摘要
[背景]反式二氢二醇环氧苯并[a]芘(BPDE)是一种环境致癌物质,可引起细胞凋亡、氧化应激等多种改变。绿原酸(CGA)具有抗氧化作用,但其对BPDE诱导的细胞凋亡的影响尚不明确。[目的]本研究首先采用BPDE染毒,模拟烟草致支气管上皮细胞损伤,然后采用CGA干预,探索其抗凋亡作用及其潜在分子机制。[方法]预实验采用终浓度为0、25、50、100、150、200μmol/L的CGA分别处理16HBE细胞2、4、8 h。根据预实验结果,选择CGA 50μmol/L预处理细胞4 h后再进行BPDE 0.5μmol/L染毒12 h。实验分为对照组、CGA单独处理组(CGA 50μmol/L)、BPDE单独处理组(BPDE 0.5μmol/L)、CGA干预组(CGA 50μmol/L+BPDE 0.5μmol/L)。采用Annexin V/PI染色及流式细胞仪检测细胞凋亡,荧光探针DCFH-DA法检测细胞内活性氧(ROS)水平,Western blot检测凋亡通路相关蛋白caspase-3的剪切体(Cleaved caspase-3)、caspase-9的剪切体(Cleaved caspase-9)、p53、p21的表达水平。[结果]CGA 50、100μmol/L组各时间点相对细胞活力与对照组相比,差异无统计学意义(P> 0.05);而150、200μmol/L组处理时长为8 h时,与对照组相比细胞活力下降(P <0.05)。与BPDE单独处理组相比,CGA干预组的细胞凋亡率下降(17.63%vs 9.21%,P <0.05),Cleaved caspase-3(1.81 vs 1.34,P <0.05)和Cleaved caspase-9(2.13 vs 1.37,P <0.05)蛋白相对表达量下降,二氯荧光素荧光强度下降(250.21±8.13 vs 199.14±6.74,P <0.05),p53(1.66 vs 1.25,P <0.05)及p21(1.64 vs 1.23,P <0.05)蛋白表达量也下降。[结论]本研究证实CGA可以抑制BPDE诱导的16HBE细胞凋亡,降低胞内ROS的水平。这种抗凋亡作用可能与caspase-9/caspase-3介导的内源性线粒体途径和抑制BPDE诱导的p53和p21的蛋白表达有关。
[Background] Benzo[a]pyrene-7, 8-diol-9, 10-epoxide (BPDE) is an environmental carcinogen that can cause cell apoptosis and oxidative stress. Chlorogenic acid (CGA) is an antioxidant, but its effect on BPDE-induced apoptosis is unclear.[Objective] BPDE exposure is administered to mimic the damage of tobacco to bronchial epithelial cells. Then CGA intervention is administered to explore its anti-apoptotic effect and its potential molecular mechanism.[Methods] In the pre-experiment, 16HBE cells were treated with CGA at final concentrations of 0, 50, 100, 150, and 200 μmol/L for 2, 4, and 8 h. Based on the results of the pre-experiment, cells were pretreated with CGA 50 μmol/L for 4 h, and then were induced by BPDE 0.5 μmol/L for 12 h. There were four groups: control group, CGA exposure group (CGA 50 μmol/L), BPDE exposure group (BPDE 0.5 μmol/L), and CGA intervention group (CGA 50 μmol/L+BPDE 0.5 μmol/L). Cell apoptosis was measured by Annexin V/PI staining and flow cytometry;intracellular reactive oxygen species (ROS) was detected with DCFH-DA probes;the expressions of apoptosis markers including Cleaved caspase-3, Cleaved caspase-9, p53, and p21 were detected by Western blot.[Results] Compared with the 0 μmol/L CGA group, the cell viabilities were not different in the 50 and 100 μmol/L CGA groups at various time points (P > 0.05), but reduced in the 150 and 200 μmol/L CGA groups at 8 h (P < 0.05). Compared with the BPDE exposure group, the CGA intervention group showed reduced cell apoptosis rate (17.63% vs 9.21%, P < 0.05), relative expression levels of Cleaved caspase-3 (1.81 vs 1.34, P < 0.05) and Cleaved caspase-9 (2.13 vs 1.37, P < 0.05), dichlorofluorescein fluorescence intensity (250.21±8.13 vs 199.14±6.74, P < 0.05), and relative expression levels of p53 (1.66 vs 1.25, P < 0.05) and p21 (1.64 vs 1.23, P < 0.05).[Conclusion] CGA can inhibit apoptosis in 16HBE cells induced by BPDE and reduce intracellular ROS level. Its mechanism may be related to caspase-9/caspase-3 mediated endogenous mitochondrial pathway and inhibited protein expressions of p53 and p21 induced by BPDE.
作者
吴静
吴诗佳
顾心培
陈汇丰
WU Jing;WU Shi-jia;GU Xin-pei;CHEN Hui-feng(School of Nursing, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China)
出处
《环境与职业医学》
CAS
CSCD
北大核心
2019年第9期818-823,共6页
Journal of Environmental and Occupational Medicine
基金
中医药大学自然科学研究项目(2016YSN23)