格尔德霉素通过诱导热休克蛋白反应抑制DA能神经元蛋白水解应激的研究

Geldanamycin activates a heat shock response and inhibits proteolytic stress in dopaminergic neurons

目的探讨格尔德霉素(geldanamycin,GA)通过诱导热休克蛋白(HSP)反应抑制α-突触核蛋白(α-syn)异常表达,加强泛素蛋白酶体降解功能的神经保护作用。方法鱼藤酮作用于NGF诱导的PC12细胞建立α-syn胞质内过表达细胞模型,MTT法检测GA不同浓度及用药时程条件下的细胞活力,应用Western blot技术及共聚焦免疫荧光双标染色观察GA预处理后细胞内HSP70和α-syn的表达。荧光分光光度计测量各组细胞内蛋白酶体水解酶活性的变化。结果MTT示GA20～300nmol·L-1预处理使细胞活力呈浓度依赖性上升,吸光度分别上升至正常的65%、76%、85%和94%(鱼藤酮组为59%,P<0·01),而GA与鱼藤酮同时给药或作用于其后20h细胞活力无明显变化。Western blot结果提示GA预处理使HSP70表达上升至正常的3倍左右。共聚焦显微镜观察显示α-syn过表达现象得以抑制,并证实两者的共定位现象。酶活性测定提示GA预处理使类胰蛋白酶,类糜蛋白酶活性明显增强,荧光指数分别上升至10·4±1·61和96·0±4·77(鱼藤酮组为7·9±0·90和82·4±3·51,P<0·05),PgH水解酶活性也轻度上升。结论格尔德霉素可诱发热休克蛋白反应,主要通过HSP70的作用抑制α-syn的异常表达,促进泛素蛋白酶体降解功能,减轻蛋白水解应激反应,发挥神经保护效应。

Aim To explore the neuroprotective effects of geldanamycin on inhibition of α-synuclein overexpression and improvement of proteasome function resulting from activation of a heat shock response.Methods α-synuclein overexpression in a cell culture model was established in rotenone-treated neuron-like PC12 cells.MTT assay was used to measure the cell viability induced by geldanamycin at different concertrations and intervals.The expression of HSP70 and α-synuclein in GA preincubation and rotenone groups were observed by Western blot and confocal techniques.Enzymatic activity of three main hydrolases in proteasome was measured by detection of the fluorophore of various cleavaged synthetic fluorogenic peptides.Results GA preincubation indicated a dose-dependent increase in cell vitality from 20 nmol·L-1 to 300 nmol·L-1,which was 65%、76%、85% and 94% of control respectively,but either co-treated or post-treated with rotenone showed no significant changes in cell vitality.Western blot and double labeling immunofluorescence showed that the protein expression of HSP70 in GA group was significantly increased while the expression of α-syn decreased(P<0.01).The colocalization positive cells with α-syn and HSP70 were further confirmed by confocal microscope technique.Trypsin-like,chymotrypsin-like activity of proteasome in GA preincubation group displayed a marked increase,fluorophore of which were 10.4±1.61 and 96.0±4.77 respectively,while those in rotenone group were 7.9±0.90 and 82.4±3.51(P<0.05).Similarly,PgH activity was also improved slightly.Conclusions Geldanamycin might provide a protective effect by activation of HSP70 to repair misfolded α-synuclein,inhibit its overexpression and promote degradation function of proteasome.