靶向探针追踪Aβ25-35的亚细胞定位并基于Nrf2通路探讨阿里红多糖对线粒体损伤的保护机制

Trace Aβ25-35 subcellular localization by targeting probes and explore Nrf2 pathway-based protective mechanism of mitochondrial damage of Fomes officinais Ames polysaccharides

用靶向探针追踪淀粉样蛋白(Aβ25-35)的亚细胞定位情况,同时基于Nrf2信号通路探讨阿里红多糖组分(FOAPs-a)和(FOAPs-b)对Aβ25-35诱导的PC12细胞线粒体损伤通路的保护作用机制。采用40μmol/L Aβ25-35诱导PC12细胞建立阿尔茨海默病(AD)细胞模型,将PC12细胞分为空白组、模型组(加40μmol/L Aβ25-35)、阳性组(加50μmol/L盐酸多奈哌齐)、不同浓度的FOAPs-a和FOAPs-b干预组(各50、100、200μg/mL)。以靶向探针追踪Aβ25-35在各组PC12细胞中的亚细胞定位情况;通过试剂盒检测PC12细胞中活性氧自由基ROS的变化情况;Western blotting法测定细胞凋亡相关蛋白Bax及Bcl-2的表达量以及和Nrf2通路相关的Nrf2、APK1和磷酸化的APK1蛋白的表达情况。结果发现,Aβ25-35处理PC12细胞会影响线粒体的完整性;在200μg/mL FOAPs-a/b预处理PC12细胞后,能够显著缓解Aβ25-35对线粒体的损伤,同时使Aβ25-35的亚细胞共定位减弱;与空白组比较,模型组细胞中ROS含量增加,与模型组比较,FOAPs-a及FOAPs-b干预组均能降低ROS的沉积,差异有统计学意义;Western blotting结果显示:与模型组比较FOAPs-a及FOAPs-b均能减少APK1的磷酸化水平,上调Nrf2的蛋白表达水平。总之Aβ25-35可进入到PC12细胞的线粒体中引起其损伤,阿里红多糖组分能够通过激活Nrf2信号通路显著缓解Aβ25-35对PC12细胞线粒体的损伤。

Targeting probes were used to track Aβ25-35 subcellular localization,while Nrf2 signaling pathway were used to investigate the protective mechanism of Fomes officinais Ames polysaccharides components(FOAPs-a) and(FOAPs-b) against β-amyloid(Aβ25-35)-induced mitochondrial damage pathways in PC12 cells.The PC12 cells were cultured and activated by Aβ25-35 in condensed state as Alzheimer’s disease cell model in vitro and were randomly divided into nine groups:control group、Aβ25-35(40 μmol/L)induced group、Aβ25-35+ FOAPs-a(50,100,200 μg/mL) groups and Aβ25-35 + FOAPs-b(50,100,200 μg/mL) groups.Targeting probes were used to track Aβ25-35 subcellular localization,The changes of the reactive oxygen species( ROS) in PC12 cells were detected by the kit.The expression of apoptosis-related proteins Bax and Bcl2 and the expression of Nrf2、ASK1 and phosphorylated ASK1 proteins related to the Nrf2-pathway were analyzed by Western boltting method.It was found that after stimulated by Aβ25-35,the cells mitochondrial integrity were changed,after the addition of 200 μg/mL FOAPs-a or b pretreated PC12 cells,it can significantly alleviate the mitochondria damage by Aβ25-35,while reducing the co-localization of mitochondria.FOAPs-a and FOAPs-b could significantly inhibit the accumulation of ROS induced by Aβ25-35 in PC12 cells in a dose-dependent manner.They could also effectively prevent Aβ25-35-stimulated cytotoxicity,which involved in attenuating cell apoptosis,increasing Nrf2 expression and the ratio of Bcl-2/Bax,as well as inhibiting ASK1 and phosphorylated ASK1 proteins levels.In conclusion FOAPs-a and FOAPs-b played neuroprotective roles against Aβ25-35-induced cytotoxicity in PC12 cells through suppressing the mitochondria-mediated apoptotic pathway.The mechanism may be related to its suppression of the activation of Nrf2 signal pathway.