研究了棒曲霉素(patulin,PAT)对人胃黏膜细胞(gastric epithelial cells,GES-1)的体外毒性作用,从细胞增殖、凋亡、氧化损伤和DNA损伤等方面对其毒性机理进行了初步探讨。采用四甲基偶氮唑盐法检测PAT对细胞增殖的毒性作用;通过荧光显微镜观察了细胞凋亡形态;利用反转录聚合酶链式扩增反应(reverse transcription-polymerase chain reaction,RT-PCR)实验考察了Bax和Bcl-2 mRNA相对表达量变化;检测了细胞内超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽(glutathione,GSH)和丙二醛(malonaldehyde,MDA)等氧化损伤指标变化;采用Western Blot技术对DNA双链断裂标志物γ-H2AX的表达量进行了测定。研究结果表明,当c(PAT)＞1.0 μmol/L时,PAT对GES-1细胞抑制率大于20%,荧光染色镜检可见明显的细胞凋亡现象;Bax mRNA转录水平表达量上调,而Bcl-2 mRNA转录水平表达量显著下调,表明GES-1细胞进入了凋亡阶段。同时,总SOD、CAT检测值降幅最高可达53.76%,GSH检测值降幅最高可达56.55%,而MDA最低增幅为139.59%;γ-H2AX表达量上调。研究结果表明PAT可显著抑制GES-1细胞增殖并诱导其凋亡,具有明显的量效关系。由研究结果推断PAT可能通过体外诱发氧化损伤和DNA损伤途径引起GES-1细胞凋亡。
The toxicity mechanism of patulin (PAT) on human gastric epithelial cells (GES-1) was studied in vitro from the aspects of cell proliferation, apoptosis, oxidative damage and DNA damage. When the concentration of PAT was above 1.0 μmol/L, the inhibition rate of PAT on GES-1 cells was greater than 20%. Meanwhile, the obvious apoptosis phenomenon was observed under fluorescence microscope. The relative expression level of Bax mRNA showed a significant up-regulation, whereas the relative expression level of Bcl-2 mRNA was distinctly down-regulated. At the same time, the activity of SOD and CAT decreased at most by 53.76% and the content of GSH reduced at most by 56.55%. The content of MDA increased at least by 139.59%. The expression of γ-H2AX was elevated with the increasing concentration of PAT. The results showed that PAT could significantly inhibit the proliferation and induce the apoptosis of GES-1 cells, with significant dose-effect relationship. It is concluded that PAT may induce apoptosis of GES-1 cells through oxidative damage and DNA damage.
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