气体信号分子硫化氢对糖尿病心肌损伤的保护作用及相关机制研究

发布时间：2018-03-08 01:27

本文选题：糖尿病心肌损伤　切入点：硫化氢　出处：《南京医科大学》2015年博士论文　论文类型：学位论文

【摘要】：背景:糖尿病心肌损伤是糖尿病的常见并发症之一,也是糖尿病患者心力衰竭发生率高和死亡率高的主要原因,其特征性病理改变包括心肌细胞肥大、肌原纤维缺失、间质纤维化、心肌内微血管病变等。当前研究表明,高血糖、脂质沉积、氧化应激、钙稳态失衡、线粒体功能障碍、肾素-血管紧张素系统激活、心肌炎症、细胞凋亡等在糖尿病心肌损伤中发挥重要作用。硫化氢(H2S)是继一氧化氮和一氧化碳以外发现的第三种气体信号分子,它是在磷酸吡哆醛-5'-磷酸依赖性酶(包括胱硫醚-β-合成酶和胱硫醚-γ-裂解酶)催化下以半胱氨酸作为底物生成的。近年来研究表明,H2S在心血管生理学和病理生理学方面发挥重要作用。H2S通过保护线粒体功能减轻心肌缺血再灌注损伤,同时能够降低缺血性心力衰竭的发生率和死亡率。内源性H2S生成减少的患者容易出现血管重构和早发动脉粥样硬化。在本实验中,我们采用链脲霉素诱导建立糖尿病大鼠模型,研究H2S对糖尿病诱导心肌损伤的保护作用及相关机制。方法:我们通过腹腔注射链脲霉素(65 mg/kg)建立糖尿病大鼠模型,然后将SD大鼠随机分成4组,共饲养16周,具体分组情况如下:1 Na HS组(正常大鼠腹腔注射Na HS溶液14μmol/kg.d);2 DM组(糖尿病大鼠腹腔注射等量的生理盐水);3 DM+Na HS组(糖尿病大鼠腹腔注射Na HS溶液14μmol/kg.d);4对照组(正常大鼠腹腔注射等量的生理盐水)。采用亚甲蓝法检测大鼠血浆和心肌组织中H2S含量;运用超声心动图检测大鼠的心脏结构和功能;取大鼠左心室组织,进行HE染色和Masson染色,分别计算心肌细胞横截面积和胶原容积分数;运用免疫组化法检测大鼠心肌组织中Ⅰ型和Ⅲ型胶原的表达;采用ELISA法检测大鼠心肌组织中肿瘤坏死因子α(TNF-α)、白介素(IL)-1β、IL-6、IL-8的含量;检测大鼠心肌组织中丙二醛(MDA)的含量及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性;应用凝胶迁移实验检测大鼠心肌组织Nrf2-ARE的结合力;运用TUNEL染色检测心肌细胞凋亡;采用ELISA法检测心肌组织中PI3K活性;运用Western blot检测心肌组织中Nrf2、HO-1、NQO1、Bax、Bcl-2、caspase-3的表达以及JNK(Thr183/Tyr185)、P38(Thr180/Tyr182)、Akt(Ser473)、Bad(Ser136)、caspase-9(Ser196)的磷酸化。心肌细胞经100μmol/L Na HS预处理30 min后,置于30 mmol/L高糖培养液中培养24 h,然后检测细胞内活性氧(ROS)含量,细胞经Annexin V/PI染色后于流式细胞仪上检测凋亡率。为了证实H2S通过激活Nrf2通路减轻高糖诱导的氧化应激,我们用Nrf2 si RNA转染心肌细胞,经Na HS预处理后置于高糖培养液中培养24 h,然后检测心肌细胞中Nrf2核蛋白表达和ROS含量。为了证实H2S通过抑制MAPK通路和激活PI3K/Akt通路而减轻高糖诱导的细胞凋亡,我们分别用JNK抑制剂SP600125(10μmol/L)、p38抑制剂SB203580(20μmol/L)和PI3K抑制剂LY294002(10μmol/L)预处理心肌细胞60 min,置于高糖培养液中培养24 h,然后检测JNK、p38、Akt的磷酸化水平和心肌细胞凋亡率。结果:1糖尿病大鼠血浆和心肌组织中H2S含量降低,而外源性补充Na HS能够增加糖尿病大鼠体内H2S浓度。2糖尿病大鼠可表现出心脏增大、室壁增厚、心肌纤维化、心功能不全等,而H2S能够减轻糖尿病大鼠心肌肥大和间质纤维化,改善左心室功能。3糖尿病大鼠心肌组织中TNF-α、IL-1β、IL-6、IL-8含量增加,而H2S能够减轻糖尿病大鼠心肌炎症反应。4糖尿病大鼠心肌组织中MDA含量增高,SOD、GSH-Px活性降低,而H2S能够减轻糖尿病大鼠心肌氧化应激水平。5H2S能够增加糖尿病大鼠心肌组织中Nrf2-ARE结合力,促进Nrf2核转移,上调抗氧化蛋白HO-1与NQO1的表达,从而减轻糖尿病大鼠心肌氧化损伤。6Na HS预处理能够激活Nrf2通路,减少高糖诱导下心肌细胞ROS产生;而Nrf2 si RNA转染的心肌细胞经Na HS预处理后不能减少高糖诱导的ROS生成,说明H2S减轻高糖诱导的氧化应激依赖Nrf2通路的激活。7糖尿病大鼠心肌细胞凋亡增加,心肌组织中Bax、caspase-3表达上调,Bcl-2表达下调;而外源性补充Na HS能够降低糖尿病大鼠心肌细胞凋亡指数及Bax/Bcl-2比率,表明H2S可能通过抑制线粒体凋亡通路而减轻高血糖诱导的心肌细胞凋亡。8糖尿病大鼠心肌组织中JNK、p38磷酸化水平增高,而H2S能够抑制糖尿病大鼠心肌组织MAPKs的磷酸化。9高糖能够诱导心肌细胞凋亡,Na HS预处理能够抑制JNK、p38通路,减少心肌细胞凋亡;心肌细胞分别经SP600125、SB203580预处理后,置于高糖培养液中培养,细胞凋亡率明显下降,表明H2S可能通过抑制JNK、p38通路而减轻高糖诱导的心肌细胞凋亡。10糖尿病大鼠心肌组织中PI3K活性以及Akt、caspase-9、Bad的磷酸化水平降低,而H2S能够激活糖尿病大鼠心肌组织PI3K/Akt通路,促进凋亡调节蛋白caspase-9、Bad的磷酸化。?Na HS预处理能够激活PI3K/Akt通路,减轻高糖诱导的心肌细胞凋亡;心肌细胞先后经LY294002、Na HS预处理后,置于高糖培养液中培养,细胞凋亡率未见明显下降,表明H2S可能通过激活PI3K/Akt通路而减轻高糖诱导的心肌细胞凋亡。结论:H2S能够抑制糖尿病大鼠心肌组织炎症反应、氧化应激和细胞凋亡,从而减轻糖尿病诱导的心肌损伤。H2S可能通过激活Nrf2/ARE通路减轻高糖诱导的氧化应激,也可通过抑制JNK、p38通路或激活PI3K/Akt通路减轻高糖诱导的细胞凋亡。
[Abstract]:Background: diabetic myocardial damage is one of the most common complications of diabetes, diabetes is the high incidence of heart failure and high mortality mainly because of its characteristic pathological changes including myocardial hypertrophy, myofibrillar loss, interstitial fibrosis, myocardial microvascular lesion. The current study shows that high blood glucose, lipid deposition, oxidative stress, calcium homeostasis, mitochondrial dysfunction, activation of the renin-angiotensin system, inflammation, cell apoptosis play an important role in myocardial injury in diabetes mellitus (H2S). Hydrogen sulfide is the third gaseous signal molecule found outside a nitric oxide and carbon monoxide, it is in the pyridoxal phosphate -5'- phosphate dependent enzymes (including cystathionine beta synthase and cystathionine gamma lyase) catalyzed by cysteine as a substrate formation. Recent studies show that H2S in cardiovascular physiology and pathology Physiological.H2S play an important role by protecting mitochondrial function in myocardial ischemia reperfusion, and can decrease the incidence and mortality of ischemic heart failure. The decrease of endogenous H2S production in patients prone to premature atherosclerosis and vascular remodeling. In this experiment, we used streptozotocin induced diabetic rat model was established and the protective effect of H2S on diabetes induced myocardial injury and the related mechanism. Methods: We performed intraperitoneal injection of streptozotocin (65 mg/kg) to establish the model of diabetic rats, and SD rats were randomly divided into 4 groups, raising a total of 16 weeks, the following specific group: 1 Na HS group (normal rats by intraperitoneal injection of Na HS solution 14 mol/kg.d 2); group DM (normal saline in diabetic rats was injected 3 DM+Na (group HS); diabetic rats by intraperitoneal injection of Na HS solution 14 mol/kg.d; 4 (normal control group) Physiological saline was injected to rats). The content of H2S with methylene blue assay in rat plasma and myocardium; cardiac structure and function in the rat detected by ultrasound echocardiography; left ventricular tissue of rats, HE staining and Masson staining were calculated, cardiomyocyte cross-sectional area and collagen volume fraction expression; using immunohistochemical method to detect type I and type III collagen in myocardium of rats; tumor necrosis factor alpha was detected by ELISA in myocardium of rats (TNF- alpha), interleukin (IL) -1 beta IL-6, IL-8, MDA content; rat myocardial tissue detection (MDA). And the content of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity; binding gel mobility assay of myocardial tissue in Nrf2-ARE rats; using TUNEL staining to detect the apoptosis of myocardial cells; detecting PI3K activity in myocardial tissue by ELISA method; transport Nrf2, Western blot in myocardial tissue were detected by HO-1, NQO1, Bax, Bcl-2, caspase-3 expression and JNK (Thr183/Tyr185), P38 (Thr180/Tyr182), Akt (Ser473), Bad (Ser136), caspase-9 (Ser196) phosphorylation of myocardial cells. By 100 mol/L Na HS 30 min after pretreatment, in 30 mmol/L high glucose cultured in 24 h, then detected the intracellular reactive oxygen species (ROS) content, V/PI cells were treated with Annexin staining to detect the apoptosis rate by flow cytometry. In order to confirm the H2S through the activation of the Nrf2 pathway of oxidative stress induced by high glucose, we use Nrf2 Si RNA Na HS after transfection of myocardial cells, pre after treatment in high glucose cultured in 24 h, then detect the expression of nuclear Nrf2 protein content and ROS content in myocardial cells. In order to confirm and decrease cell apoptosis induced by high glucose H2S by inhibiting MAPK pathway and activation of the PI3K/Akt pathway, we used JNK inhibitor SP600125 respectively (10 mol/L), P 38 inhibitor SB203580 (20 mol/L) and PI3K inhibitor LY294002 (10 mol/L) pretreatment of myocardial cells of 60 min in high glucose cultured in 24 h, and detection of JNK, p38, Akt phosphorylation and apoptosis rate. Results: 1 diabetic rats decreased plasma and myocardial tissue in H2S the content of exogenous Na and HS can increase the concentration of H2S in diabetic rats.2 diabetic rats showed cardiac enlargement, ventricular wall thickening, myocardial fibrosis, cardiac insufficiency, and H2S can reduce myocardial hypertrophy and interstitial fibrosis in diabetic rats, improve TNF- alpha, myocardial function in.3 diabetic rats left ventricular IL-1 beta, IL-6, IL-8 content increased, while H2S can increase MDA content, reduce inflammatory response in diabetic rats.4 in myocardial tissue of diabetic rats SOD, GSH-Px activity decreased, while H2S can reduce myocardial oxidative stress in diabetic rats.5H2S Nrf2-ARE can increase the myocardial tissue in diabetic rats with stress, promote Nrf2 nuclear transfer, up regulate the expression of antioxidant protein HO-1 and NQO1, thereby reducing oxidative damage in the myocardium of diabetic rats.6Na HS preconditioning can activate Nrf2 pathway, reduce myocardial cell induced by ROS and Nrf2 Si to produce high glucose; RNA transfection of myocardial cells by Na HS after pretreatment can reduce the formation of ROS induced by high glucose, indicating that the increase of H2S reduce the oxidative stress induced by High Glucose dependent activation of Nrf2 pathway of myocardial cell apoptosis in.7 diabetic rats, myocardial Bax, caspase-3 expression, Bcl-2 expression; while exogenous Na HS can reduce diabetic rat myocardial cell apoptosis index and Bax/Bcl-2 ratio, indicating that H2S may alleviate the myocardial tissue of myocardial cell apoptosis induced by high glucose in.8 diabetic rats JNK by inhibiting the mitochondrial apoptosis pathway, phosphorylation of p38 Increased phosphorylation of.9 high glucose and H2S can inhibit MAPKs myocardial tissue of diabetic rats can induce the apoptosis of myocardial cells, Na HS pretreatment could inhibit JNK and p38 pathway, reduce myocardial cell apoptosis; myocardial cells were treated with SP600125, after SB203580 pretreatment in Gao Tangpei nutrient solution culture, the apoptosis rate decreased significantly, that H2S can inhibit JNK, p38 pathway may reduce PI3K activity in myocardial tissue of myocardial cell apoptosis induced by high glucose in.10 diabetic rats, Akt, caspase-9, Bad phosphorylation decreased, while H2S can activate the diabetes rat myocardium PI3K/Akt pathway, apoptosis regulating protein caspase-9, the phosphorylation of Bad. Na HS? Preconditioning can activate PI3K/Akt pathway, reduce myocardial cell apoptosis induced by high glucose; myocardial cell has been LY294002, Na after HS pretreatment in high glucose culture medium, cell apoptosis rate was Significantly decreased, indicating that H2S may alleviate the myocardial cell apoptosis induced by high glucose through activation of the PI3K/Akt pathway. Conclusion: H2S can inhibit the inflammatory response in myocardial tissue of diabetic rats, oxidative stress and apoptosis, thereby reducing myocardial injury induced by diabetes.H2S can activate Nrf2/ARE signal pathway of oxidative stress induced by high glucose, or by inhibition of JNK. The p38 pathway or the activation of the PI3K/Akt pathway to reduce cell apoptosis induced by high glucose.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2;R542.2

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