新型孕激素衍生物3α-羟基-3β-甲氧基甲基-16,17-亚甲基-孕甾-20-酮对硝普钠损伤PC12细胞的保护作用

Protective effect of 3α-hydroxy-3β-methoxymethyl-16,17-methylenepregna-20-keto on PC12 cells injured by sodium nitroprusside

目的探讨新型孕激素衍生物3α-羟基-3β-甲氧基甲基-16,17-亚甲基-孕甾-20-酮(CPU)对硝普钠(SNP)诱导的PC12细胞损伤的神经保护作用及其机制。方法用终浓度为0.01,0.10,1.00μmol·L-1的CPU预处理30 min,然后加入SNP共同作用24 h。采用MTT法检测细胞存活率,分光光度计法检测乳酸脱氢酶(LDH)漏出率、丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性的变化;流式细胞仪检测活性氧(ROS)、线粒体膜电位和细胞凋亡率;Western蛋白质印迹法检测Bcl-2、Bax和活化的胱天蛋白酶3蛋白表达水平。结果与细胞对照组比较,SNP模型组PC12细胞的存活率显著降低,LDH的释放明显增加(P<0.01),MDA生成增多,SOD活性降低,ROS水平升高,线粒体膜电位水平降低,细胞凋亡率由细胞对照组(1.20±0.14)%增加至(55.52±3.56)%(P<0.01);Bcl-2蛋白表达降低,而Bax表达升高,Bax/Bcl-2比值升高,同时胱天蛋白酶3被激活(P<0.01)。与模型组比较,CPU 0.01,0.10和1.00μmol·L-1作用24 h后,细胞存活率显著升高,LDH的释放减少(P<0.01),MDA的生成量减少,SOD活性增加,ROS累积减少,线粒体膜电位水平升高(P<0.01);凋亡细胞分别降至(37.79±4.85)%,(22.31±4.25)%和(10.39±2.65)%;Bcl-2蛋白表达增加,Bax和活化的胱天蛋白酶3表达降低。结论 CPU对SNP所致损伤的PC12细胞有一定的保护作用,其作用机制可能与其抗氧化和抗凋亡作用有关。

OBJECTIVE To investigate the protective effects of a novel progesterone derivative, 3α-hydroxy-3β-methoxymethyl-16,17-methylene-pregna-20-keto （CPU）, on PC12 cell damage models induced by sodium nitroprusside （~SNP）. METHODS PC12 cells were pretreated with CPU 0.01, 0.10 and 1.00 μmol·L^-1 for 30 rain before SNP 100 μmol·L^-1 was added to the medium for 24 h. The colori- metric MTT assay was used to evaluate cell viability. The transudation content of lactate dehydrogenase （LDH） was observed by colorimetry reaction, the degree of injury to PC12 cells was evaluated by meas- uring the blank content of malondialdehyde （MDA） in conditioned medium, and the activity of superoxide dismutase （SOD） in cells was measured by kits. The levels of intracellular reactive oxygen species （ROS） were quantified by the Reactive Oxygen Species Assay Kit. The mitochondrial membrane poten- tial was investigated with the fluorescent probe JC-1. The apoptosis rate was assayed by FITC-Annexin V/PI staining while the protein expression of Bcl-2, Bax, and cleaved caspase 3 was measured by Western blotting. RESULTS Compared with blank control group, the survival rate of PC12 cells decreased to （40.8±4.5） % （ P〈0.01 ）, LDH release increased from （75.6±3.4） U· L^-1 to （165.2±6.4） U· L^-1 （ P〈0.01 ） ; the MDA activity significantly increased, SOD activities were significantly decreased （ P〈0.01 ）, the generation of ROS was increased and the levels of MMP were decreased, the rate of apoptosis increased from （1.20±0.14）% to （55.52±3.56）%（ P〈0.01 ）, the positive rate of Bcl-2 decreased while the positive rate of Bax and cleaved caspase 3 increased（ P〈0.01 ）, respectively, in SNP model group. Compared with SNP model group, after 24 h of incubation with CPU 0.01,0.10 and 1.00 μmol· L^-1, PC12 cell survival rate increased to （55.8±3.9）%, （66.6±5.0）% and （80.0±5.4）%（ P〈0.05）, respectively, LDH release was reduced to 137.6±21.4, 100.8±14.5 and （82.5±11.3） U· L^-1 , respectively, the activities of antioxidant enzyme SOD were increased while lipid peroxidation （MDA~） production was decreased. CPU 0.01, 0.10 and 1.00μmol·L^-1 could reduce the accumulation of ROS and increase the levels of mito- chondrial membrane potential. The rate of apoptosis decreased to （37.79±4.85）%, （22.31±4.25） % and （10.39±2.65） %（ P〈0.01 ）. CPU could up-regulate Bcl-2, down-regulate Bax and activate caspase 3（ P〈 0.01 ）. CONCLUSION CPU protects PC12 cells from SNP injury models probably via anti-oxidation and anti-apoptosis.