【摘要】：目的：隨著人民生活水平的提高、生活方式的改變以及人口老齡化進(jìn)程的加速，我國(guó)糖尿病（Diabetes Mellitus, DM）的患病率正在逐年增高，2007-2008年全國(guó)糖尿病患病率調(diào)查結(jié)果表明，我國(guó)20歲以上成人糖尿病患病率已達(dá)9.7%，據(jù)此推算，我國(guó)糖尿病總患病人數(shù)達(dá)9200萬以上，已成為世界第一糖尿病大國(guó)。糖尿病成為繼腫瘤、心血管疾病之后的第三大嚴(yán)重威脅人類健康的慢性非傳染性疾病。糖尿病的構(gòu)成以2型糖尿�。╰ype2diabetes mellitus, T2DM）為主，占90%以上。糖尿病大血管病變是指主動(dòng)脈、冠狀動(dòng)脈、腦基底動(dòng)脈、腎動(dòng)脈及周圍動(dòng)脈等發(fā)生病變，以動(dòng)脈粥樣硬化（atherosclerosis, AS）為主要病理基礎(chǔ)，它與單純的AS相比病變范圍大、程度重、發(fā)生早。大血管病變是危害最大的糖尿病慢性并發(fā)癥，是我國(guó)2型糖尿病患者主要的致殘、致死原因。約80%的2型糖尿病患者死于大血管并發(fā)癥，如心肌梗死、腦卒中等。研究顯示，AS的病理改變與絲裂素活化蛋白激酶（mitogen-activated protein kinases,MAPK）的過度激活有關(guān)，p38MAPK通路屬于MAPK家族，與內(nèi)皮細(xì)胞損傷關(guān)系密切，是參與炎癥反應(yīng)的重要的細(xì)胞內(nèi)通路，由此推測(cè)，磷酸化p38MAPK參與了2型糖尿病大血管病變的發(fā)病過程。阿托伐他汀能夠有效降低血漿膽固醇（total cholesterol, TC）水平，減少低密度脂蛋白（low density lipoprotein, LDL）的生成，除調(diào)脂作用外，阿托伐他汀還具有抗炎、改善血管內(nèi)皮功能、穩(wěn)定斑塊的作用。本課題通過高脂、高糖飼養(yǎng)wistar大鼠，建立具有2型糖尿病動(dòng)脈粥樣硬化特點(diǎn)的動(dòng)物模型，觀察wistar糖尿病大鼠胸主動(dòng)脈磷酸化p38MAPK表達(dá)水平的變化以及阿托伐他汀干預(yù)對(duì)磷酸化p38MAPK表達(dá)水平的影響，探討p38MAPK與糖尿病大血管動(dòng)脈粥樣硬化的關(guān)系及阿托伐他汀的干預(yù)作用。 方法：選用4周齡健康雄性wistar大鼠25只，適應(yīng)性飼養(yǎng)1周后，按照隨機(jī)數(shù)字表將大鼠隨機(jī)分為正常對(duì)照組（NC，n=6）和實(shí)驗(yàn)組（EX，n=19），正常對(duì)照組采用標(biāo)準(zhǔn)飼料喂養(yǎng)，實(shí)驗(yàn)組采用高脂高糖飼料喂養(yǎng)（20%的蔗糖、10%的熟豬油、2.5%的膽固醇、1%的膽酸、66.5%的標(biāo)準(zhǔn)飼料）。喂養(yǎng)4周后，實(shí)驗(yàn)組大鼠隔夜空腹腹腔注射鏈脲佐菌素（streptozotocin, STZ,30mg/kg），正常對(duì)照組僅注射等容積的檸檬酸緩沖液。于實(shí)驗(yàn)的第6周測(cè)大鼠空腹血糖（fasting blood glucose, FBG），，選擇血糖≥7.8mmol/L者為糖尿病成模標(biāo)準(zhǔn)(19只實(shí)驗(yàn)組大鼠中15只造模成功)。將成模糖尿病大鼠(n=15)隨機(jī)分為2組：2型糖尿病空白對(duì)照組（DM，n=7）、2型糖尿病+阿托伐他汀干預(yù)組（ATR，n=8）。阿托伐他汀組給予阿托伐他汀10mg/kg灌胃，每日一次，正常對(duì)照組和糖尿病組給予等量的飲用水灌胃，共8周，實(shí)驗(yàn)過程中阿托伐他汀組1只大鼠死于灌胃。于實(shí)驗(yàn)第14周末空腹后分別測(cè)量大鼠體重、血糖，麻醉后心尖取血、分離血清，測(cè)定血清TC、甘油三酯（triglyceride, TG）、LDL、高密度脂蛋白（high-density lipoprotein, HDL）、細(xì)胞間黏附分子1（intercellular adhesionmolecule1, ICAM-1）、血管細(xì)胞黏附分子1（vascular cell adhesion molecule1, VCAM-1）及核轉(zhuǎn)錄因子（nuclear transcription factor, NF）-κB水平；取胸主動(dòng)脈縱切后觀察動(dòng)脈情況，取約1cm予以4%多聚甲醛固定，進(jìn)行HE染色，光鏡下觀察血管病理變化；免疫組織化學(xué)法測(cè)胸主動(dòng)脈中磷酸化p38MAPK、NF-κB、單核細(xì)胞趨化蛋白1（monocyte chemoattractantprotein-1, MCP-1）的蛋白表達(dá)水平。以人工計(jì)數(shù)方法統(tǒng)計(jì)主動(dòng)脈組織磷酸化p38MAPK、NF-κB、MCP-1表達(dá)水平。采用SPSS16.0統(tǒng)計(jì)軟件進(jìn)行分析，所有資料均進(jìn)行正態(tài)性檢驗(yàn)及方差齊性檢驗(yàn)，正態(tài)分布數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差（x±s）表示，非正態(tài)分布數(shù)據(jù)以中位數(shù)（最小值，最大值）表示，正態(tài)分布及方差齊的資料，用t檢驗(yàn)及單因素方差分析，組間兩兩比較采用SNK-q檢驗(yàn)，不符合正態(tài)檢驗(yàn)的資料，采用非參數(shù)檢驗(yàn)，指標(biāo)間的相關(guān)性檢驗(yàn)采用pearson相關(guān)分析，以P＜0.05為有統(tǒng)計(jì)學(xué)意義 結(jié)果： 1各組大鼠一般情況 正常對(duì)照組大鼠狀態(tài)良好，飲食及尿量無明顯變化，體重呈逐漸增加趨勢(shì)。糖尿病組及阿托伐他汀組大鼠飲食、尿量有所增加，偶有毛色發(fā)黃、晦暗，毛發(fā)散亂、潮濕、體重下降者，各組大鼠體重差異無統(tǒng)計(jì)學(xué)意義（NC320.67±12.04g；DM326.29±49.74g；ATR317.43±48.67g）。 2各組大鼠血清生化指標(biāo)結(jié)果 2.1實(shí)驗(yàn)6周末數(shù)據(jù) 正常對(duì)照組大鼠空腹血糖為6.50±0.52mmol/L，實(shí)驗(yàn)組為15.90±4.85mmol/L，實(shí)驗(yàn)組大鼠空腹血糖較正常對(duì)照組明顯升高（P0.01）。 2.2實(shí)驗(yàn)14周末，三組大鼠生化指標(biāo) 正常對(duì)照組：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。 糖尿病組：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。 阿托伐他汀組：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。 糖尿病組和阿托伐他汀組大鼠血糖較正常對(duì)照組明顯升高（P0.05），糖尿病組和阿托伐他汀組大鼠血糖差別無統(tǒng)計(jì)學(xué)差異。與正常對(duì)照組相比，糖尿病組大鼠血清TG、TC、LDL、ICAM-1、VCAM-1、NF-κB顯著增高（P0.05），經(jīng)阿托伐他汀干預(yù)治療8周后，阿托伐他汀組各項(xiàng)指標(biāo)較糖尿病組明顯降低（P0.05），但仍高于正常對(duì)照組。糖尿病組及阿托伐他汀組HDL水平較正常對(duì)照組降低，阿托伐他汀組HDL水平較糖尿病組有所升高，但差異無統(tǒng)計(jì)學(xué)意義。 3胸主動(dòng)脈病理改變 胸主動(dòng)脈切片HE染色鏡下檢查：正常對(duì)照組可見血管內(nèi)皮細(xì)胞完整，排列整齊，內(nèi)膜光滑，中膜平滑肌細(xì)胞排列整齊。 糖尿病組可見主動(dòng)脈內(nèi)皮細(xì)胞腫大變性，胞核皺縮，內(nèi)膜增厚，可見泡沫細(xì)胞聚集，內(nèi)彈力板斷裂，中膜平滑肌細(xì)胞排列紊亂，可見膠原纖維增生，外膜可見新生毛細(xì)血管 阿托伐他汀組可見血管內(nèi)皮細(xì)胞基本完整，內(nèi)膜偶有增厚，中膜平滑肌細(xì)胞排列基本整齊。 4免疫組化結(jié)果 和正常對(duì)照組相比，糖尿病組血管組織磷酸化p38MAPK、NF-κB、MCP-1表達(dá)水平升高（P0.05），阿托伐他汀干預(yù)治療8周后，阿托伐他汀組血管組織磷酸化p38MAPK、NF-κB、MCP-1表達(dá)水平較糖尿病組降低（P0.05）。pearson相關(guān)分析顯示主動(dòng)脈磷酸化p38MAPK蛋白和NF-κB（r=0.406, P=0.001）、MCP-1（r=0.310, P=0.016）蛋白表達(dá)呈顯著正相關(guān)。 結(jié)論： 1p38MAPK過度激活參與了糖尿病大血管病變的發(fā)生發(fā)展。 2阿托伐他汀通過調(diào)脂、降低磷酸化p38MAPK的表達(dá)以及抗炎機(jī)制，在糖尿病大血管病變中起到防治作用。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R587.1;R543.5

【引證文獻(xiàn)】

相關(guān)期刊論文 前2條

1 黃麗;宋成文;;黃芩苷及MAPK通路在子癇前期血管內(nèi)皮保護(hù)與損傷關(guān)系中的研究概況[J];湖南中醫(yī)雜志;2017年01期

2 熊國(guó)營(yíng);童南南;;1例冠心病伴2型糖尿病患者服用阿托伐他汀鈣引發(fā)的思考[J];中國(guó)當(dāng)代醫(yī)藥;2015年16期

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