Nrf2-ARE信号通路在间歇性低氧诱导胰岛β细胞凋亡中的作用及银杏叶提取物的干预机理

发布时间：2018-07-07 19:33

本文选题：氧化应激 + 慢性间歇性低氧　；参考：《桂林医学院》2017年硕士论文

【摘要】：目的:研究银杏叶提取物(extract ginkgo biloba,EGB)通过Nrf2-ARE信号通路对慢性间歇性低氧(chronic intermittent hypoxia,CIH)大鼠胰岛β细胞凋亡中的作用及干预作用。方法:建立慢性间歇性低氧大鼠模型,将SD雄性大鼠36只随机分成两组,常氧对照组为12只,慢性间歇性低氧模型组为24只;第12周造模结束后,提取各组原代胰岛β细胞;常氧对照组细胞正常培养;慢性间歇性低氧模型组β细胞根据处理因素不同分为慢性间歇性低氧模型对照组(OSAS对照组)、抗Nrf2抗体组、Nrf2-ARE通路激活剂组、银杏叶提取物低、中、高剂量组共6组,其余组予以等量平衡盐溶液,加入含有不同处理药物的培养基相同条件培养。鉴定胰岛β细胞使用DTZ染色法,使用ELISA法检测,各组胰岛β细胞及上清液中谷胱甘肽前体(GSH-PX)、丙二醛(MAD)和活性氧(ROS)、肿瘤坏死因子(TNF-a)、白介素-8(IL-8)、8-异前列腺素(8-ISO)、超化物歧化酶(SOD)的含量,采用流式细胞仪对胰岛β细胞凋亡进行检测,使用Westerm-Blot法测定胰岛β细胞中核因子2相关因子(Nrf2)、血红素加氧酶-1(HO-1)、醌氧化还原酶(NQO1)、r-谷氨酰半胱氨酸合成酶(r-GCS)的表达水平。结果:与常氧对照组对比,OSAS对照组胰岛β细胞中ROS及上清液中MDA、TNF-a、IL-8、8-ISO水平均上升,GSH-PX、SOD表达下降,胰岛β细胞凋亡率升高,Nrf2表达升高,HO-1、NQO1、r-GCS在胰岛β细胞表达水平均下调,差异有统计学意义;与OSAS对照组对比,抗Nrf2抗体组胰岛β细胞中ROS及上清液中MDA、IL-8、TNF-a、8-ISO水平升高及SOD、GSH-PX下降更显著,胰岛β细胞凋亡率升高,HO-1、NQO1表达水平均降低,差异有统计学意义,而Nrf2、r-GCS表达水平无明显变化(P0.05);Nrf2-ARE通路激活剂组胰岛β细胞中ROS及上清液中MDA、IL-8、TNF-a、8-ISO表达水平均降低,SOD及GSH-PX表达水平均上升,胰岛β细胞凋亡率下降,Nrf2、HO-1、NQO1、r-GCS在胰岛β细胞表达均升高,差异有统计学意义;银杏叶提取物低剂量组胰岛β细胞中ROS及上清液中IL-8、8-ISO水平均下降,SOD表达水平均上升,差异有统计学意义;TNF-a、GSH-PX、MDA无差异(P0.05);胰岛β细胞凋亡率下降,Nrf2、HO-1、r-GCS在胰岛β细胞表达均升高,NQO1表达水平下调,差异有统计学意义;银杏叶提取物中、高剂量组胰岛β细胞上清液MDA、ROS、IL-8、TNF-a、8-ISO表达水平均降低,SOD及GSH-PX表达水平均上升,胰岛β细胞凋亡率下降,Nrf2、HO-1、NQO1、r-GCS在胰岛β细胞表达均升高,且高浓度组比低浓度组变化更明显,差异有统计学意义。结论:Nrf2-ARE信号通路能调节间歇性低氧所致的氧化应激及炎症因子水平,减少胰岛β细胞的凋亡,对胰岛β细胞起保护作用。银杏叶提取物通过Nrf2-ARE信号通路,调节间歇性低氧诱导胰岛β细胞凋亡中的氧化物质及炎症因子的水平,减少胰岛β细胞凋亡率,对胰岛β细胞有保护作用。
[Abstract]:Aim: to study the effect of (extract ginkgo biloba (EGB) on apoptosis of islet 尾 cells in rats with chronic intermittent hypoxia (chronic intermittent hypoxia via Nrf2-ARE signaling pathway. Methods: the chronic intermittent hypoxic rat model was established and 36 SD male rats were randomly divided into two groups: 12 rats in the normoxic control group and 24 rats in the chronic intermittent hypoxia model group. The 尾 cells of chronic intermittent hypoxia model group were divided into chronic intermittent hypoxia model control group (OSAS control group), anti-Nrf2 antibody group with Nrf2-ARE pathway activator group, ginkgo biloba leaf extract low, medium, according to the treatment factors, chronic intermittent hypoxia model group 尾 cells were divided into chronic intermittent hypoxia model control group (OSAS control group), anti-Nrf2 antibody group and Nrf2-ARE pathway activator group. The other groups were treated with the same amount of equilibrium salt solution and cultured in the same culture medium containing different treatment drugs. Islet 尾 cells were identified by DTZ staining and Elisa. The contents of glutathione precursor (GSH-PX), malondialdehyde (mad) and reactive oxygen species (Ros), tumor necrosis factor (TNF-a), interleukin-8 (IL-8) 8-isoprostaglandin (8-ISO), supernatant dismutase (SOD) in islet 尾 cells and supernatant dismutase (SOD) were detected by Elisa. Apoptosis of islet 尾 cells was detected by flow cytometry. The expression levels of nuclear factor-2 (Nrf2), heme oxygenase-1 (HO-1) and quinone redox enzyme (NQO1) r-glutamylcysteine synthase (r-GCS) in islet 尾 cells were determined by Westerm-Blot method. Results: the levels of Ros in islet 尾 cells and the levels of MDA-TNF-aIL-8 8-ISO in OSAS control group were increased and the expression of GSH-PXO SOD was decreased, and the expression of HO-1NQO1r-GCS was down-regulated in islet 尾 cells compared with normoxic control group. Compared with OSAS control group, the levels of Ros in islet 尾 cells in anti-Nrf2 antibody group and MDA-8 IL-8, TNF-aF- 8-ISO in supernatant, and the decrease of GSH-PX in islet 尾 cells were significantly higher than those in OSAS control group, and the expression level of HO-1NQO1 in islet 尾 -cell apoptosis was significantly lower than that in OSAS control group. The expression of Ros in islet 尾 cells and the expression of MDA-IL-8, TNF-aF- 8-ISO in pancreatic islet 尾 cells in Nrf2-ARE pathway activator group were decreased, and the expression of SOD and GSH-PX were increased. The apoptotic rate of islet 尾 cells was decreased, and the expression of NQO1O1r-GCS in islet 尾 cells was increased, but the expression of Nrf2-ARE pathway activator group was higher than that of Nrf2-ARE pathway activator group, while the expression of SOD and GSH-PX in pancreatic islet 尾 cells were increased in Nrf2-ARE pathway activator group. The levels of Ros in islet 尾 cells and IL-88-ISO in supernatant of ginkgo biloba extract decreased significantly. There was no significant difference in MDA between TNF-a and GSH-PXN (P0.05), the decrease of apoptosis rate of islet 尾 cells and the down-regulation of the expression of NQO1 in islet 尾 cells by Nrf2HO-1HO-1r-GCS, there was significant difference in the expression of NQO1 in ginkgo biloba leaves. In high dose group, the expression of MDA-ROSS-IL-8 and TNF-aO8-ISO in the supernatant of islet 尾 cells decreased, and the expression of SOD and GSH-PX increased, and the apoptosis rate of islet 尾 cells decreased. The expression of Nrf2HO-1NQO1O1r-GCS in islet 尾 cells increased, and the changes in high concentration group were more obvious than those in low concentration group. The difference is statistically significant. Conclusion the fraction of Nrf2-ARE signaling pathway can regulate oxidative stress and inflammatory factor level induced by intermittent hypoxia, reduce the apoptosis of islet 尾 cells, and play a protective role on islet 尾 cells. Through Nrf2-ARE signaling pathway, ginkgo biloba extract regulates the levels of oxidants and inflammatory factors in the apoptosis of islet 尾 cells induced by intermittent hypoxia, reduces the apoptosis rate of islet 尾 cells, and has protective effect on islet 尾 cells.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R766

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