环境毒素导致多巴胺能神经元蛋白水解应激的实验研究

Proteolytic stress induced by environmental toxins in dopaminergic neurons:an experimental study

目的探讨环境毒素诱发蛋白水解应激在多巴胺能神经元变性死亡中的作用。方法6-羟多巴(6-OHDA)、1-甲基4-苯基吡啶离子(MPP^+)、鱼藤酮作用于神经生长因子诱导的 PC12细胞,MTT 法检测各种药物的神经毒性作用,应用共聚焦免疫荧光双标染色观察药物处理后细胞内α-突触核蛋白及泛素化蛋白的表达及聚集。荧光分光光度计测量各组细胞蛋白酶体中各蛋白水解酶活性的变化。结果 3种神经诱变剂处理 PC12细胞后,细胞活力呈浓度依赖性下降,其中6-OHDA100 μmol/L、MPP^+75μmol/L、鱼藤酮20 nmol/L 作用24 h 使细胞活力分别下降52%、44%、40%(P<0.01)。共聚焦显微镜观察显示药物处理后细胞胞质内α-突触核蛋白及泛素表达上调,并形成蛋白颗粒物。酶活性测定提示 MPP^+75μmol/L、鱼藤酮20 nmol/L 处理使胰蛋白酶,糜蛋白酶及 PgH 水解酶活性明显下降(P<0.05),而6-OHDA 100μmol/L 处理使细胞蛋白酶水解能力轻度降低,差异无统计学意义(P>0.05),6-OHDA 200μmol/L 则诱导3种蛋白酶活性进一步下降,荧光指数分别为7.23±0.58,79.60±2.73和4.23±0.49(正常组为13.90±1.75,99.30±5.23和6.90±0.60,P<0.01)。结论神经诱变剂可导致多巴胺能神经元泛素蛋白酶体功能失调及α-突触核蛋白和泛素化蛋白的积聚,诱发蛋白水解应激异常状态。

Objective To explore the role of proteolytic stress induced by environmental toxins in degeneration and death of dopaminergic neurons. Methods Nerve growth factor-treated-rat adrenal pheochromocystoma cells of the line PC12 were co-incubated with 6-hydroxydopamine （6-OHDA）, 1- methyl-4 -phenylpyridinium ion （MPP^＋ ）, and rotenone for 24 hours. MTr assay was used to measure the cell viability induced by these neurotoxins with different concentrations. The expression levels of α-synuclein and ubiquited proteins in every group were observed with laser scanning confocal technique. The enzymatic activities of three main hydrolases in proteasome were measured by detection of the fluorophore of various cleft synthetic fluorogenic peptides. Results 6-OHDA, MPP^＋ , and rotenone decreased the activity of PC12 cells dose-dependently. Co-incubated with 100 μmol/L 6-OHDA, 75 μmol/L MPP^＋ , and 20 nmol/L rotenone, the activity of PC12 cells decreased by 52%, 44%, and 40% respectively. Immunofluorescence double labeling confirmed the overexpression of α-synuclein and ubiquitin and pellet accumulation in the cytoplasm induced by three toxins. Compared with those in the control group, the trypsin-like, chymotrypsin- like, and PgH-like activities of proteasome were markedly decreased in the MPP^＋ and rotenone groups （all P 〈 0.05 ） and slightly decreased in the 6-OHDA 100 μmol/L group （ all P 〉 0. 05）, but after the exposure to 6-OHDA 200 μmol/L for 24 h, the activities of the three enzymesl decreased rapidly, the activities of the three enzymes were 7.2 ± 0.6, 79.6 ± 2.7 and 4.2 ± 0.5 FU/100 μg respectively （vs 13.9 ± 1.8, 99.3 ± 5.2, and 6. 9±0. 6 FU/100 μg respectively in the control group, all P〈 0. 01）. Conclusion Environmental toxins induce proteolytic stress marked by dysfunction of ubiquitin proteasome and accumulation of α-synuclein and ubiquited proteins.