磷酸化p38MAPK在2型糖尿病大鼠大血管中的表达及阿托伐他汀的干预作用
发布时间：2018-09-07 21:55
【摘要】：目的：随着人民生活水平的提高、生活方式的改变以及人口老龄化进程的加速，我国糖尿病（Diabetes Mellitus, DM）的患病率正在逐年增高，2007-2008年全国糖尿病患病率调查结果表明，我国20岁以上成人糖尿病患病率已达9.7%，据此推算，我国糖尿病总患病人数达9200万以上，已成为世界第一糖尿病大国。糖尿病成为继肿瘤、心血管疾病之后的第三大严重威胁人类健康的慢性非传染性疾病。糖尿病的构成以2型糖尿病（type2diabetes mellitus, T2DM）为主，占90%以上。糖尿病大血管病变是指主动脉、冠状动脉、脑基底动脉、肾动脉及周围动脉等发生病变，以动脉粥样硬化（atherosclerosis, AS）为主要病理基础，它与单纯的AS相比病变范围大、程度重、发生早。大血管病变是危害最大的糖尿病慢性并发症，是我国2型糖尿病患者主要的致残、致死原因。约80%的2型糖尿病患者死于大血管并发症，如心肌梗死、脑卒中等。研究显示，AS的病理改变与丝裂素活化蛋白激酶（mitogen-activated protein kinases,MAPK）的过度激活有关，p38MAPK通路属于MAPK家族，与内皮细胞损伤关系密切，是参与炎症反应的重要的细胞内通路，由此推测，磷酸化p38MAPK参与了2型糖尿病大血管病变的发病过程。阿托伐他汀能够有效降低血浆胆固醇（total cholesterol, TC）水平，减少低密度脂蛋白（low density lipoprotein, LDL）的生成，除调脂作用外，阿托伐他汀还具有抗炎、改善血管内皮功能、稳定斑块的作用。本课题通过高脂、高糖饲养wistar大鼠，建立具有2型糖尿病动脉粥样硬化特点的动物模型，观察wistar糖尿病大鼠胸主动脉磷酸化p38MAPK表达水平的变化以及阿托伐他汀干预对磷酸化p38MAPK表达水平的影响，探讨p38MAPK与糖尿病大血管动脉粥样硬化的关系及阿托伐他汀的干预作用。 方法：选用4周龄健康雄性wistar大鼠25只，适应性饲养1周后，按照随机数字表将大鼠随机分为正常对照组（NC，n=6）和实验组（EX，n=19），正常对照组采用标准饲料喂养，实验组采用高脂高糖饲料喂养（20%的蔗糖、10%的熟猪油、2.5%的胆固醇、1%的胆酸、66.5%的标准饲料）。喂养4周后，实验组大鼠隔夜空腹腹腔注射链脲佐菌素（streptozotocin, STZ,30mg/kg），正常对照组仅注射等容积的柠檬酸缓冲液。于实验的第6周测大鼠空腹血糖（fasting blood glucose, FBG），，选择血糖≥7.8mmol/L者为糖尿病成模标准(19只实验组大鼠中15只造模成功)。将成模糖尿病大鼠(n=15)随机分为2组：2型糖尿病空白对照组（DM，n=7）、2型糖尿病+阿托伐他汀干预组（ATR，n=8）。阿托伐他汀组给予阿托伐他汀10mg/kg灌胃，每日一次，正常对照组和糖尿病组给予等量的饮用水灌胃，共8周，实验过程中阿托伐他汀组1只大鼠死于灌胃。于实验第14周末空腹后分别测量大鼠体重、血糖，麻醉后心尖取血、分离血清，测定血清TC、甘油三酯（triglyceride, TG）、LDL、高密度脂蛋白（high-density lipoprotein, HDL）、细胞间黏附分子1（intercellular adhesionmolecule1, ICAM-1）、血管细胞黏附分子1（vascular cell adhesion molecule1, VCAM-1）及核转录因子（nuclear transcription factor, NF）-κB水平；取胸主动脉纵切后观察动脉情况，取约1cm予以4%多聚甲醛固定，进行HE染色，光镜下观察血管病理变化；免疫组织化学法测胸主动脉中磷酸化p38MAPK、NF-κB、单核细胞趋化蛋白1（monocyte chemoattractantprotein-1, MCP-1）的蛋白表达水平。以人工计数方法统计主动脉组织磷酸化p38MAPK、NF-κB、MCP-1表达水平。采用SPSS16.0统计软件进行分析，所有资料均进行正态性检验及方差齐性检验，正态分布数据以均数±标准差（x±s）表示，非正态分布数据以中位数（最小值，最大值）表示，正态分布及方差齐的资料，用t检验及单因素方差分析，组间两两比较采用SNK-q检验，不符合正态检验的资料，采用非参数检验，指标间的相关性检验采用pearson相关分析，以P＜0.05为有统计学意义 结果： 1各组大鼠一般情况 正常对照组大鼠状态良好，饮食及尿量无明显变化，体重呈逐渐增加趋势。糖尿病组及阿托伐他汀组大鼠饮食、尿量有所增加，偶有毛色发黄、晦暗，毛发散乱、潮湿、体重下降者，各组大鼠体重差异无统计学意义（NC320.67±12.04g；DM326.29±49.74g；ATR317.43±48.67g）。 2各组大鼠血清生化指标结果 2.1实验6周末数据 正常对照组大鼠空腹血糖为6.50±0.52mmol/L，实验组为15.90±4.85mmol/L，实验组大鼠空腹血糖较正常对照组明显升高（P0.01）。 2.2实验14周末，三组大鼠生化指标 正常对照组：FBG6.33±0.69mmol/L，TG0.81±0.27mmol/L，TC2.18±0.39mmol/L，LDL1.24±0.47mmol/L，HDL1.55±0.33mmol/L，ICAM-175.63±19.52pg/ml，VCAM-1616.54±54.51ng/ml，NF-κB78.68±12.15μmol/L。 糖尿病组：FBG12.99±2.07mmol/L，TG1.83±0.40mmol/L，TC4.15±0.41mmol/L，LDL2.53±0.44mmol/L，HDL0.68±0.23mmol/L，ICAM-1130.59±16.00pg/ml，VCAM-1990.19±119.18ng/ml，NF-κB170.22±21.53μmol/L。 阿托伐他汀组：FBG12.43±2.40mmol/L，TG1.23±0.33mmol/L，TC3.07±0.51mmol/L，LDL1.86±0.28mmol/L，HDL0.98±0.33mmol/L，ICAM-1102.61±15.15pg/ml，VCAM-1809.29±150.27ng/ml，NF-κB116.13±32.63μmol/L。 糖尿病组和阿托伐他汀组大鼠血糖较正常对照组明显升高（P0.05），糖尿病组和阿托伐他汀组大鼠血糖差别无统计学差异。与正常对照组相比，糖尿病组大鼠血清TG、TC、LDL、ICAM-1、VCAM-1、NF-κB显著增高（P0.05），经阿托伐他汀干预治疗8周后，阿托伐他汀组各项指标较糖尿病组明显降低（P0.05），但仍高于正常对照组。糖尿病组及阿托伐他汀组HDL水平较正常对照组降低，阿托伐他汀组HDL水平较糖尿病组有所升高，但差异无统计学意义。 3胸主动脉病理改变 胸主动脉切片HE染色镜下检查：正常对照组可见血管内皮细胞完整，排列整齐，内膜光滑，中膜平滑肌细胞排列整齐。 糖尿病组可见主动脉内皮细胞肿大变性，胞核皱缩，内膜增厚，可见泡沫细胞聚集，内弹力板断裂，中膜平滑肌细胞排列紊乱，可见胶原纤维增生，外膜可见新生毛细血管 阿托伐他汀组可见血管内皮细胞基本完整，内膜偶有增厚，中膜平滑肌细胞排列基本整齐。 4免疫组化结果 和正常对照组相比，糖尿病组血管组织磷酸化p38MAPK、NF-κB、MCP-1表达水平升高（P0.05），阿托伐他汀干预治疗8周后，阿托伐他汀组血管组织磷酸化p38MAPK、NF-κB、MCP-1表达水平较糖尿病组降低（P0.05）。pearson相关分析显示主动脉磷酸化p38MAPK蛋白和NF-κB（r=0.406, P=0.001）、MCP-1（r=0.310, P=0.016）蛋白表达呈显著正相关。 结论： 1p38MAPK过度激活参与了糖尿病大血管病变的发生发展。 2阿托伐他汀通过调脂、降低磷酸化p38MAPK的表达以及抗炎机制，在糖尿病大血管病变中起到防治作用。
[Abstract]:Objective: With the improvement of people's living standard, the change of life style and the acceleration of population aging, the prevalence of diabetes mellitus (DM) in China is increasing year by year. The results of the national survey of diabetes prevalence from 2007 to 2008 show that the prevalence rate of diabetes mellitus in adults over 20 years old has reached 9.7%. Based on this, the sugar content in China has been estimated. Diabetes mellitus is the third most serious chronic non-communicable disease that threatens human health after cancer and cardiovascular disease. Diabetes mellitus is mainly composed of type 2 diabetes mellitus (T2DM), accounting for more than 90%. Atherosclerosis (AS) is the main pathological basis of atherosclerosis. Compared with simple AS, AS has a large range of lesions, severe degree and occurs early. Macrovascular lesions are the most harmful chronic complications of diabetes mellitus and the main type 2 diabetes mellitus in China. About 80% of type 2 diabetic patients die from macrovascular complications, such as myocardial infarction and stroke. Studies have shown that the pathological changes of AS are related to the over-activation of mitogen-activated protein kinases (MAPK). The p38 MAPK pathway belongs to the MAPK family, which is closely related to endothelial cell injury and is involved in it. Atorvastatin can effectively reduce the level of total cholesterol (TC) and the production of low density lipoprotein (LDL), in addition to its lipid-lowering effect. In this study, Wistar rats were fed with high fat and sugar to establish an animal model with atherosclerosis characteristics of type 2 diabetes mellitus. The changes of phosphorylated p38 MAPK expression in thoracic aorta of Wistar diabetic rats and the effect of atorvastatin on phosphorylated p38 MAPK were observed. To investigate the relationship between p38 MAPK and diabetic macrovascular atherosclerosis and the effect of atorvastatin.
METHODS: Twenty-five healthy male Wistar rats aged 4 weeks were randomly divided into normal control group (NC, n = 6) and experimental group (EX, n = 19) according to the random number table. The normal control group was fed with standard diet. The experimental group was fed with high fat and high sugar diet (20% sucrose, 10% cooked lard, 2.5% cholesterol, 1% cholic acid). Four weeks after feeding, the rats in the experimental group were intraperitoneally injected with streptozotocin (STZ, 30mg/kg) overnight, and the normal control group was only injected with citric acid buffer of equal volume. 15 rats in the experimental group were successfully established. The diabetic rats (n=15) were randomly divided into two groups: the blank control group of type 2 diabetes mellitus (DM, n=7), the intervention group of type 2 diabetes mellitus and atorvastatin (ATR, n=8). Atorvastatin group was given atorvastatin 10 mg/kg once a day, and the normal control group and the diabetic group were given the same amount of drinking water. At the end of the 14th week of the experiment, the body weight, blood glucose, cardiac apex blood were measured, serum TC, triglyceride (TG), LDL, high-density lipoprotein (HDL), intercellular adhesion molecule 1 (intercellular adhesion molecule 1) were measured. The levels of R adhesionmolecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and nuclear transcription factor kappa B (NF-kappa B) were measured. The expression levels of phosphorylated p38MAPK, NF-kappa B and monocyte chemoattractant protein-1 (MCP-1) in thoracic aorta were measured by histochemical method. Test and variance homogeneity test, the normal distribution data is expressed as mean (+standard deviation) (x (+s), the non-normal distribution data is expressed as median (minimum, maximum), the normal distribution and variance homogeneous data, using t test and one-way ANOVA, two-to-two comparisons between groups using SNK-q test, non-normal test data, using non-parametric test. Pearson correlation analysis was used to test the correlation between indexes, and P < 0.05 was statistically significant.
Result:
1 general condition of rats in each group
The rats in diabetes group and atorvastatin group were fed a diet with increased urine volume, occasionally yellow, dark, diffuse hair, wet, and weight loss. There was no significant difference in body weight among the groups (NC320.67 [12.04 g]; DM326.29 [49.74 g]). ; ATR317.43 + 48.67g).
2 serum biochemical indicators of rats in each group
2.1 Experiment 6 weekend data
The fasting blood glucose of the normal control group was 6.50 (+ 0.52 mmol/L) and the experimental group was 15.90 (+ 4.85 mmol/L). The fasting blood glucose of the experimental group was significantly higher than that of the normal control group (P 0.01).
2.2 experiment 14 weekend, three groups of rats biochemical indicators
Normal control group: FBG 6.33+0.69mmol/L, TG 0.81+0.27mmol/L, TC 2.18+0.39mmol/L, LDL 1.24+0.47mmol/L, HDL 1.55+0.33mmol/L, ICAM-175.63+19.52pg/ml, VCAM-1616.54+54.51ng/ml, NF-kappa B78.68+12.15mol/L
Diabetes mellitus group: FBG12.99+2.07 mmol/L, TG1.83+0.40 mmol/L, TC4.15+0.41 mmol/L, LDL 2.53+0.44 mmol/L, HDL 0.68+0.23 mmol/L, ICAM-1130.59+16.00 pg/ml, VCAM-1990.19+119.18 ng/ml, NF-kappa B170.22+21.53 umol/L
Atorvastatin group: FBG12.43 (+2.40 mmol/L), TG1.23 (+0.33 mmol/L), TC3.07 (+0.51 mmol/L), LDL 1.86 (+0.28 mmol/L), HDL 0.98 (+0.33 mmol/L), ICAM-1102.61 (+15.15 pg/ml), VCAM-1809.29 (+150.27 ng/ml, NF-kappa B16.13 (+32.63 mmol/L), HDL 0.98 (+0.33 mmol/L), ICAM-1102.61 (+15.15.15
Compared with the normal control group, the serum TG, TC, LDL, ICAM-1, VCAM-1, NF-kappa B of diabetic rats were significantly increased (P 0.05). After 8 weeks of intervention with atorvastatin, the blood glucose of the diabetic rats was significantly increased (P 0.05). The levels of HDL in the statin group were significantly lower than those in the diabetes group (P 0.05), but still higher than those in the normal control group.
3 pathological changes of thoracic aorta
HE staining of thoracic aorta slices showed that vascular endothelial cells were intact, well-arranged, smooth intima and smooth muscle cells were well-arranged in the normal control group.
In diabetic group, aortic endothelial cells were enlarged and degenerated, nucleus shrunk, intima thickened, foam cells aggregated, inner elastic plate ruptured, smooth muscle cells arranged disorderly, collagen fibers hyperplasia and new capillaries were seen in the adventitia.
Atorvastatin group showed that the vascular endothelial cells were basically intact, the intima was occasionally thickened, and the arrangement of smooth muscle cells in the mesangium was basically neat.
4 immunohistochemistry results
Compared with the normal control group, the expression levels of phosphorylated p38MAPK, NF-kappa B and MCP-1 in vascular tissue of diabetic group increased (P 0.05). After 8 weeks of atorvastatin treatment, the expression levels of phosphorylated p38MAPK, NF-kappa B and MCP-1 in vascular tissue of atorvastatin group were lower than those of diabetic group (P 0.05). Pearson correlation analysis showed that phosphorylated p38MAPK protein and N-38MAPK protein in aorta were decreased (P 0.05). F- kappa B (r=0.406, P=0.001) and MCP-1 (r=0.310, P=0.016) protein expression was positively correlated.
Conclusion:
Excessive activation of 1p38MAPK is involved in the occurrence and development of diabetic macroangiopathy.
Atorvastatin plays a preventive and therapeutic role in diabetic macroangiopathy by lowering the expression of phosphorylated p38 MAPK and anti-inflammatory mechanism through lipid regulation.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.1;R543.5
本文编号：2229527
[Abstract]:Objective: With the improvement of people's living standard, the change of life style and the acceleration of population aging, the prevalence of diabetes mellitus (DM) in China is increasing year by year. The results of the national survey of diabetes prevalence from 2007 to 2008 show that the prevalence rate of diabetes mellitus in adults over 20 years old has reached 9.7%. Based on this, the sugar content in China has been estimated. Diabetes mellitus is the third most serious chronic non-communicable disease that threatens human health after cancer and cardiovascular disease. Diabetes mellitus is mainly composed of type 2 diabetes mellitus (T2DM), accounting for more than 90%. Atherosclerosis (AS) is the main pathological basis of atherosclerosis. Compared with simple AS, AS has a large range of lesions, severe degree and occurs early. Macrovascular lesions are the most harmful chronic complications of diabetes mellitus and the main type 2 diabetes mellitus in China. About 80% of type 2 diabetic patients die from macrovascular complications, such as myocardial infarction and stroke. Studies have shown that the pathological changes of AS are related to the over-activation of mitogen-activated protein kinases (MAPK). The p38 MAPK pathway belongs to the MAPK family, which is closely related to endothelial cell injury and is involved in it. Atorvastatin can effectively reduce the level of total cholesterol (TC) and the production of low density lipoprotein (LDL), in addition to its lipid-lowering effect. In this study, Wistar rats were fed with high fat and sugar to establish an animal model with atherosclerosis characteristics of type 2 diabetes mellitus. The changes of phosphorylated p38 MAPK expression in thoracic aorta of Wistar diabetic rats and the effect of atorvastatin on phosphorylated p38 MAPK were observed. To investigate the relationship between p38 MAPK and diabetic macrovascular atherosclerosis and the effect of atorvastatin.
METHODS: Twenty-five healthy male Wistar rats aged 4 weeks were randomly divided into normal control group (NC, n = 6) and experimental group (EX, n = 19) according to the random number table. The normal control group was fed with standard diet. The experimental group was fed with high fat and high sugar diet (20% sucrose, 10% cooked lard, 2.5% cholesterol, 1% cholic acid). Four weeks after feeding, the rats in the experimental group were intraperitoneally injected with streptozotocin (STZ, 30mg/kg) overnight, and the normal control group was only injected with citric acid buffer of equal volume. 15 rats in the experimental group were successfully established. The diabetic rats (n=15) were randomly divided into two groups: the blank control group of type 2 diabetes mellitus (DM, n=7), the intervention group of type 2 diabetes mellitus and atorvastatin (ATR, n=8). Atorvastatin group was given atorvastatin 10 mg/kg once a day, and the normal control group and the diabetic group were given the same amount of drinking water. At the end of the 14th week of the experiment, the body weight, blood glucose, cardiac apex blood were measured, serum TC, triglyceride (TG), LDL, high-density lipoprotein (HDL), intercellular adhesion molecule 1 (intercellular adhesion molecule 1) were measured. The levels of R adhesionmolecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and nuclear transcription factor kappa B (NF-kappa B) were measured. The expression levels of phosphorylated p38MAPK, NF-kappa B and monocyte chemoattractant protein-1 (MCP-1) in thoracic aorta were measured by histochemical method. Test and variance homogeneity test, the normal distribution data is expressed as mean (+standard deviation) (x (+s), the non-normal distribution data is expressed as median (minimum, maximum), the normal distribution and variance homogeneous data, using t test and one-way ANOVA, two-to-two comparisons between groups using SNK-q test, non-normal test data, using non-parametric test. Pearson correlation analysis was used to test the correlation between indexes, and P < 0.05 was statistically significant.
Result:
1 general condition of rats in each group
The rats in diabetes group and atorvastatin group were fed a diet with increased urine volume, occasionally yellow, dark, diffuse hair, wet, and weight loss. There was no significant difference in body weight among the groups (NC320.67 [12.04 g]; DM326.29 [49.74 g]). ; ATR317.43 + 48.67g).
2 serum biochemical indicators of rats in each group
2.1 Experiment 6 weekend data
The fasting blood glucose of the normal control group was 6.50 (+ 0.52 mmol/L) and the experimental group was 15.90 (+ 4.85 mmol/L). The fasting blood glucose of the experimental group was significantly higher than that of the normal control group (P 0.01).
2.2 experiment 14 weekend, three groups of rats biochemical indicators
Normal control group: FBG 6.33+0.69mmol/L, TG 0.81+0.27mmol/L, TC 2.18+0.39mmol/L, LDL 1.24+0.47mmol/L, HDL 1.55+0.33mmol/L, ICAM-175.63+19.52pg/ml, VCAM-1616.54+54.51ng/ml, NF-kappa B78.68+12.15mol/L
Diabetes mellitus group: FBG12.99+2.07 mmol/L, TG1.83+0.40 mmol/L, TC4.15+0.41 mmol/L, LDL 2.53+0.44 mmol/L, HDL 0.68+0.23 mmol/L, ICAM-1130.59+16.00 pg/ml, VCAM-1990.19+119.18 ng/ml, NF-kappa B170.22+21.53 umol/L
Atorvastatin group: FBG12.43 (+2.40 mmol/L), TG1.23 (+0.33 mmol/L), TC3.07 (+0.51 mmol/L), LDL 1.86 (+0.28 mmol/L), HDL 0.98 (+0.33 mmol/L), ICAM-1102.61 (+15.15 pg/ml), VCAM-1809.29 (+150.27 ng/ml, NF-kappa B16.13 (+32.63 mmol/L), HDL 0.98 (+0.33 mmol/L), ICAM-1102.61 (+15.15.15
Compared with the normal control group, the serum TG, TC, LDL, ICAM-1, VCAM-1, NF-kappa B of diabetic rats were significantly increased (P 0.05). After 8 weeks of intervention with atorvastatin, the blood glucose of the diabetic rats was significantly increased (P 0.05). The levels of HDL in the statin group were significantly lower than those in the diabetes group (P 0.05), but still higher than those in the normal control group.
3 pathological changes of thoracic aorta
HE staining of thoracic aorta slices showed that vascular endothelial cells were intact, well-arranged, smooth intima and smooth muscle cells were well-arranged in the normal control group.
In diabetic group, aortic endothelial cells were enlarged and degenerated, nucleus shrunk, intima thickened, foam cells aggregated, inner elastic plate ruptured, smooth muscle cells arranged disorderly, collagen fibers hyperplasia and new capillaries were seen in the adventitia.
Atorvastatin group showed that the vascular endothelial cells were basically intact, the intima was occasionally thickened, and the arrangement of smooth muscle cells in the mesangium was basically neat.
4 immunohistochemistry results
Compared with the normal control group, the expression levels of phosphorylated p38MAPK, NF-kappa B and MCP-1 in vascular tissue of diabetic group increased (P 0.05). After 8 weeks of atorvastatin treatment, the expression levels of phosphorylated p38MAPK, NF-kappa B and MCP-1 in vascular tissue of atorvastatin group were lower than those of diabetic group (P 0.05). Pearson correlation analysis showed that phosphorylated p38MAPK protein and N-38MAPK protein in aorta were decreased (P 0.05). F- kappa B (r=0.406, P=0.001) and MCP-1 (r=0.310, P=0.016) protein expression was positively correlated.
Conclusion:
Excessive activation of 1p38MAPK is involved in the occurrence and development of diabetic macroangiopathy.
Atorvastatin plays a preventive and therapeutic role in diabetic macroangiopathy by lowering the expression of phosphorylated p38 MAPK and anti-inflammatory mechanism through lipid regulation.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.1;R543.5
【引证文献】
相关期刊论文 前2条
1 黄丽;宋成文;;黄芩苷及MAPK通路在子痫前期血管内皮保护与损伤关系中的研究概况[J];湖南中医杂志;2017年01期
2 熊国营;童南南;;1例冠心病伴2型糖尿病患者服用阿托伐他汀钙引发的思考[J];中国当代医药;2015年16期
本文编号：2229527
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