【作者】 陈宏；

【导师】 梅元武；

【作者基本信息】 华中科技大学 ， 神经病学， 2011， 博士

【摘要】 第一部分PGG对抗MPP+孵育所致PC12细胞的氧化应激反应摘要目的：研究1,2,3,4,6-五-氧位-没食子酰基-β-右旋葡萄糖(1,2,3,4, 6-penta-O-galloy-β-D-glucose, PGG)诱导PC12细胞中血红素加氧酶(heme oxygenase,HO-1)的产生及HO-1对抗MPP+孵育PC12所产生的氧化应激反应。方法：用不同浓度的(0、5、20、50、80、100μM) PGG处理经过神经营养因子(nerve growth factor—NGF)孵育一周的PC12细胞10小时,以及用30μM的PGG处理经过NGF孵育过一周的PC12不同时间。观察HO-1mRNA变化时所用的时间点为0、3、6、12、18、24h,观察HO-1蛋白变化时所用的时间点为-0、5、10、15、20、30h；通过免疫印迹法(Western blotting)、RT-PCR、活性氧试剂盒检测以及MTT方法观察PC12细胞中HO-1 mRNA和蛋白的合成与PGG作用浓度与作用时间的关系,PC12细胞中HO-1活力与PGG作用浓度与作用时间的关系,以及PGG对抗由于孵育MPP+所致的对PC12细胞产生的氧化应激反应。结果：PGG能促进PC12细胞中HO-1mRNA和蛋白的合成,HO-1mRNA和蛋白的合成呈浓度和时间依赖性,在PGG作用10h下,20gM的PGG便能增加HO-1mRNA和蛋白的合成,50μMPGG作用下HO-1mRNA和蛋白的合成呈现高峰,之后HO-1mRNA和蛋白合成量下降,但仍高于20μM PGG作用时HO-1mRNA和蛋白的合成量；在30μMPGG作用下,PC12细胞中HO-1mRNA和蛋白的合成均呈时间依赖性,HO-1mRNA在6h时合成增多,12h达高峰,HO-1蛋白在10h时合成增多,15时达高峰,高峰后的时间点HO-1mRNA合成量仍大于6h点时HO-1mRNA合成量,蛋白的合成量仍大于10h点时的HO-1的蛋白量。HO-1的活性变化也呈现浓度与时间依赖性,其趋势与HO-1蛋白表达的浓度与时间依赖性一致。定量与定性上均观察到PGG抑制活性氧簇(reactive oxygen species, ROS)的产生。结论：PGG促进PC12细胞中HO-1mRNA和蛋白的合成,在PGG的作用下,PC12细胞中HO-1mRNA和蛋白的合成呈现浓度和时间依赖性,HO-1的活力变化也呈现浓度和时间依赖性。PGG可通过诱导HO-1的产生对抗MPP+对PC12产生的氧化应激反应。第二部分PGG对ERKl/2信号通路诱导HO-1的影响目的：本部分研究细胞外信号调节酶1/2(extracellular singnal-regulated kinase, ERK1/2)信号通路在1,2,3,4,6-五-氧位-没食子酰基(1,2,3,4,6-penta-O-galloy-β-D-glucose,PGG)诱导PC12细胞中HO-1产生中的作用。方法：将细胞分成不同的组别,用PGG、MPP+,PD98059以及ZnPP处理PC12细胞,通过Western blotting和MTT方法,观察药物处理前后PC12细胞中ERK1/2磷酸化程度,Nrf2核转位、HO-1的表达以及细胞活力的变化。结果：PGG可促进PC12细胞中ERK1/2磷酸化增高,增高的ERK1/2磷酸化可促进PC12细胞细胞浆中的Nrf2向细胞核转位,从而促进了HO-1的合成增多,达到保护MPP+所损害的PC12细胞的作用；PD98059可通过抑制ERK1/2磷酸化而部分的抑制PC12细胞的中HO-1的表达,从而部分抑制了PGG对MPP+所损伤的PC12的保护作用。结论：ERK1/2信号通路在PGG保护MPP+损害PC12细胞中发挥重要的作用。第三部分PGG对PI3K/AKT信号通路诱导HO-1的影响目的：本部分研究磷脂酰肌醇-3激酶(P13K)／蛋白激酶AKT(phosphatidylinositol-3 kinase/protein AKT, PI3K/AKT)信号通路在PGG促进HO-1表达中的作用。方法：将细胞分成不同的组别,用PGG、MPP+, LY294002以及ZnPP处理PC12细胞,通过Western blotting和MTT方法,观察PC12细胞中AKT磷酸化程度、Nrf2核转位程度、HO-1的表达以及细胞活力的变化。结果：PGG可促进PC12细胞中AKT磷酸化增高,增高的AKT磷酸化可促进PC12细胞胞浆中的Nrf2向细胞核转位,从而促进了HO-1的合成增多,达到保护MPP+所损害的PC12细胞的作用；LY294002可通过抑制AKT磷酸化而部分的抑制PC12细胞的中HO-1的表达,从而部分抑制了PGG对MPP+所损伤的PC12的保护作用。并且LY294002与PD98059联合作用能完全阻断Nrf2的核转位与HO-1的表达,但无法完全消除PGG对与MPP+孵育PC12细胞的保护作用。结论：PI3K/AKT信号通路在PGG保护MPP+损害PC12细胞中发挥重要的作用。更多还原

【Abstract】 Part I PGG ameliorates oxidative stress.induced byMPP+in PC 12 cellsObject:To investigate whether PGG could induce the expression of HO-1 and Whether HO-1 could ameliorate oxidative stress induced by MPP+in PC 12 cells Methods:we treated the PC 12 cells,which were incubated by NGF one week before drug treatment, for 10h with various concentrations of PGG and treated the PC 12 cells, which were incubated by NGF one week before drug treatment,for various time points with 30μM PGG;we used these various time points-0、3、6、12、18、24h when we studied the production of HO-1 mRNA, and we used these various time points-0、5、10、15、20、30h when we studied the expression of HO-1 protein.Then we examined the relationship between various concentrations and various time points of PGG and the production of HO-1 mRNA and protein by Western blotting, RT-PCR, DCFH-DA and MTT assay. the relationship between activity of HO-1 and various concentrations and various time points of PGG; and whether PGG could ameliorate oxidative stress.induced by MPP+in PC12 cells.Results:The production of HO-1 mRNA and protein is in dose-dependent concentration-dependent manners. HO-1 mRNA levels showed an initial rise at 20μM,a peak induction at 50μM,and a decrease by 80μM. Induction of the HO-1 mRNA in PC12 cells is a time-dependent manner, HO-1 mRNA levels showed an initial rise at 6h,a peak induction at 12h,followed by a decrease at 18h. Exposure of PC 12 cells to various concentrations of PGG for 10h resulted in a concentration-dependent increase in HO-1 protein expression,and the increase trend is similar to production of HO-1 mRNA.And the trend of HO-1 protein levels showed an initial rise atlOh,a peak induction at 15h,followed by a decrease at 20h. HO-1 activity was also increased in dose-dependent and time-dependent manners which is similar to the dose-dependent and time-dependent manners of HO-1 protein expression. And PGG could ameliorate induced byMPP+in PC 12 cells. Part II Effect of PGG on the expression of HO-1 via ERK1/2 signaling pathwayObject:Here, we study the role of ERK1/2 signaling pathway in induction of HO-1 by PGG in the PC 12 cells.. Methods:The PC12 cells were divided into different groups,and then were incubated by PGG, PD98059 and ZnPP according to different groups. And then Western blotting and MTT assay were used to investigate the difference of p-ERK1/2,Nrf2、HO-1 expression,and activity of the PC 12 cells after the treatment with different drugs.Results: PGG could promote the phosphorylation of ERK1/2 which promoted Nrf2 nuclear translocation which promoted the expression of HO-1 and thus protected PC 12 from damage by MPP+.But PD98059 produced opposite role in the process that PGG protected PC 12 cells-PD98059 suppressed the protection of PC 12 cells by PGG partly.Conclusion:ERK1/2 signaling pathway played a role in the protection of PC12 cells by PGG. PartⅢEffect of PGG on the expression of HO-1 via PI3K/AKT signaling pathwayObject:Here, we study the role of PI3K/AKT signaling pathway in induction of HO-1 by PGG in the PC 12 cells.Methods:The PC12 cells were divided into different groups,and then were incubated by PGG, PD98059 and ZnPP according to different groups. And then Western blotting and MTT assay were used to investigate the difference of p-AKt,Nrf2、HO-1 xpression,and activity of the PC 12 cells after the treatment with different drugs.Results:PGG could promote the phosphorylation of AKt which promoted Nrf2 nuclear translocation which promoted the expression of HO-1 and thus protected PC 12 from damage by MPP+。But LY294002 produced opposite role in the process that PGG protected PC 12 cells-LY294002 suppressed the protection of PC 12 cells by PGG partly.LY294002 and PD98059 together could blocked Nrf2 nuclear translocation and expression of HO-1 completely, but could not abolish completely the protection of PC 12 cells by PGG.Conclusion:PI3K/AKT signaling pathway played a role in the protection of PC 12 cells by PGG.更多还原