基于PERK-eIF2α-NF-κB信号通路研究甘草酸苷对小鼠肠上皮细胞内质网应激的保护作用

Protective Effect of Glycyrrhizin on Endoplasmic Reticulum Stress in Mouse Intestinal Epithelial Cells Based on PERK-eIF2α-NF-κB Signaling Pathway

关键词:  甘草酸苷  溃疡性结肠炎  肠上皮细胞  内质网应激  PERK-eIF2α-NF-κB信号通路

Key words:  glycyrrhizin  ulcerative colitis  intestinal epithelial cells  endoplasmic reticulum stress  PERK-eIF2α-NF-κB signaling pathway

摘要:

目的 从蛋白激酶R样内质网激酶（protein kinase R-like endoplasmic reticulum kinase，PERK）-真核细胞起始因子2α（eukaryotic initiation factor 2α，eIF2α）-核因子κB （nuclear factor kappa B，NF-κB）信号通路角度观察甘草酸苷（glycyrrhizin，GL）对肠上皮细胞（intestinal epithelial cells，IECs）的保护作用，并探讨其治疗溃疡性结肠炎（ulcerative colitis，UC）的可能作用机制。方法 体外培养并通过免疫荧光法鉴定小鼠IECs，H₂O₂刺激法建立细胞内质网应激模型，GL组于造模同时给予不同剂量的GL干预。采用CCK8法检测细胞的存活率；流式细胞术检测细胞的凋亡水平；细胞跨膜电阻抗和异硫氰酸荧光素标记的右旋糖酐（fluorescein isothiocyanate-dextran，FITC-dextran）法共同明确细胞屏障的通透性；Western blotting检测细胞中PERK-eIF2α-NF-κB通路核心蛋白的表达水平。结果 与模型对照组相比，GL中、高剂量组的存活率均升高（P<0.05或P<0.01），凋亡率均下降（P<0.05或P<0.01）；GL各剂量组单层膜细胞的细胞跨膜电阻抗值均显著升高（P<0.01）、FITC-dextran浓度均显著下降（P<0.01），p-PERK、p-eIF2α和NF-κB蛋白的水平均下降（P<0.05或P<0.01）。结论 GL通过抑制PERK-eIF2α-NF-κB信号通路的活化，从而提高小鼠体外细胞内质网应激状态下IECs的存活率，降低其凋亡水平，并改善细胞屏障的通透性，这可能是GL保护IECs、治疗UC的部分作用机制。

Abstract:

OBJECTIVE To observe the protective effect of glycyrrhizin(GL) on intestinal epithelial cells(IECs) from the perspective of protein kinase R-like endoplasmic reticulum kinase(PERK)-eukaryotic initiation factor 2α(eIF2α)-nuclear factor kappa B(NF-κB) signaling pathway, and to explore the possible therapeutic mechanism of GL on treating ulcerative colitis(UC). METHODS Mice's IECs were cultured and identified by immunofluorescence assay in vitro, endoplasmic reticulum stress model was established by H₂O₂ stimulation, and GL groups were given different doses of GL intervention simultaneously while modeling. The cell's survival rate was assessed by CCK8 method. The apoptosis rate was tested by flow cytometry. The cell barrier permeability was determined by transepithelial resistance and fluorescein isothiocyanate-dextran(FITC-dextran) method. And the proteins' expression levels of PERK-eIF2α-NF-κB pathway were detected by Western blotting. RESULTS Compared with model control group, the survival rates of GL middle and high dose groups were increased(P<0.05 or P<0.01), their apoptosis rates were decreased(P<0.05 or P<0.01). The transepithelial resistance values in GL groups were all distinctly elevated(P<0.01), their FITC-dextran concentration were all markedly declined(P<0.01), and their expression levels of p-PERK, p-eIF2α and NF-κB were all descended(P<0.05 or P<0.01). CONCLUSION GL can increase the survival rate and reduce the apoptosis rate of IECs in vitro under endoplasmic reticulum stress state in mice, and improve the permeability of cell barrier by inhibiting the activation of PERK- eIF2α-NF-κB signaling pathway, which might be partial mechanism of GL protecting IECs and treating UC.