硫辛酸抑制高糖诱导的人主动脉内皮细胞氧化应激和细胞凋亡

发布时间：2018-08-03 12:12

【摘要】：目的:作为一种由多种原因引起的以慢性高血糖为特征的终身代谢性疾病,糖尿病已经成为21世纪人类所面临的最具挑战性的疾病之一。长期持续的血糖增高会导致大血管、微血管损害而导致心、脑、肾、周围神经、眼睛、足等发生病变。据估计,糖尿病患者中75%的死亡率都是由于心血管疾病所引起的。越来越多的研究表明炎症和氧化应激在糖尿病血管系统并发症的形成和发展的起着重要作用。α-硫辛酸(LA),是一种线粒体中的辅酶,因有很强的还原性,被称为“万能抗氧化剂”。本课题通过高浓度葡萄糖诱导人主动脉内皮细胞损伤,建立糖尿病细胞模型,用不同剂量的α-硫辛酸进行干预处理,探讨α-硫辛酸对高糖诱导的人主动脉内皮细胞炎症反应和氧化应激作用的影响并揭示其相关机制。方法:体外建立高糖(30mM)诱导的人主动脉细胞(HAECs)损伤模型,分别加入不同浓度硫辛酸(50,100,200μM)。用还原性谷胱甘肽(GSH)测试盒检测细胞中GSH的含量。采用流式细胞技术检测硫辛酸对高糖诱导的HAECs内活性氧(ROS)产生量的影响。利用蛋白质印迹法(Western Blot)方法检测HAECs中NADPH氧化酶-4(Nox4)、p22phox、Caspase-3、Bcl-2蛋白的表达水平,细胞色素C的线粒体释放以及Nuclear Factor-κB(NF-κB)信号通路的激活情况。采用实时荧光测定PCR(Real-time RT-PCR)检测Nox4、p22phox mRNA的表达水平。结果:1.与空白组相比较,高糖诱导后HAECs中GSH含量明显降低(p0.01),硫辛酸给予后可以剂量依赖性的增加人细胞中GSH的表达量(p0.01);2.与空白组相比较,高糖增加HAECs中ROS的过量产生(p0.01)、增加Nox4和p22phox mRNA和蛋白的过量表达(p0.01,p0.01),加入不同浓度的硫辛酸后高糖诱导人主动脉内皮细胞中活性氧ROS的产生量明显减少(p0.05),且硫辛酸抑制高糖诱导的Nox4和p22phox mRNA和蛋白的过量表达(p0.01,p0.01);3.与空白组相比较,高糖显著增加I-κB的降解,促进P65的核转位,从而增加NF-κB的转录活性,而硫辛酸可以抑制高糖诱导的细胞中I-κB的降解,以及P65的核转位,进而抑制NF-κB信号通路的激活(p0.05);4.高糖诱导后,凋亡相关蛋白Caspcase-3表达上调而抗凋亡蛋白Bcl-2表达明显下降(p0.01),硫辛酸干预后使抗凋亡蛋白Bcl-2的表达呈浓度依赖性增加(p0.01),同时硫辛酸还能下调凋亡蛋白Caspcase-3的表达(p0.01);5.与空白组相比较,高糖使人主动脉内皮细胞中细胞色素C的线粒体释放量增加,而加入硫辛酸后减少高糖诱导的细胞色素C的线粒体释放(p0.05)。结论:硫辛酸可以抑制高糖诱导的HAECs中ROS过量产生,抑制还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)家族中Nox4、p22phox亚基的mRNA和蛋白的过量表达;硫辛酸能够显著降低高糖诱导的Caspase-3的表达,抑制由高糖导致的Bcl-2蛋白表达量的下降。硫辛酸抑制NF-κB信号通路的转录活性,同时还可以抑制HAECs中细胞色素C的线粒体释放,由此来抑制高糖诱导的人主动脉内皮细胞的氧化应激和炎症反应以及抑制细胞凋亡。我们推测硫辛酸可能是通过阻断Nox4的表达来抑制细胞中ROS的过量产生,并通过影响NF-κB信号通路的转录活性从而减弱高糖诱导的HAECs中的氧化应激和炎症反应。
[Abstract]:Objective: as a life-long metabolic disease characterized by chronic hyperglycemia, diabetes has become one of the most challenging diseases in twenty-first Century. Prolonged continuous hyperglycemia can lead to large blood vessels and microvascular damage and cause lesions in the heart, brain, kidney, peripheral nerves, eyes, and feet. It is estimated that 75% of the mortality rates in diabetic patients are caused by cardiovascular disease. More and more studies have shown that inflammation and oxidative stress play an important role in the formation and development of diabetic vascular system complications. Alpha lipoic acid (LA), a cofactor in the mitochondria, is called "universal anti oxygen" because of its strong reducibility. Using high concentration glucose to induce the injury of human aortic endothelial cells, the diabetic cell model was established. The effects of alpha lipoic acid on the inflammatory response and oxidative stress induced by high glucose induced human aortic endothelial cells were investigated by interfering with different doses of alpha lipoic acid, and the mechanism was revealed. A model of human aortic cell (HAECs) injury induced by high glucose (30mM) was established with different concentrations of lipoic acid (50100200 u M). The content of GSH in cells was detected by a reduced glutathione (GSH) test box. The effect of thioctanoic acid on the production of ROS in high glucose induced HAECs was detected by flow cytometry. Western Blot method was used to detect the expression level of NADPH oxidase -4 (Nox4), p22phox, Caspase-3, Bcl-2 protein, mitochondrial release of cytochrome C, and activation of Nuclear Factor- kappa signaling pathway. Results: 1. and blank Compared with the group, the content of GSH in HAECs decreased significantly (P0.01) after high glucose induction. The dose dependent increase of GSH expression in human cells (P0.01) after the administration of lipoic acid was increased. 2. compared with the blank group, high glucose increased the excessive production of ROS in HAECs (P0.01), increased Nox4 and p22phox mRNA and protein overexpression (P0.01,), and added different concentrations. High glucose induced the production of reactive oxygen ROS in human aortic endothelial cells (P0.05), and lipoic acid inhibited the excessive expression of Nox4 and p22phox mRNA and protein (P0.01, P0.01) induced by high glucose (P0.01, P0.01). 3. compared with the blank group, high glucose significantly increased the degradation of I- kappa B and promoted the nuclear transposition of P65, thus increasing the transcriptional activity of NF- kappa B. Lipoic acid can inhibit the degradation of I- kappa B in high glucose induced cells, and the nuclear transposition of P65, and then inhibit the activation of NF- kappa B signaling pathway (P0.05). After the induction of 4. high glucose, the expression of apoptosis related protein Caspcase-3 is up and the expression of anti apoptotic protein Bcl-2 is obviously decreased (P0.01), and the expression of anti apoptotic protein Bcl-2 is thicker after the intervention of lipoic acid. Degree dependence increased (P0.01), while lipoic acid also lowered the expression of apoptotic protein Caspcase-3 (P0.01); 5. compared with the blank group, high glucose increased the mitochondrial release of cytochrome C in human aortic endothelial cells, and after adding lipoic acid, it reduced the mitochondrial release of hyperpigmented C induced by high glucose (P0.05). Conclusion: lipoic acid can inhibit the mitochondrial release of cytochrome C (P0.05). The excessive production of ROS induced by high glucose induced HAECs inhibited the overexpression of mRNA and protein in the Nox4, p22phox subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) family; thioctanoic acid could significantly reduce the expression of high glucose induced Caspase-3 and inhibit the decrease of Bcl-2 protein expression caused by high sugar. Thioctanoic acid inhibited NF- kappa B The transcriptional activity of the signal pathway also inhibits the mitochondrial release of cytochrome C in HAECs, which inhibits oxidative stress and inflammatory response and inhibits apoptosis of human aortic endothelial cells induced by high glucose. We speculate that lipoic acid may inhibit the excessive production of ROS in cells by blocking the expression of Nox4 and through shadow The transcriptional activity of the NF- - kappa B signaling pathway weakens the oxidative stress and inflammatory response in HAECs induced by high glucose.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2

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