地黄中松果菊苷对皮质酮诱导PC-12细胞凋亡的抑制作用及机制研究

Inhibitory effect and mechanism of Echinacoside isolated from Rehmannia glutinosa Libosch on corticosterone-induced apoptosis in PC-12 cell line

目的观察松果菊苷对于皮质酮诱导的PC-12细胞损伤的保护作用及机制。方法将PC-12细胞分为6组:正常组,模型组(皮质酮500μmol·L^(-1)),阳性对照组(氟西汀0.3μmol·L^(-1))和低、中、高3个剂量实验组(松果菊苷:5,10,20μmol·L^(-1))。24 h后,用噻唑蓝法检测细胞存活率,用流式细胞术检测细胞凋亡率和线粒体膜电位水平,用蛋白质印迹法检测细胞内胱天蛋白酶3(Caspase-3)、剪切的胱天蛋白酶3(Cleaved Caspase-3)、B细胞淋巴瘤-2(Bcl-2)和B细胞淋巴瘤-2蛋白相关X蛋白(Bax)的蛋白表达水平。结果正常组、模型组、阳性对照组和低、中、高3个剂量实验组的细胞存活率分别为(100.0±3.1)%,(88.7±1.9)%,(94.3±3.4)%,(101.3±3.1)%,(102.1±1.9)%和(104.3±1.6)%。选择松果菊苷中剂量进行后续实验研究。正常组、模型组、阳性对照组和中剂量实验组的凋亡水平分别为(10.7±0.9)%,(25.5±1.3)%,(17.7±1.7)%和(19.7±0.8)%;这4组的JC-1单体比例分别为(18.7±0.6)%,(52.1±1.4)%,(25.6±0.9)%和(22.6±1.0)%;这4组的Cleaved caspase 3/Caspase 3比值分别为1.01±0.17,1.98±0.29,1.43±0.29和1.34±0.12;这4组的Bax/Bcl-2比值分别为1.23±0.21,3.01±0.81,1.64±0.39和0.91±0.29。上述指标:模型组与正常组比较,差异均有统计学意义(P<0.05,P<0.01);阳性对照组和中剂量实验组与模型组比较,差异均有统计学意义(P<0.05,P<0.01)。结论松果菊苷可能是通过抑制线粒体凋亡信号通路降低凋亡水平来改善皮质酮诱导的PC-12细胞损伤。松果菊苷可能是地黄发挥抗抑郁作用的物质基础之一。

Objective To observe the inhibitory effect and mechanism of Echinacoside isolated from Rehmannia glutinosa Libosch on PC-12cells damaged by corticosterone.Methods The PC-12 cells were divided into six groups:normal group,model group(treated with 500μmol·L^(-1)corticosterone),positive control group(treated with 0.3μmol·L^(-1)fluoxetine in injured cells)and experimental-L,experimental-M,experimental-H groups(treated with 5,10,20μmol·L^(-1)Echinacoside in injured cells).After 24 h,the cell survival rates were measured by the methyl thiazolyl tetrazolium(MTT)method.The levels of intracellular apoptosis and mitochondrial membrane potential(MMP)were detected by flow cytometry.Protein expression of Cleaved caspase 3/Caspase 3 and Bax/Bcl-2 were determined by Western blot.Results The cell survival rates of the normal group,model group,positive control group and experimental-L,experimental-M,experimental-H groups were(100.0±3.1)%,(88.7±1.9)%,(94.3±3.4)%,(101.3±3.1)%,(102.1±1.9)%and(104.3±1.6)%,respectively.The middle dose of echinacoside was selected for follow-up experimental research.The level of intracellular apoptosis of the normal group,model group,positive control group and experimental-M group were(10.7±0.9)%,(25.5±1.3)%,(17.7±1.7)%and(19.7±0.8)%,respectively;the JC-1 monomer ratios in the 4 groups were(18.7±0.6)%,(52.1±1.4)%,(25.6±0.9)%and(22.6±1.0)%,respectively;the ratios of Cleaved caspase 3/Caspase 3 protein in the 4 groups were 1.01±0.17,1.98±0.29,1.43±0.29 and 1.34±0.12;and the ratios of Bax/Bcl-2 protein in the 4 groups were 1.23±0.21,3.01±0.81,1.64±0.39 and 0.91±0.29.Comparison between model group and the normal group,the difference of the factors were significant(P<0.05,P<0.01);comparison between positive control group,experimental-M group and model group,the difference of the factors were significant(P<0.05,P<0.01).Conclusion Echinacoside can effectively alleviate the damage of PC-12 cells induced by corticosterone by inhibiting the mitochondrial apoptosis signal pathway and reducing the level of apoptosis.Echinacoside may be one of the effective components of Rehmannia glutinosa Libosch to exert antidepressant effect.