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

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

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。

Objective To study the effects of allitridum on the antioxidative capability of rat lung epithelial L2 eels （ CCL-149 cell line ） by observing the changes of glutathione （ GSH ）, malondialdehyde （MDA） content, and the expressing level of T-glutamyl-eysteine synthetase （ T-C, CS） 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. 1,1.0,5.0μg/m] of allitridum for 6,12,24,48h. After treatment, GSH content and MDA formation were measured by speetrophotometic assay, and the T-C, CS protein was semi-quantified by Western blot. Results Allitridum （ 0. 1,1.0,5. 0 μg/m] ） 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 plot when CCL-149 cells were treatment with 0,0, 1, 1. 0,5.0 μg/m] 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 γ-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 ,γGCS protein and the content of GSH.