood pressure and the maximum descending rate of left ventricular systolic blood pressure in each group were statistically significant (P0.05), and the results showed that the above indexes of group C were higher than group A and group B. The indexes of group B were the lowest and the difference was statistically significant (P0.05). Compared with group A, group C and group C, there was significant difference between group B and group C (P0.05). After myocardial ischemia/ reperfusion, the expression of caspase-3 protein was detected by immunofluorescence using frozen section. The group B was significantly higher than that in group A, group C and group C, and the difference was statistically significant (P0.05). The level of myocardial infarction was detected by western blot after myocardial ischemia/ reperfusion. The results showed that group B was significantly higher than that in group A, group C and group C, and the difference was statistically significant (P0.05). Compared with group A, group B and group B, there was significant difference between group B and group B (P0.05). Compared with group A, group C and group C, there was significant difference between group B and group C (P0.05). Conclusion: 1. In normal adult rats, the expression level of SIRT3 was down-regulated after ischemia/ reperfusion, and the level of myocardial ischemia/ reperfusion increased significantly. Mechanisms for protecting diabetic myocardium may be associated with inhibition of myocardial oxidative stress.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R542.2
本文编号：2254988
[Abstract]:Purpose: 1. The changes of the expression of SIRT3 after myocardial ischemia/ reperfusion and the changes of myocardial calcium level were studied in the model of heart perfusion. To study the effect of SIRT3 in myocardial ischemia/ reperfusion model of diabetic rats and to verify the protective effect of SIRT3 on myocardial ischemia/ reperfusion injury in diabetic rats. Methods: The first part: 10 male SD rats, 2 ~ 3 months old and 220-250 g, randomly divided into control group (group A, n = 5), experimental group (group B, n = 5). Langendorff perfusion model was used in both groups. In group A, the heart function was stable after 20min, and then perfused with 60min in parallel. After the cardiac function of 20min in group B was stable, the whole heart stopped perfusion for 40min and then poured for 60min to prepare the model of myocardial ischemia/ reperfusion. At the end of perfusion, the expression of Srt3 and the level of myocardial ischemia were detected by fluorescence quantitative PCR (RT-q PCR) and frozen section immunofluorescence assay, respectively. Part 2: 24 male SD rats, 2-3 months old and 250-300 g body weight (250-300) g were induced by Streptozoocin (STZ), and blood glucose was monitored at 72h, 7, 14 and 28 days after injection. More than + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + DM rats were randomly divided into control group (group A, n = 8), SIRT3 inhibitor group (group B, n = 8) and SIRT3 activator group (group C, n = 8). After 5 weeks of feeding, group A was given normal saline for 4 weeks, and the group B was given intragastric intragastric administration for 4 weeks, and the group C was given intragastric intragastric intragastric administration for 4 weeks. Three groups of DM rats were perfused with Langendorff's heart-to-body perfusion model, perfused in parallel for 20min, the whole heart was stopped for 40min, and the reperfusion was 60min to prepare the model of myocardial ischemia/ reperfusion. The cardiac function changes of different experimental groups were recorded before and after myocardial ischemia, including heart rate (HR), left ventricular development pressure (LVDP), maximum systolic blood pressure rising/ descending rate (Vdp/ dtmax), etc. The levels of troponin T (Tn-T) were measured by ELISA in 20 min parallel perfusion and 1h reperfusion respectively. At the end of perfusion, the area of myocardial infarction was determined by TCTC staining, and the expression level of caspase3 was measured by using frozen section immunofluorescence and western boldt, respectively, to reflect the apoptosis of atrial cells, and to determine the level of myocardial infarction and the levels of SOD and MDA. Results: In the first part, the expression of Sirt3 mRNA in myocardium was compared with that of group B, and group A was 1. 7 times of group B. The difference was statistically significant (P0.05). Compared with group A, group B was significantly higher than group A in group B. In the second part, the heart rate, left ventricular development pressure, maximum left ventricular systolic blood pressure and the maximum descending rate of left ventricular systolic blood pressure in each group were statistically significant (P0.05), and the results showed that the above indexes of group C were higher than group A and group B. The indexes of group B were the lowest and the difference was statistically significant (P0.05). Compared with group A, group C and group C, there was significant difference between group B and group C (P0.05). After myocardial ischemia/ reperfusion, the expression of caspase-3 protein was detected by immunofluorescence using frozen section. The group B was significantly higher than that in group A, group C and group C, and the difference was statistically significant (P0.05). The level of myocardial infarction was detected by western blot after myocardial ischemia/ reperfusion. The results showed that group B was significantly higher than that in group A, group C and group C, and the difference was statistically significant (P0.05). Compared with group A, group B and group B, there was significant difference between group B and group B (P0.05). Compared with group A, group C and group C, there was significant difference between group B and group C (P0.05). Conclusion: 1. In normal adult rats, the expression level of SIRT3 was down-regulated after ischemia/ reperfusion, and the level of myocardial ischemia/ reperfusion increased significantly. Mechanisms for protecting diabetic myocardium may be associated with inhibition of myocardial oxidative stress.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R542.2
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