褪黑素对糖尿病大鼠心肌细胞凋亡的影响及其机制的研究
发布时间：2018-09-03 07:56
【摘要】：目的糖尿病心肌病是在糖尿病患者尤其是2型糖尿病患者的基础上发病的,且具有病情重、预后差和病死率高的特点。为寻求有效的治疗方法,现本文就褪黑素(MLT)对长期患2型糖尿病(T2DM)大鼠心肌细胞凋亡的影响及其可能的机制进行研究和探讨。方法(1)T2DM大鼠模型:在使用基础饲料喂养一周后,36只SPF级健康雄性SD大鼠随机分为对照组(Control,n=12),模型组(DM,n=12),治疗组(DM+MLT,n=12)。采用高脂饮食喂养联合腹腔注射链脲佐菌素(STZ)25 mg/kg的方法诱导2型糖尿病模型。造模成功后,治疗组给予MLT(10 mg/(kg·d))灌胃治疗。24周后,动脉插管检测大鼠心功能;收集大鼠腹主动脉血和心肌组织,并检测大鼠空腹血糖(FBG),血脂(TC、LDL-c、TG)和胰岛素水平;HE染色和Masson染色分别观察大鼠心肌的形态结构变化和胶原纤维的堆积情况;原位末端标记法(TUNEL)观察心肌组织细胞凋亡;免疫组化(IHC)检测Caspase-3的表达水平。此外,为探讨其可能的分子机制,Western blot法检测内质网应激相关蛋白(GRP78,CHOP,PERK,ATF-6α,IRE1α,Caspase-12),丝裂原活化蛋白激酶(MAPK)通路相关蛋白(JNK,p38,ERK)和凋亡相关蛋白(Caspase-9,Caspase-3,Bcl-2,Bax)的表达情况。(2)高糖培养大鼠心肌细胞H9C2为细胞模型:不同糖浓度培养基培养细胞48 h后,Hoechst 33258染色检测细胞凋亡,western blot检测MAPK通路相关蛋白JNK、p38和ERK的磷酸化水平。结果(1)T2DM大鼠模型:模型组大鼠的FBG为(19.27±1.03)mmol/L,胰岛素水平为(34.79±7.97μIU/m L),均显著高于对照组大鼠,且胰岛素敏感性指数(ln ISI)为-6.48±0.23明显比对照组大鼠低,表明存在胰岛素抵抗(IR)。这些结果都说明已成功构建T2DM大鼠模型。MLT不仅能降低FBG和血脂(TC、LDL-c、TG)水平,还能增强胰岛素的敏感性。HE染色结果提示MLT能改善心肌组织形态的紊乱情况,Masson染色结果表明MLT能减少心肌组织胶原纤维的堆积。模型组大鼠的心肌细胞凋亡细胞明显增多,且心功能严重受损,经MLT治疗后均明显改善。IHC显示MLT能降低T2DM大鼠Caspase-3的表达。Western blot结果显示模型组大鼠心肌组织内质网应激相关蛋白GRP78、CHOP、PERK、ATF-6α、IRE1α和Caspase-12的表达均明显上升,且JNK的磷酸化水平也明显上调。T2DM大鼠经MLT治疗后,内质网应激相关蛋白表达和JNK的磷酸化水平均下调。(2)高糖培养H9C2细胞模型:Hoechst 33258染色显示高糖能诱导H9C2细胞凋亡;Western blot结果显示高糖能上调H9C2细胞中JNK的磷酸化水平,并具有一定的浓度依赖性。结论T2DM大鼠心肌细胞凋亡增多,其机制可能是血糖血脂升高、经由内质网应激介导的JNK通路活化调控下游相关凋亡蛋白所致;而MLT能通过降低血糖血脂、增强胰岛素敏感性和降低心肌细胞凋亡对T2DM大鼠心脏有一定的保护作用。在H9C2细胞中,高糖能上调MAPK通路中JNK的磷酸化水平,诱导细胞凋亡,并具有一定的浓度依赖性。
[Abstract]:Objective Diabetic cardiomyopathy is developed on the basis of diabetes, especially type 2 diabetes, and has the characteristics of severe disease, poor prognosis and high mortality. In order to find an effective treatment method, the effect of melatonin (MLT) on cardiomyocyte apoptosis and its possible mechanism in chronic type 2 diabetes mellitus (T2DM) rats were studied and discussed. Methods (1) T2DM rat model: after feeding with basic diet for one week, 36 SPF grade healthy male SD rats were randomly divided into three groups: control group (Control,n=12), model group (DM,n=12) and treatment group (DM MLT,n=12). Type 2 diabetic model was induced by high fat diet and intraperitoneal injection of streptozotocin (STZ) 25 mg/kg. After the model was successfully established, the treatment group was given MLT (10 mg/ (kg d) intragastric administration for .24 weeks), the cardiac function was measured by arterial catheterization, and the blood and myocardial tissues of abdominal aorta were collected. Fasting blood glucose, (FBG), lipids (TC,LDL-c,TG) and insulin levels were detected by HE staining and Masson staining respectively. The expression of Caspase-3 was detected by immunohistochemical (IHC). In addition, In order to investigate its possible molecular mechanism, the expression of endoplasmic reticulum stress-related protein (GRP78,CHOP,PERK,ATF-6 伪), mitogen-activated protein kinase (MAPK) pathway protein (JNK,p38,ERK) and apoptosis-related protein (Caspase-9,Caspase-3,Bcl-2,Bax) was detected by Western blot method. (2) the expression of H9C2 in rat cardiomyocytes cultured with high glucose was fine. Cell model: after cultured in different glucose concentration medium for 48 h, Hoechst 33258 staining was used to detect the phosphorylation of MAPK pathway related proteins JNK,p38 and ERK by western blot. Results (1) T2DM rat model: FBG of the model group was (19.27 卤1.03) mmol/L, insulin level was significantly higher than that of the control group (34.79 卤7.97 渭 IU/m L), and the insulin sensitivity index (ln ISI) was -6.48 卤0.23 significantly lower than that of the control group, indicating the existence of insulin resistance (IR). These results suggest that the successful establishment of T2DM rat model. MLT can not only reduce the levels of FBG and TC,LDL-c,TG. The results of HE staining showed that MLT could improve the disorder of myocardial morphology. The results showed that MLT could reduce the accumulation of collagen fibers in myocardial tissue. In the model group, the number of cardiomyocyte apoptosis was significantly increased, and the cardiac function was seriously damaged. After treatment with MLT, the results showed that MLT could decrease the expression of Caspase-3 in T2DM rats. Western blot showed that the expression of ER stress-related protein GRP78,CHOP,PERK,ATF-6 伪, IRE1 伪 and Caspase-12 in myocardial tissue of the model group were significantly increased. Moreover, the phosphorylation level of JNK was also up-regulated in rats treated with MLT. The expression of ER stress-related protein and the phosphorylation level of JNK were down-regulated. (2) in high glucose culture H9C2 cell model: Hoechst 33258 staining showed that high glucose could induce apoptosis of H9C2 cells. Western blot results showed that high glucose could up-regulate the phosphorylation of JNK in H9C2 cells. And it has a certain concentration dependence. Conclusion the increase of cardiomyocyte apoptosis in T2DM rats may be due to the elevation of blood glucose and lipids, which may be caused by activation of JNK pathway mediated by endoplasmic reticulum stress to regulate downstream apoptotic proteins, while MLT can reduce blood glucose and blood lipid. Increasing insulin sensitivity and decreasing cardiac myocyte apoptosis may protect the heart of T2DM rats. In H9C2 cells, high glucose can up-regulate the level of JNK phosphorylation in MAPK pathway and induce apoptosis in a dose-dependent manner.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R542.2
本文编号：2219374
[Abstract]:Objective Diabetic cardiomyopathy is developed on the basis of diabetes, especially type 2 diabetes, and has the characteristics of severe disease, poor prognosis and high mortality. In order to find an effective treatment method, the effect of melatonin (MLT) on cardiomyocyte apoptosis and its possible mechanism in chronic type 2 diabetes mellitus (T2DM) rats were studied and discussed. Methods (1) T2DM rat model: after feeding with basic diet for one week, 36 SPF grade healthy male SD rats were randomly divided into three groups: control group (Control,n=12), model group (DM,n=12) and treatment group (DM MLT,n=12). Type 2 diabetic model was induced by high fat diet and intraperitoneal injection of streptozotocin (STZ) 25 mg/kg. After the model was successfully established, the treatment group was given MLT (10 mg/ (kg d) intragastric administration for .24 weeks), the cardiac function was measured by arterial catheterization, and the blood and myocardial tissues of abdominal aorta were collected. Fasting blood glucose, (FBG), lipids (TC,LDL-c,TG) and insulin levels were detected by HE staining and Masson staining respectively. The expression of Caspase-3 was detected by immunohistochemical (IHC). In addition, In order to investigate its possible molecular mechanism, the expression of endoplasmic reticulum stress-related protein (GRP78,CHOP,PERK,ATF-6 伪), mitogen-activated protein kinase (MAPK) pathway protein (JNK,p38,ERK) and apoptosis-related protein (Caspase-9,Caspase-3,Bcl-2,Bax) was detected by Western blot method. (2) the expression of H9C2 in rat cardiomyocytes cultured with high glucose was fine. Cell model: after cultured in different glucose concentration medium for 48 h, Hoechst 33258 staining was used to detect the phosphorylation of MAPK pathway related proteins JNK,p38 and ERK by western blot. Results (1) T2DM rat model: FBG of the model group was (19.27 卤1.03) mmol/L, insulin level was significantly higher than that of the control group (34.79 卤7.97 渭 IU/m L), and the insulin sensitivity index (ln ISI) was -6.48 卤0.23 significantly lower than that of the control group, indicating the existence of insulin resistance (IR). These results suggest that the successful establishment of T2DM rat model. MLT can not only reduce the levels of FBG and TC,LDL-c,TG. The results of HE staining showed that MLT could improve the disorder of myocardial morphology. The results showed that MLT could reduce the accumulation of collagen fibers in myocardial tissue. In the model group, the number of cardiomyocyte apoptosis was significantly increased, and the cardiac function was seriously damaged. After treatment with MLT, the results showed that MLT could decrease the expression of Caspase-3 in T2DM rats. Western blot showed that the expression of ER stress-related protein GRP78,CHOP,PERK,ATF-6 伪, IRE1 伪 and Caspase-12 in myocardial tissue of the model group were significantly increased. Moreover, the phosphorylation level of JNK was also up-regulated in rats treated with MLT. The expression of ER stress-related protein and the phosphorylation level of JNK were down-regulated. (2) in high glucose culture H9C2 cell model: Hoechst 33258 staining showed that high glucose could induce apoptosis of H9C2 cells. Western blot results showed that high glucose could up-regulate the phosphorylation of JNK in H9C2 cells. And it has a certain concentration dependence. Conclusion the increase of cardiomyocyte apoptosis in T2DM rats may be due to the elevation of blood glucose and lipids, which may be caused by activation of JNK pathway mediated by endoplasmic reticulum stress to regulate downstream apoptotic proteins, while MLT can reduce blood glucose and blood lipid. Increasing insulin sensitivity and decreasing cardiac myocyte apoptosis may protect the heart of T2DM rats. In H9C2 cells, high glucose can up-regulate the level of JNK phosphorylation in MAPK pathway and induce apoptosis in a dose-dependent manner.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R542.2
【参考文献】
相关期刊论文 前1条
1 魏娜;贺海波;张长城;袁丁;王婷;;JNK信号通路与细胞凋亡关系的研究进展[J];中国临床药理学与治疗学;2013年07期
，本文编号：2219374
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