Salusin-β在糖尿病心肌病的炎症和氧化应激中的作用与机制

发布时间：2018-05-09 03:19

本文选题：Salusin-β + 糖尿病心肌病　；参考：《南京医科大学》2017年硕士论文

【摘要】：背景糖尿病心肌病(Diabetic Cardiomyopathy,DCM)是指发生于糖尿病患者,而不能用高血压性心脏病、冠心病、心脏瓣膜病以及动脉粥样硬化性疾病来解释的心脏结构和功能异常性疾病。患者在糖尿病的基础上,出现心脏微血管病变和心肌代谢紊乱,进而发生心脏广泛局灶性坏死,重症患者甚至猝死。糖尿病心肌病的病理特点主要包括心肌炎症、心肌肥大、心肌纤维化、心肌凋亡和自噬以及心功能障碍等。糖尿病是心力衰竭的一个独立危险因素,男性糖尿病患者发生心力衰竭的的风险高出同性别正常人两倍,而女性患者则高出同性别正常人五倍。糖尿病使心力衰竭的患病率提高约2.5倍,15%-35%的糖尿病患者发生心力衰竭,66%的糖尿病患者死于心脏疾病。Salusins是2003年从编码人类扭转应力障碍基因TOR2A进行选择性剪切而来的产物,包括salusin-α和salusin-β,分别由28个和20个氨基酸残基组成。Salusin-β是一种血管活性肽,在人、大鼠和小鼠的心血管系统、免疫系统以及中枢神经系统中均有广泛表达。我们实验室前期研究发现活性氧(Reactive oxygen species,ROS)的产生介导了 salusin-β诱导的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的泡沫细胞形成和单核细胞粘附,并涉及血管损伤引起的VSMCs的迁移和内膜增生。干涉salusin-β表达能有效降低血管内ROS的产生。糖尿病患者血浆salusin-β水平升高,抑制内源性salusin-β改善心肌缺血引起的心肌重塑。然而salusin-β在糖尿病心肌病中是否发挥作用尚不明确,本研究主要探讨salusin-β在糖尿病心肌病中的作用及其分子机制。目的1.探讨salusin-β在糖尿病心肌病的心肌炎症和氧化应激中的作用与机制;2.探讨salusin-β干涉能否改善糖尿病心肌病的心肌炎症和氧化应激。方法联合应用高脂饮食(high fat diet,HFD)和小剂量链脲霉素(streptozocin,STZ)来诱导2型糖尿病模型:将SD大鼠随机分为4组,每组6只。其中一组用来做为对照组,另外3组用来诱导2型糖尿病。2型糖尿病具体诱导方法是:先给予高脂饮食(34.5%脂肪,17.5%蛋白质、48%碳水化合物)喂养4周,在禁食12h后,腹腔单次注射小剂量链脲霉素(27.5mg/kg体重,溶解于柠檬酸盐缓冲液中,pH=4.5)。在注射链脲霉素12周后,检测大鼠空腹血糖(禁食12h),血糖大于11.lmmol/L的大鼠视为糖尿病大鼠。这3组糖尿病大鼠通过尾静脉分别注射磷酸盐缓冲溶液(PBS)、空病毒以及salusin-β干涉病毒(1.0 × 1011 PFU),2周后,重复此项操作一次。大鼠的心肌细胞系H9c2细胞在含体积分数为10%胎牛血清(FBS)的DMEM培养基中进行连续传代培养。用酶消化法和差速离心法从出生后1-3天内的新生大鼠的心肌内获得原代心肌细胞。用含糖量为33.3mmol/L的培养基来模拟体外高糖环境,培养H9c2细胞和原代心肌细胞,正常组的H9c2细胞和原代心肌细胞使用含糖量为5.5mmol/L的培养基培养。大鼠禁食4h后,腹腔注射胰岛素(1单位/kg体重),检测胰岛素抵抗。禁食12h后,腹腔注射葡萄糖(lg/kg体重),检测糖耐量。超声心动图检测大鼠心功能。酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测大鼠心肌、血浆和细胞中salusin-β、白介素-1β(interleukin-1β,IL-lβ)、白介素-6(interleukin-6,IL-6)、以及肿瘤坏死因子(tumor necrosis factor-α,TNF-α)的含量。蛋白质免疫分析技术(Western blotting)检测心肌组织及细胞中prosalusin、VCAM-1、NOX2、NOX4、4-HNE、p65、IL-lβ、IL-6、和 TNF-α 的蛋白含量。用 real-time PCR(RT-PCR)法检测细胞及心肌组织中 salusin-β、VCAM-1、NOX2、NOX4、4-HNE、p65、IL-1β、IL-6、和 TNF-α 的基因表达水平。Dihydroethidium(DHE)作为超氧化物阴离子荧光探针用来检测心肌组织的活性氧水平。免疫荧光染色(immunofluorescence,IF)用来检测大鼠心肌组织中salusin-β的表达水平。结果1.Salusin-β增强H9c2细胞的炎症和氧化应激。2.NADPH氧化酶抑制剂二苯乙内酰脲(diphenyleneiodonium,DPI)和抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine,NAC)都能阻断salusin-β和高糖诱导的H9c2细胞炎症和氧化应激以及NFκB的激活。3.NFκB抑制剂Bay11-7082能够阻断salusin-β及高糖诱导的H9c2细胞炎症,但不能阻断salusin-β及高糖诱导的H9c2细胞氧化应激。4干涉salusin-β改善高糖诱导的心肌细胞炎症、氧化应激以及NFκB激活。5糖尿病大鼠的血浆以及心肌中salusin-β表达增加。6干涉salusin-β改善糖尿病大鼠的心肌炎症、氧化应激、NFκB激活以及心功能障碍。结论1.Salusin-β通过NOX2/ROS/NFKB引起糖尿病心肌病的心肌炎症;2.干涉salusin-β减轻糖尿病的心肌炎症和氧化应激,并改善心功能。
[Abstract]:Diabetic Cardiomyopathy (DCM) is an abnormal cardiac and functional disorder that occurs in patients with diabetes and cannot be explained by hypertensive heart disease, coronary heart disease, valvular heart disease, and atherosclerotic diseases. The pathological features of diabetic cardiomyopathy mainly include myocarditis, myocardial hypertrophy, myocardial fibrosis, myocardial apoptosis and autophagy, and cardiac dysfunction. Diabetes is an independent risk factor for heart failure, and heart failure in male diabetic patients. The risk was two times higher than normal homosexual people, while women were five times higher than normal homosexual people. Diabetes increased the incidence of heart failure about 2.5 times, 15%-35% diabetic patients had heart failure, and 66% of diabetic patients died from heart disease.Salusins in 2003 from the coding of human torsion stress disorder gene TOR2A The products of sexual shear, including salusin- alpha and salusin- beta, are composed of 28 and 20 amino acid residues,.Salusin- beta, a vasoactive peptide, which are widely expressed in the cardiovascular system, the immune system and the central nervous system in human, rat and mice. We found reactive oxygen species (Reactive oxygen species) in the early laboratory. The production of ROS) mediates the formation of vascular smooth muscle cells (VSMCs) foams and monocyte adhesion to salusin- beta induced vascular smooth muscle cells, and involves the migration of VSMCs and intimal hyperplasia caused by vascular damage. Interference of salusin- beta expression can effectively reduce the production of ROS in the blood tube. The plasma salusin- beta level of diabetic patients Increase, inhibit endogenous salusin- beta to improve myocardial remodeling caused by myocardial ischemia. However, it is not clear whether salusin- beta plays a role in diabetic cardiomyopathy. This study mainly discusses the role and molecular mechanism of salusin- beta in diabetic cardiomyopathy. Objective 1. to explore the myocarditis and oxidation of salusin- beta in diabetic cardiomyopathy. 2. to explore whether salusin- beta interference can improve myocarditis and oxidative stress in diabetic cardiomyopathy. Methods combined use of high fat diet (high fat diet, HFD) and small dose of streptozotocin (streptozocin, STZ) to induce type 2 diabetes mellitus model: the rats were randomly divided into 4 groups, each group was 6. One group was used as a control. In the other 3 groups, the other 3 groups used to induce type 2 diabetes mellitus (type 2 diabetes) to induce the specific induction of diabetes: first feeding the high fat diet (34.5% fat, 17.5% protein, 48% carbohydrate) for 4 weeks. After fasting, the abdominal cavity was injected with small dose of streptozotocin (27.5mg/kg weight, dissolved in citrate buffer, pH=4.5). After injection of streptozotocin for 12 weeks, Rats with fasting blood glucose (fasting 12h) and rats with blood glucose greater than 11.lmmol/L were treated as diabetic rats. The 3 groups of diabetic rats were injected with phosphate buffer solution (PBS), empty virus and salusin- beta interfero virus (1 x 1011 PFU) in the tail vein. After 2 weeks, this operation was repeated. The rat's myocardial cell line H9c2 cells were contained in the volume. Continuous subculture was carried out in the DMEM medium of 10% fetal bovine serum (FBS). Primary cardiomyocytes were obtained by enzyme digestion and differential centrifugation from the myocardium of newborn rats at 1-3 days after birth. The high glucose environment in vitro was simulated with the medium containing sugar content of 33.3mmol/L, cultured H9c2 cells and primary cardiomyocytes, and H9c2 of the normal group. Cells and primary cardiomyocytes were cultured in medium with sugar content of 5.5mmol/L. After fasting 4h, rats were intraperitoneally injected with insulin (1 units of /kg body weight) to detect insulin resistance. After fasting 12h, glucose tolerance was detected by intraperitoneal injection of glucose (lg/kg weight). Cardiac function of rats was detected by echocardiography. Enzyme linked immunosorbent assay (enzyme-linked IMM) Unosorbent assay, ELISA) detection of rat myocardium, plasma and cell salusin- beta, interleukins -1 beta (interleukin-1 beta, IL-l beta), interleukins -6 (interleukin-6, IL-6), and tumor necrosis factor (tumor necrosis alpha, alpha). Protein immunoassay technique for detection of myocardial tissue and cells 1, NOX2, NOX4,4-HNE, p65, IL-l beta, IL-6, and TNF- alpha protein content. Use real-time PCR (RT-PCR) method to detect the gene expression level of salusin- beta, VCAM-1, NOX2, and alpha, as a superoxide anion fluorescent probe used to detect reactive oxygen species in myocardial tissue. Level. Immunofluorescence (IF) was used to detect the expression level of salusin- beta in rat myocardial tissue. Results 1.Salusin- beta enhanced H9c2 cell inflammation and oxidative stress.2.NADPH oxidase inhibitor two Phenylacetyl urea (diphenyleneiodonium, DPI) and the antioxidant N- acetyl cysteine (N-acetylcysteine, NAC). Blocking salusin- beta and high glucose induced H9c2 cell inflammation and oxidative stress, and the activation of.3.NF kappa B inhibitor Bay11-7082 in NF kappa B can block salusin- beta and high glucose induced H9c2 cell inflammation, but can not block salusin- beta and high glucose induced H9c2 cell oxidative stress.4 interfere with the high glucose induced cardiomyocyte inflammation and oxidation Stress and NF kappa B activated the plasma of.5 diabetic rats and the expression of salusin- beta in myocardium increased.6 interference salusin- beta to improve myocarditis, oxidative stress, activation of NF kappa B and cardiac dysfunction in diabetic rats. Conclusion 1.Salusin- beta induces myocarditis in diabetic cardiomyopathy by NOX2/ROS/NFKB; 2. interfering with salusin- beta to alleviate diabetes Cardiac myositis and oxidative stress and the improvement of cardiac function.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R542.2

【参考文献】