褐藻多糖硫酸酯对组织蛋白酶B活性影响的实验研究

Fucoidan alleviates apoptosis by inhibiting cathepsin B and oxidative stress

目的研究褐藻多糖硫酸酯(Fucoidan)对氧化应激损伤及溶酶体组织蛋白酶B活性的影响,探讨Fucoidan用于中枢神经系统疾病的作用机制。方法 50μg.L-1神经生长因子(nerve growth factor,NGF)分化大鼠肾上腺嗜铬细胞瘤PC12细胞(differentiated PC12,dPC12)7 d,0.5 mmol/L H2O2刺激30 min,给予10 mg.L-1Fucoidan干预。酶标仪检测细胞内超氧化物歧化酶(superoxide dismutase,SOD)及活性氧(reactive oxygen species,ROS)活性;荧光底物法检测细胞质中溶酶体组织蛋白酶B的活性;酶-底物法检测不同浓度Fucoidan(0.1、1、10、50、100 mg.L-1)对人肝脏溶酶体组织蛋白酶B活性的影响。结果氧化损伤后细胞中ROS显著升高达正常水平的185.2%,伴有SOD下降,溶酶体组织蛋白酶B活性上升至正常水平的236.7%;Fucoidan对SOD活性没有明显影响,能降低ROS水平,且对细胞质内和人肝脏溶酶体组织蛋白酶B的活性均具有抑制作用。结论 Fucoidan降低活性氧含量抗氧化损伤的作用可能与其抑制溶酶体组织蛋白酶B的活性有关。

Objective To study mechanism of Fucoidan in neurodegenerative disease by observing its effect on cathepsin B activities and oxidative stress.Methods After 7 days＇ differentiation by 50 μg·L-1 nerve growth factor（NGF）,PC12 cells were exposed to 0.5 mmol/L H2O2 for 30 min,and then treated with Fucoidan（10 mg·L-1）.The activities of superoxide dismutase（SOD） and reactive oxygen species（ROS） inside cells of each group were assayed.Cathepsin B activities inside cells were determined after being extracted from dPC12.Human liver cathepsin B activities were tested after being exposed to different concentrations of Fucoidan（0.1,1,10,50,100 mg·L-1）.Results After oxidative stress,the activities of ROS inside cells increased to 185.2% of normal control,accompanied by lower SOD activities and higher activities of cathepsin B（236.7%） in cytoplasm.Fucoidan treatment decreased ROS levels without any effect on SOD;furthermore,Fucoidan could inhibit both dPC12 cytoplasm cathepsin B activities and human liver cathepsin B activities.Conclusion Fucoidan＇s suppression on cathepsin B activities contributes to its protective effect against oxidative stress which leads to cell apoptosis.