摘要
目的研究谷氨酰胺(Gln)调节内毒素(LPS)诱导的大鼠肺泡Ⅱ型上皮细胞(AECⅡ)肿瘤坏死因子(TNF)-α分泌及谷胱甘肽(GSH)在其中的作用。方法原代培养成年雄性SD大鼠AECⅡ细胞,原代培养24h后,分为空白对照、10.0mmol/L Gln,1μg/mL LPS、LPS+(0.5、2.0和10.0mmol/L)Gln六组,每组3个孔。LPS刺激24h,Gin在LPS刺激前8h加入;另一组实验分为1μg/mL LPS,LPS+10.0mmol/L Gln,LPS+100μmol/L丁硫氨酸亚砜胺(BSO)、LPS+Gln+(1、10和100μmol/L)BSO六组,每组3个孔。LPS刺激24h,BSO和Gln在LPS刺激前8h加入。以二硫双硝基苯甲酸(DTNB)法测定细胞内GSH浓度,酶联免疫(EHSA)法测定细胞上清TNF-α水平。统计学方法用方差分析(LDS-t检验)比较各组间差异。结果谷氨酰胺可以提高IPS诱导大鼠AECⅡ细胞的GSH浓度,降低TNF-α的水平,其作用随浓度的增加而增加,在10mmol/L浓度最为显著,GSH水平从(50.69±3.04)pmol/mg cell上升到(126.74±7.13)pmol/mg cell(P〈0.01),TNF-α水平从(1104.5±48.8)pg/mL下降到(329.67±48.27)pg/mL(P〈0.01);同时,谷氨酰胺的作用可以被10μmol/L以上浓度BSO显著阻断(P〈0.01)。结论谷氨酰胺可以抑制LPS诱导的大鼠AECⅡ细胞TNF-α的释放,其作用可能是通过促进GSH的合成而实现的。
Objective To investigate the role of glutathione (GSH) synthesis in the regulation of Glutamine (Gin) on TNF-α release in lipopolysaceharide (LPS)-stimulated alveolar epithelial type Ⅱ cells (AEC Ⅱ ). Method Primary cultured AEC Ⅱ were divided into six groups, including control, 10.0 mmol/L Gin, 1μg/mL LPS and LPS+ (0.5, 2.0 and 10.0 mmol/L) Gln. In another set of experiments, AECI] were divided into six groups including 1μg/mL LPS, LPS + 10.0 mmol/L Gin, LPS + 100 μmol/L L-buthionine-(S, R)-sulfoximine (BSO), LPS + Gln + ( 1,10 and 1130/maol/L) BSO. Each group had three samples. BSO and Gln were added at 8 hours before LPS challenge. After 24 hours of LPS stimulation, calls were obtained for GSH measurement by 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB) method. TNF-α level in the supernatant was determined by enzyme- linked immunosorbent assay (ELISA). ANOVA (LSD- t test) was used for statistical analysis. Results Supple- mentation of Gin increased the GSH level and attenuated TNF-α release in LPS-stimulated AEC II in a dose-depen- dant manner. GSH level increased from (50.69±3.04) pmoL/mg cell (LPS group) to ( 126.74 ±7.13) pmol/mg cell (LPS + 10.0 mmol/L group) ( P 〈 0.01 ) and TNF-α level decreased from ( 1104.5 ± 48.8) pg/mL ( LPS group) to (329.67 ± 48.27) pg/mL (LPS±10.0 mmol/L group) ( P 〈 0.01). BSO, an GSH synthesis blocker, at doses greater than 10μmol/L reversed the effect of Gln significantly ( P 〈 0.01 ). Conclusions As a precursor of GSH, glutamine could attenuate TNF-α release in LPS-stimulated AEC Ⅱ, and the with may be mediated via GSH synthesis.
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2009年第5期462-465,共4页
Chinese Journal of Emergency Medicine
基金
国家自然科学基金资助项目(30500404)
江苏省自然科学基金资助项目(BK2006530)
关键词
谷氨酰胺
谷胱甘肽
肺泡Ⅱ型上皮细胞
内毒素
肿瘤坏死因子
Glutamine
Glutathione
Alveolar epithelial type Ⅱ ceils
Lipopolysaccharide
Tumor necrosis factor