低聚糖复合物对ARPE-19细胞及小鼠的抗氧化活性研究

发布时间：2019-04-02 15:25

【摘要】：视网膜色素上皮(RPE)细胞的退化是年龄相关性黄斑变性(AMD)的临床标志。随着线粒体氧化磷酸化减少,RPE细胞代谢障碍及功能异常,从而导致AMD发生发展。RPE细胞的氧化损伤是AMD病理机制中的主要原因。本论文首先利用H_2O_2建立ARPE-19细胞氧化损伤模型,通过细胞活力检测,细胞凋亡形态学观察,SOD活性、GSH和MDA含量测定,ROS及线粒体膜电位检测,探究了低聚糖复合物(OSC)在ARPE-19细胞内的抗氧化能力。实验结果表明,当利用不同浓度的OSC作用后,H_2O_2引起的ARPE-19细胞的氧化应激水平明显减弱,并呈剂量依赖性。当OSC浓度为250μg/mL时,细胞活力升高了19%,凋亡形态明显减弱。SOD活性和GSH含量分别恢复至对照组的94.8%和90.1%,MDA含量和ROS含量分别下降至对照组的1.47倍和1.4倍,线粒体膜电位显著升高。这一研究表明OSC在ARPE-19细胞内具有一定的抗氧化能力。然后,利用乙醇建立小鼠氧化损伤模型,通过血清中SOD的活性、GSH和MDA的含量测定,探究了OSC对乙醇损伤小鼠的抗氧化作用。实验结果表明,当给予0.8g/kg的OSC时,SOD活性和GSH含量分别恢复至对照组的94.1%和100.1%,而MDA含量下降至对照组的1.49倍。这种作用结果说明OSC在小鼠体内也有很好的抗氧化功能,可以抵御乙醇对小鼠造成的氧化损伤。最后,为了研究OSC的抗氧化机制,我们利用Western Blot检测ARPE-19细胞和小鼠的肝脏及眼球中的SIRT1及PGC-1α蛋白表达水平。实验发现,当OSC浓度为250μg/m L时,ARPE-19细胞中SIRT1及PGC-1α的表达量分别比H_2O_2损伤组增加了31.6%和23.7%。当给予0.8g/kg的OSC喂食小鼠时,小鼠肝脏和眼球的SIRT1蛋白表达量分别比对照组上调了23.3%和35.2%,PGC-1α蛋白表达量分别比对照组上调了13.6%和11.5%。说明OSC可以显著活化SIRT1/PGC-1α信号通路,从而抵御氧化损伤。综上所述,OSC具有保护H_2O_2氧化损伤的ARPE-19细胞和乙醇氧化损伤的小鼠的功能,通过活化SIRT1/PGC-1α信号通路,抵御氧化损伤。因此,OSC可以成为预防和治疗AMD的一种潜在药物。
[Abstract]:The degeneration of the retinal pigment epithelium (RPE) cells is a clinical sign of age-related macular degeneration (AMD). With the decrease of mitochondrial oxidative phosphorylation, the metabolic disorder and functional abnormality of RPE cells resulted in the development of AMD. The oxidative damage of RPE cells is the main cause of AMD's pathological mechanism. In this paper, an ARPE-19 cell oxidative damage model was established by H _ 2O _ 2, and the anti-oxidation ability of the oligosaccharide complex (OSC) in the ARPE-19 cells was investigated by the cell viability test, the morphological observation of apoptosis, the activity of SOD, the content of GSH and MDA, the detection of ROS and the mitochondrial membrane potential. The results showed that the oxidative stress level of ARPE-19 cells induced by H _ 2O _ 2 was significantly reduced and dose-dependent after using the OSC of different concentrations. When the OSC concentration was 250. m u.g/ mL, the cell viability was increased by 19% and the apoptosis was significantly reduced. The activity of SOD and the content of GSH were 94.8% and 90.1% in the control group, respectively. The content of MDA and ROS decreased to 1.47 and 1.4 times in the control group, respectively. This study shows that the OSC has a certain antioxidant capacity in the ARPE-19 cells. Then, the oxidative damage model of mice was established by using ethanol, the activity of SOD in serum, the content of GSH and MDA were measured, and the anti-oxidation effect of OSC on the mice with ethanol injury was investigated. The results showed that when the OSC of 0.8 g/ kg was given, the activity of SOD and the content of GSH were 94.1% and 100.1%, respectively, while the content of MDA decreased to 1.49 times in the control group. The results show that the OSC has good anti-oxidation function in the mouse, and can resist the oxidative damage of the ethanol to the mice. Finally, in order to study the anti-oxidation mechanism of OSC, we used the Western Blot to detect the expression levels of SIRT1 and PGC-1 in the liver and the eye of ARPE-19 cells and mice. The results showed that the expression of SIRT1 and PGC-1 in ARPE-19 cells increased by 31.6% and 23.7%, respectively, when the OSC concentration was 250 & mu; g/ m L. When 0.8 g/ kg of OSC was administered to the mice, the expression of the SIRT1 protein in the liver and the eye of the mice increased by 23.3% and 35.2%, respectively, compared with the control group, and the expression of the PGC-1 protein was 13.6% and 11.5% higher than that of the control group, respectively. The OSC can significantly activate the SIRT1/ PGC-1 signal path to resist oxidative damage. In conclusion, the OSC has the function of protecting the mice with the ARPE-19 cell and the ethanol oxidative damage of the H _ 2O _ 2 oxidative damage, and resisting the oxidative damage by activating the SIRT1/ PGC-1 signal path. Thus, the OSC can be a potential drug for the prevention and treatment of AMD.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R774.5

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