丁苯酞对β淀粉样蛋白诱导的U87细胞自噬性死亡的影响

Effect of dl-3-n-butylphthalide on autophagic cell death in Aβ-treated U87 cells

目的观察丁苯酞对β淀粉样蛋白(Aβ1-42)处理后U87细胞自噬性死亡的影响。方法方法将U87细胞分为Aβ组、Aβ+丁苯酞组和对照组(空白对照)。Aβ组细胞用Aβ1-42(20μmol/L)处理24 h;Aβ+丁苯酞组细胞用丁苯酞(10μmol/L)预处理0.5 h后再加入Aβ1-42(20μmol/L)处理24 h。采用MTT法测定细胞活力,检测Caspase 3活性,采用CM-H2 DCFDA检测胞内活性氧(ROS)水平,Western blotting检测LC3-Ⅰ和LC3-Ⅱ蛋白表达。结果与对照组比较,Aβ组U87细胞活力显著下降(P<0.01);Aβ+丁苯酞组U87细胞活力显著高于Aβ组(P<0.05)。对照组、Aβ组和Aβ+丁苯酞组之间Caspase 3活性的差异均无统计学意义(P>0.05)。与对照组比较,Aβ组U87细胞ROS水平显著升高(P<0.01);Aβ+丁苯酞组U87细胞ROS水平显著低于Aβ组(P<0.01)。与对照组比较,Aβ组U87细胞LC3-Ⅱ/LC3-Ⅰ比值显著升高(P<0.01);Aβ+丁苯酞组U87细胞LC3-Ⅱ/LC3-Ⅰ比值显著低于Aβ组(P<0.01)。结论丁苯酞通过抑制ROS介导的自噬性死亡发挥神经保护作用。

Objective To observe the effect of dl-3-n-butylphthalide on the autophagic cell death in β-amyloid （Aβ1-42）- treated U87 cells. Methods U87 cells were divided into Aβ group, Aβ ＋ dl-3-n-butylphthalide group and control group （blank control）. Cells in AI3 group were treated with Aβ1 -42（20 μmol/L） for 24 h, and cells in Aβ ＋ dl-3-n-butylphthalide group were pre-incubated with dl-3-n-butylphthalide （ 10 μmol/L） for 0.5 h prior to treatment with 20 μmol/L Aβ1 -42 for 24 h. Cell viability was detected by MTT, Caspase-3 activity of was examined, intracellular reactive oxygen species （ROS） was determined by CM-H2 DCFDA, and the expression of LC3- I and LC3-11 protein was detected by Western blotting. Results The viability of U87 cells in AI3 group was significantly lower than that in control group （P 〈 0.01）, and the viability of U87 cells in AI3 ＋ dl-3-n-butylphthalide group was significantly higher than that in AI3 group （ P 〈 0.05）. There was no significant difference in the Caspase 3 activity among Aβ group, Aβ ＋ di-3-n-butylphthalide group and control group （P 〉 0.05）. The ROS level in U87 cells in AI3 group was significantly higher than that in control group （P 〈 0.01）, and the ROS level in U87 cells in Aβ ＋ dl-3-n-butylphthalide group was significantly lower than that in AI3 group （P 〈 0.01）. The LC3-II/LC3- I ratio in U87 cells in AI3 group was significantly higher than that in control group （P 〈 0.01）, and the LC3- U/LC3- I ratio in U87 cells in Aβ ＋ dl-3-n-butylphthalide group was significantly lower than that in Aβ group （P 〈 0.01）. Conclusion dl-3-n-butylphthalide exhibits neuroproteetive effect through inhibition of ROS-mediated autophagic cell death in Aβ-treated U87 cells.