低聚糖復合物對ARPE-19細胞及小鼠的抗氧化活性研究
發(fā)布時間：2019-04-02 15:25
【摘要】：視網(wǎng)膜色素上皮(RPE)細胞的退化是年齡相關(guān)性黃斑變性(AMD)的臨床標志。隨著線粒體氧化磷酸化減少,RPE細胞代謝障礙及功能異常,從而導致AMD發(fā)生發(fā)展。RPE細胞的氧化損傷是AMD病理機制中的主要原因。本論文首先利用H_2O_2建立ARPE-19細胞氧化損傷模型,通過細胞活力檢測,細胞凋亡形態(tài)學觀察,SOD活性、GSH和MDA含量測定,ROS及線粒體膜電位檢測,探究了低聚糖復合物(OSC)在ARPE-19細胞內(nèi)的抗氧化能力。實驗結(jié)果表明,當利用不同濃度的OSC作用后,H_2O_2引起的ARPE-19細胞的氧化應(yīng)激水平明顯減弱,并呈劑量依賴性。當OSC濃度為250μg/mL時,細胞活力升高了19%,凋亡形態(tài)明顯減弱。SOD活性和GSH含量分別恢復至對照組的94.8%和90.1%,MDA含量和ROS含量分別下降至對照組的1.47倍和1.4倍,線粒體膜電位顯著升高。這一研究表明OSC在ARPE-19細胞內(nèi)具有一定的抗氧化能力。然后,利用乙醇建立小鼠氧化損傷模型,通過血清中SOD的活性、GSH和MDA的含量測定,探究了OSC對乙醇損傷小鼠的抗氧化作用。實驗結(jié)果表明,當給予0.8g/kg的OSC時,SOD活性和GSH含量分別恢復至對照組的94.1%和100.1%,而MDA含量下降至對照組的1.49倍。這種作用結(jié)果說明OSC在小鼠體內(nèi)也有很好的抗氧化功能,可以抵御乙醇對小鼠造成的氧化損傷。最后,為了研究OSC的抗氧化機制,我們利用Western Blot檢測ARPE-19細胞和小鼠的肝臟及眼球中的SIRT1及PGC-1α蛋白表達水平。實驗發(fā)現(xiàn),當OSC濃度為250μg/m L時,ARPE-19細胞中SIRT1及PGC-1α的表達量分別比H_2O_2損傷組增加了31.6%和23.7%。當給予0.8g/kg的OSC喂食小鼠時,小鼠肝臟和眼球的SIRT1蛋白表達量分別比對照組上調(diào)了23.3%和35.2%,PGC-1α蛋白表達量分別比對照組上調(diào)了13.6%和11.5%。說明OSC可以顯著活化SIRT1/PGC-1α信號通路,從而抵御氧化損傷。綜上所述,OSC具有保護H_2O_2氧化損傷的ARPE-19細胞和乙醇氧化損傷的小鼠的功能,通過活化SIRT1/PGC-1α信號通路,抵御氧化損傷。因此,OSC可以成為預(yù)防和治療AMD的一種潛在藥物。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R774.5
本文編號：2452665
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R774.5
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