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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 investigate the effect of arginine glutamine (Gln) on the inflammatory reaction of Caco-2, cell viability and barrier dysfunction in intestinal epithelial cells induced by lipopolysaccharide (LPS) and its possible mechanism. Methods Caco-2 cells were cultured with different concentrations of Arg-Gln in Gln-free medium for 24 h with or without addition of Gln synthesis inhibitor 4.0 mmol.L- 1. Then the following experiments were carried out: (1) 0.05gL-1 LPS was added and cultured for another 24 h, the levels of IL-8 and MTS were measured respectively; .L-1) Anti-inflammatory effects of Arg-Gln and Gln, Arg. (2) The trans-epithelial resistance (TER) of Caco-2 cells was determined by adding LPS0.4 g.L-1 for 24 h. (3) The LPS (0.05 g.L-1) was added to culture for another 5 h, and the changes of NF-κB and its inhibitor I-κB were detected by Western blotting. Results Compared with the cells without LPS, LPS supplemented with 0.05 g L-1 for 24 h significantly stimulated Caco-2 cells to produce IL-8 and inhibited cell viability (Pa <0.05) -1) resulted in a significant decrease of TER (P <0.05). Glutamate could inhibit the level of IL-8 induced by LPS (P <0.05) at the concentration of 1.25-5.50 mmol.L-1 Arg-Gln, while Gln inhibited IL-8 at the same dose (2.5 mmol.L-1) Gln and Arg. 2.0 ~ 4.0 mmol.L-1Arg-Gln significantly promoted the recovery of LPS-inhibited Caco-2 cell viability (P <0.05); Arg-Gln inhibited the LPS-induced TER decline (P <0.05). LPS (0.05 g.L-1) or Arg-Gln (2.0 mmol.L-1) had no significant effect on the expression of NF-κB and I-κB. Conclusion Arg-Gln can reduce the intestinal barrier and inflammatory response induced by LPS and restore cell viability. The protective mechanism of Arg-Gln may not be related to NF-κB pathway.