吴静, 吴诗佳, 顾心培, 陈汇丰. 绿原酸对反式二氢二醇环氧苯并[a]芘诱导的细胞凋亡的保护作用和相关分子机制[J]. 环境与职业医学, 2019, 36(9): 818-823. DOI: 10.13213/j.cnki.jeom.2019.19091
引用本文: 吴静, 吴诗佳, 顾心培, 陈汇丰. 绿原酸对反式二氢二醇环氧苯并[a]芘诱导的细胞凋亡的保护作用和相关分子机制[J]. 环境与职业医学, 2019, 36(9): 818-823. DOI: 10.13213/j.cnki.jeom.2019.19091
WU Jing, WU Shi-jia, GU Xin-pei, CHEN Hui-feng. Protective effect of chlorogenic acid on BPDE-induced cell apoptosis and its molecular mechanism[J]. Journal of Environmental and Occupational Medicine, 2019, 36(9): 818-823. DOI: 10.13213/j.cnki.jeom.2019.19091
Citation: WU Jing, WU Shi-jia, GU Xin-pei, CHEN Hui-feng. Protective effect of chlorogenic acid on BPDE-induced cell apoptosis and its molecular mechanism[J]. Journal of Environmental and Occupational Medicine, 2019, 36(9): 818-823. DOI: 10.13213/j.cnki.jeom.2019.19091

绿原酸对反式二氢二醇环氧苯并a芘诱导的细胞凋亡的保护作用和相关分子机制

Protective effect of chlorogenic acid on BPDE-induced cell apoptosis and its molecular mechanism

  • 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、200 μmol/L的CGA分别处理16HBE细胞2、4、8h。根据预实验结果,选择CGA 50μmol/L预处理细胞4h后再进行BPDE 0.5μmol/L染毒12h。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 50 μmol/L+BPDE 0.5 μmol/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、100 μmol/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、200 μmol/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),Cleavedcaspase-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。

     

    Abstract: Background Benzoapyrene-7, 8-diol-9, 10-epoxide (BPDE) is an environmental carcinogen that can cause cell apoptosis and oxidatve stress. Chlorogenic acid (CGA) is an antoxidant, 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 interventon is administered to explore its ant-apoptotc effect and its potental molecular mechanism.

    Methods In the pre-experiment, 16HBE cells were treated with CGA at fnal concentratons 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 50μmol/L for 4h, and then were induced by BPDE 0.5μmol/L for 12h. There were four groups:control group, CGA exposure group (CGA 50 μmol/L), BPDE exposure group (BPDE 0.5 μmol/L), and CGA interventon group (CGA 50 μmol/L+BPDE 0.5 μmol/L). Cell apoptosis was measured by Annexin V/PI staining and flow cytometry; intracellular reactve 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 viabilites were not different in the 50 and 100 μmol/L CGA groups at various tme 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 interventon group showed reduced cell apoptosis rate (17.63% vs 9.21%, P < 0.05), relatve 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 relatve 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.

     

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