CAJ | 학술논문

目的研究谷氨酰胺(Gin)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24 h后,分为空白对照、10.0 mmol/L Gin,1 μg/mL LPS、LPS+(0.5、2.0和10.0 mmol/L)Gln六组,每组3个孔.LPS刺激24 h,Gln在LPS刺激前8 h加入;另一组实验分为l μg/mL LPS,LPS+10.0 mmol/L Gln,LPS+100 μmol/L丁硫氨酸哑砜胺(BSO)、LPS+Gln+(1、10和100 μmol/L)BSO六组,每组3个孔.LPS刺激24 h,BSO和Gln在LPS刺激前8 h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(ELISA)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LPS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10 mmol/L浓度最为显著,GSH水平从(50.69±3.04)pmol/mgcell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10 μmol/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.
목적 연구곡안선알(Gin)조절내독소(LPs)유도적대서폐포Ⅱ형상피세포(AECⅡ)종류배사인자(TNF)-α분비급곡광감태(GSH)재기중적작용.방법 원대배양성년웅성SD대서AECⅡ세포,원대배양24 h후,분위공백대조、10.0 mmol/L Gin,1 μg/mL LPS、LPS+(0.5、2.0화10.0 mmol/L)Gln륙조,매조3개공.LPS자격24 h,Gln재LPS자격전8 h가입;령일조실험분위l μg/mL LPS,LPS+10.0 mmol/L Gln,LPS+100 μmol/L정류안산아풍알(BSO)、LPS+Gln+(1、10화100 μmol/L)BSO륙조,매조3개공.LPS자격24 h,BSO화Gln재LPS자격전8 h가입.이이류쌍초기분갑산(DTNB)법측정세포내GSH농도,매련면역(ELISA)법측정세포상청TNF-α수평.통계학방법용방차분석(LDS-t검험)비교각조간차이.결과 곡안선알가이제고LPS유도대서AECⅡ세포적GSH농도,강저TNF-α적수평,기작용수농도적증가이증가,재10 mmol/L농도최위현저,GSH수평종(50.69±3.04)pmol/mgcell상승도(126.74±7.13)pmol/mg cell(P<0.01),TNF-α수평종(1104.5±48.8)pg/mL하강도(329.67±48.27)pg/mL(P<0.01);동시,곡안선알적작용가이피10 μmol/L이상농도BSO현저조단(P<0.01).결론 곡안선알가이억제LPS유도적대서AECⅡ세포TNF-α적석방,기작용가능시통과촉진GSH적합성이실현적.
Objective To investigate the role of glutathione (GSH) synthesis in the regulation of Glutamine (Gin) on TNF-α release in lipopolysaccharide (LPS)-stimulated alveolar epithelial type Ⅱ cells (AEC Ⅱ). Method Primary cultured AECⅡ were divided into six groups, including control, 10.0 mmol/L Gln, 1 μg/mL LPS and LPS+(0.5, 2.0 and 10.0 mmol/L) Gln. In another set of experiments, AECⅡ were divided into six groups including 1 μg/mL LPS, LPS+10.0 mmol/L Gln, LPS+100 μmol/L L-buthionine-(S, R)-sulfoximine (BSO), LPS+Gln+(1,10 and 100 μmol/L) BSO. Each group had three samples. BSO and Gln were added at 8 hours before LPS challenge. After 24 hours of LPS stimulation, cells were obtained for GSH measurement by 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB) method. TNF-α level in the supematant was determined by enzyme-hnked immunesorbent 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-dam 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 10 μmol/L reversed the effect of Gin significantly (P<0.01). Conclusions As a precursor ofGSH, glutsmine could attenuate TNT-α release in LPS-stimulated AECⅡ,and the with may be mediated via GSH synthesis.