摘要
目的本文拟采用体外实验,探讨脑细胞色素P450 2E1(cytochrome P450 2E1,CYP 2E1)在尼古丁致神经细胞氧化损伤中的可能作用。方法实验选用IMR-32人神经母细胞瘤细胞系,设置对照组,尼古丁小剂量组(0.1 n M)、中剂量组(1 n M)和大剂量组(10 n M),并选用中剂量尼古丁加入不同剂量CYP2E1特异性抑制剂二丙烯基硫醚(diallyl sulfide,DAS)(0.025、0.05、0.075 n M)。尼古丁或尼古丁与二丙烯基硫醚处理细胞48 h。取处理后细胞,分别检测乳酸脱氢酶(lactate dehydrogenase,LDH)、超氧化物歧化酶(superoxide dismutase,SOD)、活性氧(reactive oxygen species,ROS)、CYP2E1mRNA和蛋白水平的变化,并对CYP2E1蛋白表达水平与LDH活力、SOD活力、ROS含量进行相关性分析。结果与对照组相比,经低、中、高剂量尼古丁处理后,IMR-32细胞增殖受抑制,抑制率分别为39%、47%、52%(均P<0.05);细胞受到损伤,培养基LDH活力分别升高14%、50%、70%(均P<0.05);SOD活力下降至对照组的76%、58%、44%(均P<0.05);细胞CYP2E1蛋白表达水平显著升高至2.19、2.65及2.76倍(均P<0.05),未见CYP2E1 mRNA水平改变;ROS生成量升高48%、65%、136%(均P<0.05)。与尼古丁(1 nm)组相比,加入低、中、高剂量抑制剂后,SOD活力升高24%、47%、52%(均P<0.05);ROS含量下降至77.8%、57.8%、44.3%(均P<0.05)。相关性分析显示,CYP2E1含量与LDH活力呈正相关,与SOD活力呈负相关,相关系数分别为0.740和-0.584,与ROS含量呈正相关,相关系数为0.695(均P<0.01)。结论尼古丁处理可抑制IMR-32细胞增殖,诱导脑CYP2E1表达,明显诱导ROS生成,致神经细胞损伤。
Objective To investigate roles of CYP2E1 in neural cell damage induced by nicotine treatment. Methods In this study,IMR-32 neuroblastoma cells were cultured. Those cells were ramdomly divided into control group,low dose of nicotine group,middle dose of nicotine group,and high dose of nicotine group. Those nicotine groups were treated with nicotine(0. 1,1 and 10 n M),while middle dose of nicotine group was co-treated with nicotine(1 n M) and dially sulfide(0. 025,0. 05 and 0. 075 n M) for 48 hours. After treatment,levels of ROS,SOD,CYP2E1 protein and CYP2E1 mRNA were detected. The relationship between CYP2E1 protein levels and LDH, SOD, ROS levels were analysed by correlation analysis.Results Compared with control group,the results of nicotine groups showed that proliferation of IMR-32 neuroblastoma cells were suppressed after the treatment of low dose of nicotine,middle dose of nicotine and high dose of nicotine,and the suppression ratios were 39%,47%,52% respectively(all P < 0. 05). The lactate dehydrogenase significantly increased(1. 14-fold,1. 5-fold,1. 7-fold,all P < 0. 05),and superoxide dismutase significantly decreased to 76%,58% and 44% of control group(all P < 0. 05). The expression of CYP2E1 protein significantly increased(2. 19-fold,2. 65-fold,2. 76-fold),and the levels of ROS significantly increased(1. 48-fold,1. 65-fold,2. 36-fold)(all P < 0. 05),while CYP2E1 mRNA did not change significantly. Compared with middle dose of nicotine group(1 n M),the levels of ROS decreasd(88. 5%,79. 9%,51. 2%) and SOD increased(1. 24-fold,1. 47-fold,1. 52-fold) after treatment with DAS(0. 025,0. 05 and 0. 075 n M). Correlation analysis demonstrated that CYP2E1 level was correlated to LDH(0. 740),SOD(-0. 584) and ROS(0. 695) levels(all P < 0. 01). Conclusion Nicotine treatment could inhibit the proliferation of IMR-32 cells,induce brain CYP2E1 expression,induce ROS generation,and induce damage to nerve cells.
出处
《中国生化药物杂志》
CAS
北大核心
2014年第8期34-37,共4页
Chinese Journal of Biochemical Pharmaceutics
基金
高等学校博士学科点专项科研基金(20091417120003)
