丁氟螨酯对SH-SY5Y细胞的毒性作用及其机制

Cytotoxicity of cyflumetofen on SH-SY5Y cells and possible mechanism

目的研究丁氟螨酯对人经神经母细胞瘤细胞SH-SY5Y细胞的毒性作用及其机制。方法加入丁氟螨酯0.03,0.06,0.125,0.25,0.5,1,2,2.6,4,6,8和16 mmol·L^(-1)处理SH-SY5Y细胞48 h,MTT法测定细胞存活;DCFH-DA荧光探针标记法检测细胞内活性氧(ROS)水平;JC-1标记法检测细胞线粒体膜电位;Hoechst 33258染色观察细胞核形态;碘化丙啶(PI)染色和流式细胞仪检测细胞周期和细胞凋亡;Western蛋白印迹法检测磷酸化P38蛋白(p-P38)和磷酸化Jun激酶(p-JNK)表达水平。结果与溶剂(DMSO)对照组相比,共孵育48 h后,丁氟螨酯≥0.06 mmol·L^(-1)时可降低细胞存活率(P<0.05),且随浓度增高细胞存活率有降低趋势,IC_(50)为2.6 mmol·L^(-1);丁氟螨酯1,2,4和6 mmol·L^(-1)组细胞ROS水平升高(P<0.01),线粒体膜电位下降(P<0.01)。Hoechst 33258染色结果显示,丁氟螨酯2,4和6 mmol·L^(-1)组SH-SY5Y细胞出现颗粒状荧光,细胞核固缩和崩解;流式细胞仪检测结果显示,丁氟螨酯2,4和6 mmol·L^(-1)组细胞凋亡率由DMSO对照组的(0.7±0.1)%分别上升至(6.7±0.1)%,(72.4±8.6)%和(90.7±3.2)%(P<0.01);丁氟螨酯4和6 mmol·L^(-1)组G_1期细胞较DMSO对照组明显增加(P<0.01);Western蛋白印迹结果表明,丁氟螨酯4和6 mmol·L^(-1)组p-JNK表达均升高(P<0.01),丁氟螨酯6 mmol·L^(-1)组p-P38表达水平增加(P<0.01)。结论丁氟螨酯可能通过氧化损伤、激活P38蛋白和JNK蛋白诱导SH-SY5Y细胞G_1期阻滞和凋亡。

OBJECTIVE To investigate the cytotoxicity of cyflumetofen for SH-SY5Y cells and the mechanism. METHODS SH-SY5Y cells treated with cyflumetofen 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2, 2.6, 4, 6, 8 and 16 mmol. L-1 for 48 h. Cell survival was measured with M-IF assay. The reactive oxygen species （ROS） was determined with the DCFH-DA probe, and mitochondrial membrane potential （MMP） was detected by JC-1 staining. The morphological changes in cell nuclei were observed with Hoechst33258 staining. Cell cycle and apoptosis were determined by flow cytometry. The protein levels of phosphorylated Jun Kinase （p-JNK） and p-P38 were measured by Western blotting. RESULTS Compared with solvent （DMSO） control group, cyflumetofen （≥0.06 retool-L-1） inhibited the proliferation of SH-SY5Y cells obviously （P〈0.05）, and the IC50 was 2.6 retool. L-1. MMP declined and ROS levels increased significantly in cyflumetofen 1, 2, 4 and 6 mmol. L-1 groups （P〈0.01）. Cyflumetofen 2, 4 and 6 mmol. L-1 induced nucleic accumulation, nuclear shrinkage and disintegration in SH-SY5Y cells. Apoptosis rates of cyflu- metofen 2, 4 and 6 mmol. L-1 groups increased from （0.7＋0.1）% in DMSO control group to （6.7±0.1）%, （72.4±8.6）% and （90.7±3.2）% （P〈0.01）. Cyflumetofen 4 and 6 rnmol. L-1 induced G1 phase cell cycle arrest （P〈0.01）. In addition, Western blotting showed that cyflumetofen 4 and 6 mmol. L-1 up-regulated the expression of p-JNK （P〈0.01）, while the level of p-P38 in SH-SY5Y cells was increased in cyflumetofen 6mmol. L-1 group （P〈0.01）. CONCLUSION Cyflumetofen induces cell damage, apoptosis and G1 phase cell cycle arrest in SH-SY5Y cells. The mechanism may be associated with oxidative damage, and activation of P38 and JNK stress-response pathways.