DHA保护H9C2心肌细胞脂超载损伤的机制研究
发布时间：2018-09-10 12:50
【摘要】：目的:近年文献报道2型糖尿病(type 2 diabetes mellitus,T2DM)患者数量呈逐年上升的趋势,糖尿病相关心脏并发症是2型糖尿病患者晚期最主要的死亡原因。临床及动物实验发现,食物中补充n-3系多不饱和脂肪酸(n-3 Polyunsaturated fatty acids,n-3 PUFAs),可以对抗心肌肥大,改善心功能。本课题组前期研究发现高脂饮食并注射小剂量STZ(链脲佐菌素)诱导的2型糖尿病模型大鼠心肌n-3 PUFAs二十二碳六烯酸(docosahexaenoic acid,DHA)含量明显下降,并伴有左心肥大,左心室心肌收缩功能明显下降,而饮食中补充DHA的2型糖尿病大鼠上述情况均有所改善。细胞凋亡是指为维持内环境的稳态由基因控制的细胞自主有序的死亡,与细胞坏死不同,凋亡涉及一系列基因的激活,表达及调控。前期实验已发现在整体水平2型糖尿病大鼠心肌内细胞凋亡增加,饮食补充DHA的2型糖尿病大鼠细胞凋亡有所改善。但DHA及其它n-3 PUFAs是否确可对抗心肌细胞凋亡,作用及机制又如何?需要在细胞水平进行研究。细胞骨架是指真核细胞中的蛋白纤维网络结构。细胞骨架不仅在维持细胞形态,承受外力,保持内部结构有序性方面起重要作用,还参与许多重要的生命活动。在心肌细胞中,细胞骨架和它的结合蛋白组成动力系统。文献报道,在心肌肥大和心力衰竭的动物的心肌细胞中可见细胞骨架改变。前期实验已发现T2DM大鼠肌原纤维纹理模糊,纤维束排列不规则,出现断裂溶解,补充DHA可阻止T2DM大鼠心肌肌原纤维的断裂和溶解。DHA等n-3 PUFAs确对心肌细胞骨架有什么影响吗?需在细胞水平证实。基于上述目的,本实验以H9C2大鼠心肌细胞株作为研究对象,给予高浓度软脂酸模拟糖尿病心肌所处的高脂环境,造成细胞损伤后,补充DHA等n-3 PUFAs,观察H9C2心肌细胞凋亡情况及细胞骨架改变,探究DHA的保护机制。方法:1 MTS实验对细胞活性进行检测,确定可使细胞活力下降的C16:0浓度以及dha的保护浓度。2westernblot检测细胞凋亡相关的cleavedcaspase3,cleavedparp,parp和fadd蛋白表达。3realtime-rtpcr检测细胞凋亡相关基因caspase3,9,12,炎症相关基因tnf-α以及氧化应激相关基因ho-1等的mrna表达量。4tunel法检测不同浓度c16:0,不同浓度n-3系pufas以及不同浓度n-6系aa处理后的细胞凋亡情况。5鬼笔环肽染色法观察c16:0对h9c2心肌细胞骨架影响,以及给予dha等n-3系pufas或者n-6系的aa后能否逆转c16:0导致的细胞骨架的改变。6westernblot检测高c16:0对肌钙蛋白tnnt2,肌钙蛋白酶calpain1,calpain2,calpains1的蛋白量的影响,以及补充dha后tnnt2,calpain1,calpain2,calpains1蛋白量的变化。7realtime-rtpcr检测补充dha之后细胞calpain1mrna表达量的变化。8结果数据以x±sd表示,采用spss17.0统计学软件统计学分析,两组之间的差异采用配对t检验进行分析,以p0.05为差异有统计学意义。结果:1脂质超载引起了h9c2心肌细胞的活力下降,dha则可以改善脂质超载损伤的细胞活力给予150-400μmol/l浓度范围内的c16:0处理细胞24小时后,细胞活性明显低于bsa溶剂对照组(p0.05),并且c16:0浓度越高h9c2心肌细胞活性越低。单纯dha处理细胞24小时后,细胞活性与对照组无统计学差别,但在c16:0200μmol/l基础上补充10、100μmol/ldha后,c16:0导致的细胞活力的下降得以改善。2c16:0引起的心肌细胞活力下降与氧化应激及炎症反应的增加有关随着c16:0浓度的增加,h9c2心肌细胞氧化应激相关因子ho-1和炎症相关因子tnf-α的mrna表达量明显增加,表明c16:0引起的心肌细胞活力下降与心肌细胞氧化应激及炎症反应的发生有关。3脂超载引起的细胞活力下降与内质网凋亡途径介导的凋亡有关c16:0处理h9c2细胞24小时后,随着c16:0浓度的逐渐升高,细胞凋亡的数量也出现增加。与此同时,细胞凋亡关的caspase3的mrna表达量,以及cleavedcaspase3和cleavedparp蛋白量明显增加,而细胞坏死的标志性蛋白fadd的表达量却没有出现明显变化,表明c16:0引起心肌细胞活性降低主要是凋亡而不是坏死所致。因为内质网凋亡途径的重要起始子caspase12而不是细胞线粒体凋亡途径的上游分子caspase9的mrna表达量表达增加,表明c16:0超载引起的细胞凋亡与内质网凋亡途径有关。4dha等n-3pufas改善脂超载损伤的心肌细胞活力与其降低细胞凋亡有关给予200μmol/l16:0处理24小时后,凋亡细胞数量增多,但同时给予1、10、100μmol/ldha、epa和lna处理后,则可降低由c16:0上调的凋亡细胞的数量;dha、epa、lna均可以逆转c16:0导致的cleavedcaspase3和cleavedparp蛋白量的增加。但是,n-6pufas花生四烯酸aa本身却可增加细胞凋亡。5脂超载引起h9c2心肌细胞活下降,与其引起心肌细胞细胞骨架改变有关随着c16:0浓度增加,心肌细胞内的肌丝增粗,折叠,致使细胞由规则的长梭形变成有多处凹陷的不规则多边形。随着细胞肌丝形态的改变,细胞的肌钙蛋白酶calpain1,calpain2和calpains1蛋白表达量增加,而肌钙蛋白troponint(tnnt2)的蛋白量逐渐下降。表明c16:0超载引起的细胞活性下降与其肌钙蛋白酶calpain表达增加,从而引起肌钙蛋白troponint的降解,导致细胞骨架形态改变有关。6dha等n-3pufas能够抑制脂超载引起的细胞骨架的改变,进而改善脂超载所致的心肌细胞活力下降给h9c2补充1、10、100μmol/ldha、epa和lna后,可逆转c16:0所致的h9c2细胞骨架改变。但是n-6pufa花生四烯酸aa本身即可造成细胞骨架翻折,并且随着aa浓度的增加,形态异常细胞数量逐渐增加。给h9c2补充dha能降低c16:0导致的肌钙蛋白酶calpain1,2,S1表达上升和TNNT2蛋白量的下降。补充100μmol/L DHA后,C16:0引起的Calpain 1 mRNA的表达上升,被缓解。结论:1 DHA可以改善C16:0脂超载导致的H9C2心肌细胞细胞活力下降。2 DHA可能的作用机制:(1)通过抑制内质网凋亡途径从而抑制细胞凋亡;(2)通过下调肌钙蛋白酶Calpain表达,减少肌钙蛋白TNNT2降解,进而改善细胞骨架的损伤。
[Abstract]:Objective: Recent literatures have reported that the number of type 2 diabetes mellitus (T2DM) patients is increasing year by year. Diabetes-related cardiac complications are the leading cause of death in patients with advanced type 2 diabetes mellitus. Our previous study found that the content of n-3 PUFAs docosahexaenoic acid (DHA) in myocardium of type 2 diabetic rats induced by high-fat diet and low-dose STZ (streptozotocin) significantly decreased, accompanied by left ventricular hypertrophy, and left ventricular systolic function significantly decreased. Apoptosis refers to the autonomous and orderly death of cells controlled by genes in order to maintain homeostasis of the internal environment. Unlike cell necrosis, apoptosis involves the activation, expression and regulation of a series of genes. Previous experiments have found that the heart of type 2 diabetic rats at the overall level is involved. Intramuscular apoptosis was increased in type 2 diabetic rats fed with DHA. However, whether DHA and other n-3 PUFAs can indeed prevent cardiomyocyte apoptosis, and what are their effects and mechanisms? Cellular level studies are needed. Cytoskeleton refers to the structure of protein fiber network in eukaryotic cells. Cytoskeleton is not only in maintaining fine structure. Cytoskeleton and its binding proteins constitute a dynamic system in cardiomyocytes. Cytoskeleton alterations have been reported in cardiomyocytes of hypertrophic and heart failure animals. Previous experiments have found that T2D is involved in many important life activities. Does n-3 PUFAs, such as DHA, have any effect on the cytoskeleton of cardiomyocytes in T2DM rats? It needs to be confirmed at the cellular level. The apoptosis and cytoskeleton of H9C2 myocardial cells were observed and the protective mechanism of DHA was explored after high concentration palmitic acid was given to simulate the hyperlipidemic environment of diabetic myocardium. Methods: 1 MTS assay was used to detect the cell viability and to determine the concentration of C16:0 which could decrease the cell viability and the protection of dha. 2 Western blot was used to detect the expression of cleaved caspase 3, cleaved parp, PARP and FADD protein. 3 realtime-rtpcr was used to detect the expression of caspase 3, 9, 12, inflammation-related gene TNF-a and oxidative stress-related gene ho-1. 5 ghost-pencil peptide staining was used to observe the effect of c16:0 on the cytoskeleton of H9c2 myocardial cells and whether the changes of cytoskeleton induced by c16:0 could be reversed by DHA and other n-3 PUFAs or n-6 aa. The changes of tnnt 2, calpain 1, calpain 2 and calpain 1 protein after DHA supplementation were observed. 7 realtime-rtpcr was used to detect the changes of calpain 1 mRNA expression after DHA supplementation. Results: 1. Lipid overload caused the decrease of H9c2 myocardial cell viability, DHA could improve the cell viability of lipid overload injury, and the activity of H9c2 myocardial cells treated with c16:0 in the concentration range of 150-400 micromol/l for 24 hours was significantly lower than that of BSA solvent control group (p0.05). The higher the concentration of c16:0, the lower the activity of H9c2 myocardial cells. There was no significant difference in cell viability between the control group and the DHA treated cells 24 hours later. However, the decrease of cell viability induced by c16:0 was ameliorated by supplementation of 10,100 micromol/l DHA with c16:0200 micromol/l. The decrease of cardiomyocyte viability induced by c16:0 was related to the increase of oxidative stress and inflammatory reaction with the increase of c16:0 concentration. The increase of the degree of H9c2 myocardial cell oxidative stress-related factors HO-1 and inflammation-related factors TNF-a mRNA expression increased significantly, indicating that c16:0-induced decline in cardiomyocyte viability is related to oxidative stress and inflammation. 3 lipid overload-induced cell viability decline is related to endoplasmic reticulum apoptosis pathway-mediated apoptosis At the same time, the expression of Caspase-3 mrna, cleaved caspase-3 and cleaved PARP protein were significantly increased, while the expression of fadd, a marker of cell necrosis, did not change significantly. 6:0 decreased cardiomyocyte viability was mainly due to apoptosis rather than necrosis, because the expression of caspase 12, an important initiator of the endoplasmic reticulum apoptosis pathway, was higher than that of caspase 9, an upstream molecule of the mitochondrial apoptosis pathway, suggesting that apoptosis induced by c16:0 overload was related to the apoptosis pathway of endoplasmic reticulum. The viability of cardiomyocytes injured by hyperlipidemia was related to the decrease of apoptosis. The number of apoptotic cells increased 24 hours after treatment with 200 micromol/l16:0, but the number of apoptotic cells increased by 1,10,100 micromol/ldha, EPA and LNA at the same time could be decreased by treatment with 1,10,100 micromol/ldha, EPA and lna. However, n-6 PUFAs arachidonic acid AA itself can increase cell apoptosis. 5 lipid overload leads to the decrease of H9c2 cardiomyocyte viability, which is related to the cytoskeleton change of cardiomyocyte. with the increase of c16:0 concentration, the myofilament in cardiomyocyte thickens and folds, resulting in the cells from regular long shuttle to multiple depressions. The expression of calpain-1, calpain-2 and calpain-1 protein increased with the change of myofibril morphology, while the expression of troponin-2 protein decreased gradually. It indicated that the decrease of cell activity and the increase of calpain expression induced by c16:0 overload could result in troponin troponin expression. 6 DHA and other n-3 PUFAs can inhibit the changes of cytoskeleton induced by lipid overload, and then improve the decrease of cardiomyocyte viability induced by lipid overload. When H9c2 is supplemented with 1,10,100 micromol/ldha, EPA and lna, the cytoskeleton changes of H9c2 induced by c16:0 can be reversed. The expression of calpain 1,2,S1 and the decrease of TNNT2 protein induced by c16:0 could be decreased by DHA supplementation. The expression of calpain 1 mRNA induced by C16:0 increased and was alleviated by 100 micromol/L DHA supplementation. To improve the activity of H9C2 cardiomyocytes induced by C16:0 lipid overload, the possible mechanisms of 2-DHA are: (1) inhibiting apoptosis by inhibiting the endoplasmic reticulum apoptosis pathway; (2) reducing the expression of calpain and the degradation of troponin TNNT2, thereby improving the cytoskeleton damage.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R54
本文编号：2234492
[Abstract]:Objective: Recent literatures have reported that the number of type 2 diabetes mellitus (T2DM) patients is increasing year by year. Diabetes-related cardiac complications are the leading cause of death in patients with advanced type 2 diabetes mellitus. Our previous study found that the content of n-3 PUFAs docosahexaenoic acid (DHA) in myocardium of type 2 diabetic rats induced by high-fat diet and low-dose STZ (streptozotocin) significantly decreased, accompanied by left ventricular hypertrophy, and left ventricular systolic function significantly decreased. Apoptosis refers to the autonomous and orderly death of cells controlled by genes in order to maintain homeostasis of the internal environment. Unlike cell necrosis, apoptosis involves the activation, expression and regulation of a series of genes. Previous experiments have found that the heart of type 2 diabetic rats at the overall level is involved. Intramuscular apoptosis was increased in type 2 diabetic rats fed with DHA. However, whether DHA and other n-3 PUFAs can indeed prevent cardiomyocyte apoptosis, and what are their effects and mechanisms? Cellular level studies are needed. Cytoskeleton refers to the structure of protein fiber network in eukaryotic cells. Cytoskeleton is not only in maintaining fine structure. Cytoskeleton and its binding proteins constitute a dynamic system in cardiomyocytes. Cytoskeleton alterations have been reported in cardiomyocytes of hypertrophic and heart failure animals. Previous experiments have found that T2D is involved in many important life activities. Does n-3 PUFAs, such as DHA, have any effect on the cytoskeleton of cardiomyocytes in T2DM rats? It needs to be confirmed at the cellular level. The apoptosis and cytoskeleton of H9C2 myocardial cells were observed and the protective mechanism of DHA was explored after high concentration palmitic acid was given to simulate the hyperlipidemic environment of diabetic myocardium. Methods: 1 MTS assay was used to detect the cell viability and to determine the concentration of C16:0 which could decrease the cell viability and the protection of dha. 2 Western blot was used to detect the expression of cleaved caspase 3, cleaved parp, PARP and FADD protein. 3 realtime-rtpcr was used to detect the expression of caspase 3, 9, 12, inflammation-related gene TNF-a and oxidative stress-related gene ho-1. 5 ghost-pencil peptide staining was used to observe the effect of c16:0 on the cytoskeleton of H9c2 myocardial cells and whether the changes of cytoskeleton induced by c16:0 could be reversed by DHA and other n-3 PUFAs or n-6 aa. The changes of tnnt 2, calpain 1, calpain 2 and calpain 1 protein after DHA supplementation were observed. 7 realtime-rtpcr was used to detect the changes of calpain 1 mRNA expression after DHA supplementation. Results: 1. Lipid overload caused the decrease of H9c2 myocardial cell viability, DHA could improve the cell viability of lipid overload injury, and the activity of H9c2 myocardial cells treated with c16:0 in the concentration range of 150-400 micromol/l for 24 hours was significantly lower than that of BSA solvent control group (p0.05). The higher the concentration of c16:0, the lower the activity of H9c2 myocardial cells. There was no significant difference in cell viability between the control group and the DHA treated cells 24 hours later. However, the decrease of cell viability induced by c16:0 was ameliorated by supplementation of 10,100 micromol/l DHA with c16:0200 micromol/l. The decrease of cardiomyocyte viability induced by c16:0 was related to the increase of oxidative stress and inflammatory reaction with the increase of c16:0 concentration. The increase of the degree of H9c2 myocardial cell oxidative stress-related factors HO-1 and inflammation-related factors TNF-a mRNA expression increased significantly, indicating that c16:0-induced decline in cardiomyocyte viability is related to oxidative stress and inflammation. 3 lipid overload-induced cell viability decline is related to endoplasmic reticulum apoptosis pathway-mediated apoptosis At the same time, the expression of Caspase-3 mrna, cleaved caspase-3 and cleaved PARP protein were significantly increased, while the expression of fadd, a marker of cell necrosis, did not change significantly. 6:0 decreased cardiomyocyte viability was mainly due to apoptosis rather than necrosis, because the expression of caspase 12, an important initiator of the endoplasmic reticulum apoptosis pathway, was higher than that of caspase 9, an upstream molecule of the mitochondrial apoptosis pathway, suggesting that apoptosis induced by c16:0 overload was related to the apoptosis pathway of endoplasmic reticulum. The viability of cardiomyocytes injured by hyperlipidemia was related to the decrease of apoptosis. The number of apoptotic cells increased 24 hours after treatment with 200 micromol/l16:0, but the number of apoptotic cells increased by 1,10,100 micromol/ldha, EPA and LNA at the same time could be decreased by treatment with 1,10,100 micromol/ldha, EPA and lna. However, n-6 PUFAs arachidonic acid AA itself can increase cell apoptosis. 5 lipid overload leads to the decrease of H9c2 cardiomyocyte viability, which is related to the cytoskeleton change of cardiomyocyte. with the increase of c16:0 concentration, the myofilament in cardiomyocyte thickens and folds, resulting in the cells from regular long shuttle to multiple depressions. The expression of calpain-1, calpain-2 and calpain-1 protein increased with the change of myofibril morphology, while the expression of troponin-2 protein decreased gradually. It indicated that the decrease of cell activity and the increase of calpain expression induced by c16:0 overload could result in troponin troponin expression. 6 DHA and other n-3 PUFAs can inhibit the changes of cytoskeleton induced by lipid overload, and then improve the decrease of cardiomyocyte viability induced by lipid overload. When H9c2 is supplemented with 1,10,100 micromol/ldha, EPA and lna, the cytoskeleton changes of H9c2 induced by c16:0 can be reversed. The expression of calpain 1,2,S1 and the decrease of TNNT2 protein induced by c16:0 could be decreased by DHA supplementation. The expression of calpain 1 mRNA induced by C16:0 increased and was alleviated by 100 micromol/L DHA supplementation. To improve the activity of H9C2 cardiomyocytes induced by C16:0 lipid overload, the possible mechanisms of 2-DHA are: (1) inhibiting apoptosis by inhibiting the endoplasmic reticulum apoptosis pathway; (2) reducing the expression of calpain and the degradation of troponin TNNT2, thereby improving the cytoskeleton damage.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R54
【参考文献】
相关博士学位论文 前1条
1 侯连国;DHA保护2型糖尿病心肌的分子机制研究[D];河北医科大学;2013年
，本文编号：2234492
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