大蒜素对大鼠肺泡上皮细胞γ谷氨酰半胱氨酸合成酶表达的影响

目的观察大鼠肺泡上皮细胞(CCL149细胞系)谷胱甘肽(GSH)、丙二醛(MDA)及γ谷氨酰半胱氨酸合成酶(γGCS)等表达变化,了解大蒜素对CCL149细胞抗氧化能力的影响。方法在以四甲基偶氮唑盐(MTT)法确定大蒜素适当作用浓度的基础上,用不同浓度大蒜素(0、0.1、1.0、5.0μg/ml)作用CCL149不同时间(6、12、24、48h),用分光光度法检测细胞内GSH、MDA的变化;用Westernblot法检测细胞内γGCS蛋白的表达,分析不同处理剂量、不同处理时间大蒜素对细胞内GSH、MDA及γGCS的影响。结果(1)大蒜素浓度≤5μg/ml不影响细胞活力。(2)大蒜素0.1、1.0、5.0μg/ml组在处理6h后细胞内GSH含量6h组GSH含量分别为(16.45±0.69)、(16.81±0.79)、(17.80±1.10)mg/g蛋白]较对照组(13.38±1.16)mg/g蛋白]显著增加(t=3.92～4.78,P均<0.05),MDA含量(1.07±0.02)、(1.02±0.06)、(1.00±0.05)nmol/mg蛋白]较对照组(1.23±0.05)nmol/mg蛋白]下降(t=5.75～6.34,P均<0.05)。细胞内GSH水平升高至24h达顶峰,48h有所下降,仍较对照组高,相同时间点不同大蒜素浓度组分别与对照组比较,差异均有统计学意义(P均<0.05)。(3)大蒜素0.1、1.0、5.0μg/ml组在刺激6、12、24h细胞内γGCS蛋白表达(24h为0.693±0.027、0.646±0.081、0.667±0.077)较对照组(0.531±0.007)显著增强(t=2.82～9.92,P<0.05),各观察指标未呈现剂量依赖性。结论大蒜素能增强大鼠肺泡上皮细胞的抗氧化作用,可能与其促进γGCS的表达有关。

Effect of allitridum on expression of the γ-glutamyl-cysteine synthetase in rat lung epithelial L2 cells

OBJECTIVE: To study the effects of allitridum on the antioxidative capability of rat lung epithelial L2 cells (CCL-149 cell line) by observing the changes of glutathione (GSH), malondialdehyde (MDA) content, and the expressing level of gamma-glutamyl-cysteine synthetase (gamma-GCS) protein. METHODS: The cell viability of CCL-149 cells treated with allitridum was detected by MTT assay. CCL-149 cells were incubated with 0, 0.1, 1.0, 5.0 microg/ml of allitridum for 6, 12, 24, 48 h. After treatment, GSH content and MDA formation were measured by spectrophotometic assay, and the gamma-GCS protein was semi-quantified by Western blot. RESULTS: Allitridum (0.1, 1.0, 5.0 microg/ml) showed no side effects on cell growth (P > 0.05). Treatment with allitridum increased GSH levels and decreased MDA formation in CCL-149, the most significant time being incubation at 24 h. The GSH content were (13.34 +/- 0.62), (27.67 +/- 2.39), (29.54 +/- 0.71), (30.25 +/- 1.05) mg/g prot, and the MDA content were (1.25 +/- 0.08), (0.90 +/- 0.06), (0.84 +/- 0.06), (0.81 +/- 0.02) nmol/mg prot when CCL-149 cells were treatment with 0, 0.1, 1.0, 5.0 microg/ml of allitridum for 24 h, respectively. The effect showed no dose dependent effects (P > 0.05). After 6, 12, 24 h incubation, the expression of gamma-GCS protein was increased as compared to the control cells (t = 2.82 - 9.92, P < 0.05). CONCLUSION: Allitidum may increase the antioxidative ability of CCL-149 cells by increasing the expression of gamma-GCS protein and the content of GSH.