【摘要】 目的:观察海洋药物提取剂藻酸双酯钠对血红素所致神经细胞损伤的保护作用。方法:实验于1999-01/2000-01在河南医科大学药理实验室完成。体外培养大鼠胚胎皮质神经细胞,血红素组加入100mg/L血红素建立神经细胞血红素损伤模型;正常对照组不加血红素,其余处理同血红素组。藻酸双酯钠组在血红素处理前后24h及处理过程中加入50,100,150mg/L藻酸双酯钠。采用噻唑蓝微量自动比色测定细胞死亡数;采用酶联免疫吸附法测定损伤神经细胞的吸光度值;测定乳酸脱氢酶漏出量、丙二醛含量、超氧化物歧化酶活性;Fura-2负载,以荧光分光光度计测定细胞内游离钙的变化;以碘化丙啶染色,流式细胞仪定量分析细胞凋亡百分率。结果:①噻唑蓝测定显示50,100,150mg/L藻酸双酯钠均可显著减少细胞死亡数;血红素组神经细胞乳酸脱氢酶漏出量高于正常对照组[分别为(1025.20±125.86),(383.41±53.34)kat/g],50,100,150mg/L藻酸双酯钠组乳酸脱氢酶漏出量低于血红素组[分别为(843.50±25.67),(740.15±79.02),(551.44±83.02),(1025.20±125.86)μkat/g]。②血红素组丙二醛含量与正常对照组比较显著增加[分别为(14.9±1.1),(5.1±0.6)μmol/g],超氧化物歧化酶活性显著降低[分别为(9.50±0.08),(31.17±2.83)μkat/g;50,100,150mg/L藻酸双酯钠组丙二醛含量与血红素组比较均显著降低[分别为(11.62±0.50),(10.70±0.37),(8.76±0.78),(14.9±1.1)μmol/g]、超氧化物歧化酶活性显著升高[分别为(16.00±1.67),(22.00±1.00),(24.50±0.83),(9.50±0.08)μkat/g]。③神经细胞损伤后血红素组[Ca2+]i含量高于正常对照组[分别为(579±39),(145±35)nmol/L,50,100,150mg/L藻酸双酯钠组[Ca2+]i含量低于血红素组[分别为(432±40),(376±47),(302±64),(579±39)nmol/L],抑制率分别为23%,35%,48%。④50,100,150mg/L藻酸双酯钠组神经细胞平均凋亡率低于血红素组[分别为(25.3±5.3)%,(15.2±4.1)%,(8.9±3.1)%,(36.8±6.5)%]。结论:藻酸双酯钠对血红素所致神经细胞损伤具有显著的保护作用,机制可能与藻酸双酯钠降低[Ca2+]i累积及抗过氧化作用有关。更多还原

【Abstract】 AIM: To investigate the protective effects of marine-drug extract propylene glycol alginate sodium sulfate (PSS) on hemoglobin-induced neurocyte injury. METHODS: The experiment was conducted in the Laboratory of Pharmacology, Henan Medical University between January 1999 and January 2000. The cortical neurons of fetal in rats were cultured in vitro, and subjects in the hemoglobin group were added into 100 mg/L hemoglobin to establish neurocyte model of hemoglobin-induced injury, while subjects in the normal control group received no hemoglobin, and others were treated the same as the hemoglobin group. 50, 100 and 150 mg/L of PSS was added into the PSS group respectively at 24 hours before and after the administration with hemoglobin and the processing. MTT was adopted to determine the number of cell death. Enzyme linked immunosorbent assay was used to detect the absorption of injured neurocytes. The leaking amount of lactate dehydrogenase (LAD), content of malonaldehyde (MDA) and activity of superoxide dismutase (SOD) were measured. The fluorospectrophotometer (SPF) was used to determine the changes of free calcium in cells with Fura-2 loading. The Propidium Iodide was used for staining, and flow cytometry was used to quantitatively analyze the apoptotic percentage of cells. RESULTS: MMT manifested that 50, 100 and 150 mg/L PSS could significantly reduce the number of cell death, and the leaking amount of LAD in neurocytes of the hemoglobin group was higher than the normal control group respectively were(1025.20±125.86),(383.41±53.34) kat/g , and that in the 50, 100and 150 mg/L PSS group were lower than the hemoglobin group respectively were(843.50±25.67),(740.15±79.02),(551.44±83.02),(1 025.20±125.86) μkat/g .②The content of MDA in the hemoglobin group was obviously increased in comparison with the normal control group (14.9±1.1),(5.1±0.6) μmol/g , and the SOD activity was remarkably decreased (9.50±0.08),(31.17±2.83)μkat/g . The content of MDA in the 50,100 and 150 mg/L PSS group were obviously reduced comparing with the hemoglobin group respectively were (11.62±0.50),(10.70±0.37),(8.76±0.78),(14.9±1.1) μmol/g . The SOD activity was greatly enhanced respectively were(16.00±1.67),(22.00±1.00),(24.50±0.83),(9.50±0.08) μkat/g .③The content of Ca2+ i in hemoglobin group after neurocyte injury was higher than the normal control group respectively were (579±39),(145±35) nmol/L , and that in 50, 100 and 150 mg/L PSS group were lower than the hemoglobin group (432±40),(376±47),(302±64),(579±39) nmol/L with the inhibition ratio of 23%,35% and 48% separately.④The average apoptotic rate of neurocytes in PSS group was lower than the hemoglobin group respectively were (25.3 ±5.3)%,(15.2±4.1)%,(8.9±3.1)%,(36.8±6.5)% . CONCLUSION: PSS has a significant protective effect on hemoglobin-induced neurocyte injury, the mechanism of which may be in relation to the decrease of Ca2+ i by PSS and the anti-peroxidation.更多还原