谷氨酰胺对热打击下单层肠上皮Caco-2细胞屏障功能的影响

目的 探讨谷氨酰胺(Gln)对热打击后肠黏膜单层上皮细胞屏障通透性改变的影响及可能机制.方法 应用Caco-2细胞株建立肠上皮机械屏障模型,加入Gln培养24h,43℃持续1h热打击.以CCK-8法检测不同浓度Gln(0.4、0.7、1.4、2.1、2.8mmol/L)对细胞增殖的影响,为后续实验选择最适合浓度;Transwell法测定单层跨膜电阻抗(TEER)值和对辣根过氧化物酶(HRP)的通透性;Western blotting检测紧密连接蛋白occludin和ZO-1的表达;采用考马斯亮蓝对细胞骨架进行染色观察.结果 0.7mmol/L的Gln促进细胞增殖的效果最强,与其他浓度相比,差异均有统计学意义(P<0.05).相较于单纯43℃热打击组,0.7mmol/L的Gln可抑制单层上皮细胞TEER的下降和HRP通过率的升高(P<0.01),增加occludin和ZO-1的表达,有益于维持细胞骨架的正常结构.结论 0.7mmol/L的Gln可减轻热打击对单层肠上皮细胞结构的破坏,保护屏障功能.

Influence of glutamine on barrier function of heat-stressed intestinal epithelial Caco-2 cells

Objective To investigate the effect of Glutamine (Gln) on heat stress-induced dysfunction of intestinal epithelial barrier. Methods Human intestinal epithelial Caco-2 cells were pre-incubated with Gln for 24h and then exposed to heat 43℃ for 1h. Cell counting kit-8 (CCK-8) was used to detect the cellular proliferation with various concentrations of Gln and choose an optimum concentration for subsequent experiments. The barrier integrity was measured by transepithelial electrical resistance (TEER) and horseradish peroxidase (HRP) permeability. Levels of tight junction protein occludin and ZO-1 were analyzed by Western blotting. Cytoskeleton using Coomassie blue staining was observed by microscopy. Results At 0.7mmol/L concentration, Gln showed the most effective cell proliferation compared with other concentration groups (P<0.05). Therefore, 0.7mmol/L Gln was used as effective concentration in following experiments. Gln attenuated the TEER decrease and impairment of intestinal permeability induced by heat exposure compared with 43℃ group (P<0.01). The expressions of occludin and ZO-1 were significantly elevated by pretreatment with Gln. The distortion of cytoskeleton was also effectively prevented. Conclusion 0.7mmol/L Gln is potentially beneficial for protecting against heat stress-induced permeability dysfunction and epithelial barrier damage.