七氟醚后处理对糖尿病大鼠心肌保护失效的机制与Drp1活性的关系
发布时间：2018-08-30 17:49
【摘要】：目的:干预动力相关蛋白-1(dynamin related protein-1,Drp1)的活性,探究糖尿病大鼠心肌缺血再灌注损伤(myocardial ischemia reperfusion injury,MIRI)模型不可逆损伤程度之差别;探求七氟醚后处理对糖尿病大鼠心肌保护失效的机制是否与Drp1的活性有关。方法:健康成年雄性SD大鼠,净重为225~275 g,以高碳水化合物-高脂肪饲料饲喂并行链脲佐菌素30 mg/kg腹腔内给药制备II型糖尿病(T2DM)模型。对大鼠每个星期测定-次空腹血糖,连续四星期不低于300 mgl/dL者认为模型合格。取T2DM模型大鼠40只,依据计算机随机数划分为4组(n=10):(1)单纯穿线组(Sham组)、(2)缺血/再灌注组(I/R组)、(3)七氟醚后处理组(SP组)、(4)Drp1活性抑制剂Mdivi-1+七氟醚后处理组(M-SP组)。采取在体阻断左前降支(left anterior descending,LAD)血流0.5 h,再灌注2 h法建立MIRI模型,Sham组单纯穿线而不阻断LAD血流。缺血前15 min M-SP组静脉注射Mdivi-1 1.2 mg/kg,再灌注初期5 min内SP组、M-SP组吸入2.5%七氟醚进行后处理。再灌注2 h时采集大鼠右颈内静脉血样,高速离心后取血清,采用ELISA法检测并计算血清cTnI浓度;随后处死各组大鼠并取其心肌组织,采用TTC法测定心肌梗死区面积(infarct size,IS)和缺血危险区(area at risk,AAR)面积,采用TUNEL法检测凋亡并计算细胞凋亡指数(apoptotic index,AI);采用western blot法测定心肌组织中活化型caspase-3表达水平。结果:1.AAR和IS测定结果:与Sham组比较,其余3组心肌AAR面积增大很显著(P0.01),但3组之间两两比较AAR面积不构成统计学差异(P0.05)。与Sham组比较,其余3组IS增大(P0.01);与I/R组比较,M-SP组IS减小(P0.05),而SP组IS差异无统计学意义(P0.05);与SP组比较,M-SP组IS减小(P0.05)。2.血清cTnI浓度检测结果:与Sham组比较,其余3组血清cTnI浓度升高(P0.05);与I/R组比较,M-SP组血清cTnI浓度降低(P0.05),而SP组血清cTnI浓度差异无统计学意义(P0.05);与SP组比较,M-SP组血清cTnI浓度降低(P0.05)。3.TUNEL法检测结果:与Sham组比较,其余3组心肌组织AI值升高(P0.05);与I/R组比较,M-SP组心肌组织AI值降低(P0.05),而SP组心肌组织AI值差异无统计学意义(P0.05);与SP组比较,M-SP组心肌组织AI值降低(P0.05)。4.western blot法检测结果:与Sham组比较,其余3组活化型caspase-3表达上调(P0.05);与I/R组比较,M-SP组活化型caspase-3表达下调(P0.05),SP组活化型caspase-3表达差异无统计学意义(P0.05);与SP组比较,M-SP组活化型caspase-3表达下调(P0.05)。结论:在糖尿病条件下,七氟醚后处理对大鼠心肌保护作用失效,抑制Drp1活性后T2DM大鼠MIRI模型的心肌不可逆损伤程度有所减轻;提示七氟醚后处理对糖尿病大鼠心肌保护失效的机制可能与Drp1活性有关。
[Abstract]:Objective: to investigate the irreversible degree of myocardial ischemia-reperfusion injury (myocardial ischemia reperfusion injury,MIRI) model in diabetic rats by interfering with the activity of dynamic associated protein 1 (dynamin related protein-1,Drp1). To explore whether the mechanism of myocardial protection failure after sevoflurane treatment in diabetic rats is related to the activity of Drp1. Methods: healthy adult male SD rats with a net weight of 225? 275g were fed with high carbohydrate-high fat diet and intraperitoneal administration of streptozotocin for 30 mg/kg to establish II diabetes mellitus (T2DM) model. Rats who measured fasting blood glucose for four weeks or more per week were considered to be eligible for the model. Forty T2DM model rats were randomly divided into 4 groups (n = 10): (1, Sham group (), (2), I / R group (), (3), SP group), (4, Drp1 activity inhibitor Mdivi-1 sevoflurane post-treatment group, M-SP group). The left anterior descending branch (left anterior descending,LAD) was occluded in vivo for 0.5 h, and the MIRI model was established by reperfusion for 2 h. In the sham group, the blood flow of LAD was not blocked. 15 min M-SP before ischemia, 2.5% sevoflurane was inhaled into the SP group within 5 min after Mdivi-1 1.2 mg/kg, reperfusion. The blood samples of right internal jugular vein were collected at 2 h after reperfusion, the serum samples were collected after high speed centrifugation, the serum cTnI concentration was detected and calculated by ELISA method, then the rats in each group were killed and their myocardial tissues were taken. The area of myocardial infarction area (infarct size,IS) and ischemic risk area (area at risk,AAR) were measured by TTC method, apoptosis index (apoptotic index,AI) was calculated by TUNEL method, and the expression of activated caspase-3 was measured by western blot method. Results 1. The results of AAR and IS: compared with Sham group, the area of myocardial AAR in the other three groups increased significantly (P0.01), but there was no statistical difference in AAR area between the three groups (P0.05). Compared with Sham group, IS in the other three groups increased (P0.01), IS in M-SP group decreased (P0.05) compared with I / R group, but IS in SP group was not significantly different (P0.05), IS in M-SP group decreased (P0.05) compared with SP group (P0.05). Results of serum cTnI concentration: compared with Sham group, The serum cTnI concentration in the other three groups was increased (P0.05), the serum cTnI concentration in the M-SP group was lower than that in the I / R group (P0.05), but the serum cTnI concentration in the SP group was not significantly different (P0.05), and the serum cTnI concentration in the M-SP group was lower than that in the SP group (P0.05). 3. Tunel method: compared with the Sham group, the serum cTnI concentration in the M-SP group was lower than that in the Sham group. Compared with the I / R group, the AI value of myocardial tissue in M-SP group decreased (P0.05), but there was no significant difference in AI value of myocardial tissue in SP group (P0.05). Compared with SP group, the AI value of myocardial tissue in M-SP group decreased (P0.05). 4. Western blot assay results: compared with Sham group, the myocardial tissue AI value of M-SP group decreased (P0.05). The expression of activated caspase-3 in the other three groups was up-regulated (P0.05), the expression of activated caspase-3 in M-SP group was down-regulated (P0.05), the expression of activated caspase-3 in M-SP group was not significantly higher than that in SP group (P0.05), and the expression of activated caspase-3 in M-SP group was lower than that in SP group (P0.05). Conclusion: under the condition of diabetes, the protective effect of sevoflurane on myocardium of rats was ineffective, and the degree of irreversible myocardial damage of MIRI model of T2DM rats after inhibiting the activity of Drp1 was alleviated. The results suggest that the mechanism of myocardial protection failure after sevoflurane treatment in diabetic rats may be related to the activity of Drp1.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614
本文编号：2213818
[Abstract]:Objective: to investigate the irreversible degree of myocardial ischemia-reperfusion injury (myocardial ischemia reperfusion injury,MIRI) model in diabetic rats by interfering with the activity of dynamic associated protein 1 (dynamin related protein-1,Drp1). To explore whether the mechanism of myocardial protection failure after sevoflurane treatment in diabetic rats is related to the activity of Drp1. Methods: healthy adult male SD rats with a net weight of 225? 275g were fed with high carbohydrate-high fat diet and intraperitoneal administration of streptozotocin for 30 mg/kg to establish II diabetes mellitus (T2DM) model. Rats who measured fasting blood glucose for four weeks or more per week were considered to be eligible for the model. Forty T2DM model rats were randomly divided into 4 groups (n = 10): (1, Sham group (), (2), I / R group (), (3), SP group), (4, Drp1 activity inhibitor Mdivi-1 sevoflurane post-treatment group, M-SP group). The left anterior descending branch (left anterior descending,LAD) was occluded in vivo for 0.5 h, and the MIRI model was established by reperfusion for 2 h. In the sham group, the blood flow of LAD was not blocked. 15 min M-SP before ischemia, 2.5% sevoflurane was inhaled into the SP group within 5 min after Mdivi-1 1.2 mg/kg, reperfusion. The blood samples of right internal jugular vein were collected at 2 h after reperfusion, the serum samples were collected after high speed centrifugation, the serum cTnI concentration was detected and calculated by ELISA method, then the rats in each group were killed and their myocardial tissues were taken. The area of myocardial infarction area (infarct size,IS) and ischemic risk area (area at risk,AAR) were measured by TTC method, apoptosis index (apoptotic index,AI) was calculated by TUNEL method, and the expression of activated caspase-3 was measured by western blot method. Results 1. The results of AAR and IS: compared with Sham group, the area of myocardial AAR in the other three groups increased significantly (P0.01), but there was no statistical difference in AAR area between the three groups (P0.05). Compared with Sham group, IS in the other three groups increased (P0.01), IS in M-SP group decreased (P0.05) compared with I / R group, but IS in SP group was not significantly different (P0.05), IS in M-SP group decreased (P0.05) compared with SP group (P0.05). Results of serum cTnI concentration: compared with Sham group, The serum cTnI concentration in the other three groups was increased (P0.05), the serum cTnI concentration in the M-SP group was lower than that in the I / R group (P0.05), but the serum cTnI concentration in the SP group was not significantly different (P0.05), and the serum cTnI concentration in the M-SP group was lower than that in the SP group (P0.05). 3. Tunel method: compared with the Sham group, the serum cTnI concentration in the M-SP group was lower than that in the Sham group. Compared with the I / R group, the AI value of myocardial tissue in M-SP group decreased (P0.05), but there was no significant difference in AI value of myocardial tissue in SP group (P0.05). Compared with SP group, the AI value of myocardial tissue in M-SP group decreased (P0.05). 4. Western blot assay results: compared with Sham group, the myocardial tissue AI value of M-SP group decreased (P0.05). The expression of activated caspase-3 in the other three groups was up-regulated (P0.05), the expression of activated caspase-3 in M-SP group was down-regulated (P0.05), the expression of activated caspase-3 in M-SP group was not significantly higher than that in SP group (P0.05), and the expression of activated caspase-3 in M-SP group was lower than that in SP group (P0.05). Conclusion: under the condition of diabetes, the protective effect of sevoflurane on myocardium of rats was ineffective, and the degree of irreversible myocardial damage of MIRI model of T2DM rats after inhibiting the activity of Drp1 was alleviated. The results suggest that the mechanism of myocardial protection failure after sevoflurane treatment in diabetic rats may be related to the activity of Drp1.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614
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