二肽精氨酰-谷氨酰胺对脂多糖致肠道上皮细胞损伤的保护作用

Protective Effect of Dipeptide Arginyl-Glutamine on Intestinal Epithelial Cells Damage Induced by Lipopolysaccharide

目的探讨二肽精氨酰-谷氨酰胺(Arg-Gln)对脂多糖(LPS)致肠道上皮细胞Caco-2炎性反应、细胞活力及屏障功能损伤的影响及其可能的机制。方法用不含Gln的培养液加入不同水平的Arg-Gln培养Caco-2细胞24 h,同时加或不加Gln合成抑制剂———甲硫氨酸亚矾胺(MS)4.0 mmol.L-1。然后分别进行如下实验:(1)加入LPS 0.05 g.L-1,再培养24 h,分别测定促炎细胞因子IL-8水平(ELISA法)、细胞活力(MTS法)变化;比较相同水平(2.5 mmol.L-1)Arg-Gln和Gln、Arg的抗炎效果。(2)加入LPS0.4 g.L-1,再培养24 h,测定Caco-2细胞跨上皮电阻(TER)变化。(3)加入LPS 0.05 g.L-1再培养5 h,测定细胞核因子-κB(NF-κB)及其抑制因子I-κB表达的变化(免疫印迹法)。结果与不加LPS的细胞相比,加入LPS 0.05 g.L-1培养24 h可显著刺激Caco-2细胞产生IL-8,抑制细胞活力(Pa<0.05);加MS时,大剂量LPS(0.4 g.L-1)导致TER明显下降(P<0.05)。1.25～5.00 mmol.L-1 Arg-Gln可抑制LPS诱导的IL-8水平(P<0.05);同样剂量时(2.5 mmol.L-1),Gln对IL-8的抑制效果优于Arg-Gln和Arg。2.0～4.0 mmol.L-1Arg-Gln明显促进LPS抑制的Caco-2细胞活力的恢复(Pa<0.05);Arg-Gln可抑制LPS诱导的TER下降(P<0.05)。LPS(0.05 g.L-1)或Arg-Gln(2.0 mmol.L-1)对NF-κB和I-κB表达均无明显影响。结论 Arg-Gln可减轻LPS诱导的肠道上皮细胞屏障障碍及炎性反应,恢复细胞活力;Arg-Gln的保护机制可能与NF-κB途径无明显关系。

Objective To explore the effects of dipeptide arginyl - glutamine （ Arg -Gln ） on inflammation, cell viability and barrier function damage induced by lipopolysaccharide （LPS） in Caco -2 cells and its potential mechanism. Methods Caco -2 cells were incuba- ted in Gln free medium in different concentrations of Arg -Glli and with or without methionine sulfoximine（ MS, an inhibitor of glutamine syn- thetase） for 24 h. Then 0.05 g ·L^-1 LPS was added for another 24 h. IL -8 levels in the medium and cell viability were determined by en- zyme - linked immunosorbent assay or MTS method. The same dose （2.5 mmol·L^-1） of Arg -Gln, Gln or Arg was used and the anti - in- flammation effects of 3 reagents were compared, and 0.4 g ·L^-1 LPS was added for another 24 h. Transepithelial electrical resistance （TER） was determined. 0.05 g ·L^-1 LPS was added for 5 h. Nuclear factor kappa B （ NF - κB） and inhibitor of nuclear factor kappa B （ I - κB ） were determined by Western blot analysis. Results LPS （0.05 g ·L^-1） induced significantly IL - 8 production in Caco - 2 ceils, depressed cell viability. In the presence of MS,LPS（0.4 g ·L^-1） remarkably induced TER decrease in Caco -2 cells（ P 〈 0.05 ）. Pro -treatment with Arg - Gin decreased IL - 8 production induced by LPS in the presence of MS（Pa 〈 0.05 ）. The anti - inflammation effect of Gin was better than that of Arg - Gin and Arg in the same dose. When stimulated with LPS in the presence of MS,Arg - Gin at concentrations of 2.0 - 4.0 mmol ·L^-1 increased cell viability（ Pa 〈 0.05 ）. Pre -treatment with Arg -Gin significantly increased TER in LPS stimulated Caco -2 cells （ Pa 〈 0.05 ）. Neither LPS nor Arg - Gin treatment induced significantly changes of either NF - κB or I - κB in Caco - 2 cells. Conclusions Arg - Gin pre - treatment is capable of reducing mucosal damage induced by LPS, improving cell viability and reducing IL - 8 production in Caco -2 cells. However,these effects do not seem to be directly associated with the NF - κB pathway in this study.