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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 observe the expression changes of glutathione (GSH), malondialdehyde (MDA) and γ-glutamylcysteine synthetase (γGCS) in rat alveolar epithelial cells (CCL149) Effect of antioxidant capacity. Methods Allicin (0, 0.1, 1.0, 5.0 μg / ml) was used to treat CCL149 at various times (6, 12, 24 h and 48 h). The changes of intracellular GSH and MDA were detected by spectrophotometry. The expression of intracellular GGCS protein was detected by Western blot. The effect of allicin on the intracellular GSH, MDA and GGCS was analyzed. Results (1) allicin concentration ≤ 5μg / ml does not affect cell viability. (2) The contents of GSH in the groups of 0.1,1.0,5.0μg / ml allicin after 6h treatment were (16.45 ± 0.69), (16.81 ± 0.79) and (17.80 ± 1.10) mg / g protein (1.07 ± 0.02), (1.02 ± 0.06) and (1.00 ± 0.05), respectively, compared with the control group [(13.38 ± 1.16) mg / g protein] nmol / mg protein] compared with control group [(1.23 ± 0.05) nmol / mg protein] (t = 5.75 ~ 6.34, P <0.05). The intracellular GSH level reached the peak at 24 h and decreased at 48 h, but still higher than that of the control group. The differences were statistically significant (P <0.05) in different allicin concentrations at the same time points compared with the control group. (3) Allicin 0.1, 1.0 and 5.0 μg / ml groups showed significantly higher expression of γGCS protein at 6, 12 and 24 hours (24h, 0.693 ± 0.027, 0.646 ± 0.081 and 0.667 ± 0.077) than that of the control group (0.531 ± 0.007) (T = 2.82 ~ 9.92, P <0.05). All the indexes did not show dose-dependent. Conclusion Allicin can enhance the anti-oxidative effect of alveolar epithelial cells in rats, which may be related to the promotion of γGCS expression.