大气细颗粒物对CHO细胞的损伤作用及天然物质的拮抗机制  

The Detrimental Effects of PM_(2.5) and the Protective Effects of Bioactive Compounds on CHO Cell

作　　者：郭辰[1] 毕婷婷[1] 陈世杰[1] 赵晓红[1] 刘涛[2] 孙健[1] 

机构地区：[1]北京联合大学功能食品科学技术研究院,北京100191 [2]首都师范大学生命科学学院,北京100048

出　　处：《生态毒理学报》2015年第3期101-111,共11页Asian Journal of Ecotoxicology

基　　金：北京市教育委员会科技计划重点项目(KZ201211417041)

摘　　要：PM2.5引起的健康危害日益受到广泛关注,但其确切的损伤机理仍不完全清楚,目前也缺乏有效拮抗PM2.5损伤的天然活性物质。因此本文对北京市PM2.5造成CHO细胞的损伤作用及天然物质的拮抗作用进行研究,结果可为PM2.5污染治理和疾病的预防提供科学依据。通过CCK-8法测定细胞存活率;通过流式细胞仪测定细胞凋亡;用荧光探针DCFH-DA法测定ROS;提取细胞内总蛋白并测定其中SOD含量;以及通过Western Blot法分别测定了p-akt/akt、p65及Bad的相对表达量。结果显示：（1）10~40μg·m L-1的PM2.5可明显降低CHO细胞存活率,并呈现极显著的剂量-效应关系（r=-0.964,P〈0.01）;15μg·m L-1PM2.5可以显著增加细胞凋亡、引起细胞内ROS增加、SOD含量下降;p-akt/akt、p65表达量增加说明Akt通路与NF-κB通路均被激活。（2）20μmol·L-1阿魏酸、50μmol·L-1绿原酸和10μmol·L-1荭草素预处理可拮抗PM2.5引起的细胞存活率下降以及细胞凋亡率增加,同时降低细胞内ROS并增加SOD含量,下调p-akt/akt、p65及Bad的相对表达量。其中20μmol·L-1阿魏酸的保护作用最佳。结果说明,PM2.5引起的CHO细胞损伤与细胞凋亡与Akt和NF-κB通路的激活有关;三种天然活性物质具有拮抗PM2.5造成的细胞损伤的能力,其保护机理是通过其降低PM2.5引起的细胞内氧化应激反应,进而降低被PM2.5激活的Akt通路与NF-κB通路和下调Bad蛋白表达量来实现的。Fine particulate matters （PM2.5） cause health problems that have brought great deal of concern. However, the mechanisms by which PM2.5 involved casus detrimental effects and the antagonism of bioactive compounds are still unclearcurrently unknown. The detrimental effects of PM2.5 collected in Beijing and the antagonism of Ferulic acid （FA）, Chlorogenic acid （CA） and Orientin （ORI） on CHO cell were studied in this research and provided basic information for controlling the air pollution control and the subsequent respiratory diseases. CCK-8 assay was used to detect the cell viability, while cell apoptosis was detected with a Flow Cytometry. The gene expression ofp-akt, akg p65 and Bad were analyzeddetected with a Western Blot analysis. Intracellular ROS and SOD level were meas- ured with the fluorescent probe DCFH-DA and WST-1, respectively. Results showed that 10~40μg. mE1 PMz5 re- duced cell viability in a dose dependent manner （r=-0.964, P〈0.01）. 15 μg · mL-1 of PMz5 dramatically remarkebly reduces the cell viability and as well as SOD level andas well asincreased the apoptotic rate and the intracellular ROS in CHO cells. It also up-regulated the gene expressions ofp-akt/akt and p65 which indicated the activation of Akt and NF-xB signaling pathways. Pretreatments with 20 μmol · E-1 FA, 50 μmol · L -1 CA and 10 μmol · L-1 ORI maintained CHO cell viability and reduced PMzs-induced apoptosisthrough the down-regulate reduction of the in- tracellular ROS, up-regulated SOD, and the down-regulated p-akt/akt, p65 and Bad. Taking together, PM2.5 causes damage on CHO cells through a variety of mechanisms, and the bioactive compounds can attenuated these effects by reducing oxidative stress and the over- up-regulatedinduction of Akt and NF-kB pathways.

关 键 词：细颗粒物(PM2.5) 天然活性物质 细胞毒性 氧化应激 AKT信号通路 NF-ΚB信号通路 BAD蛋白 

分 类 号：Q78[生物学—分子生物学]