甘草酸对LPS诱导的IEC-6细胞NF-κB通路及炎症因子表达的影响
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摘要
目的:研究甘草酸(GA)对脂多糖(LPS)诱导肠上皮细胞IEC-6发生炎症应激的影响,并进一步探讨其炎症调控的作用机制。方法:以IEC-6细胞株为研究对象,实验分Control组、LPS模型组、LPS+GA组、GA组,Western blot法检测TLR4、CD14、MyD88、IκBα、p-IκBα、NF-κB p65表达变化; RT-qPCR和Western blot检测下游炎症基因的mRNA水平及蛋白表达。结果:与Control组相比,LPS诱导后IEC-6细胞中TLR4、CD14、MyD88的蛋白表达水平上调,IκBα的磷酸化水平显著升高及NF-κB p65的核转位增加(P<0. 05),同时上调ICAM-1、COX-2、i NOS和IL-6炎症相关基因表达,并减少IL-10表达(P<0. 05),甘草酸干预后可显著逆转上述改变,结果均具有统计学意义(P<0. 05)。结论:甘草酸能抑制LPS活化的TLR4/NF-κB信号通路,进而下调LPS诱导的促炎基因表达,减轻肠上皮细胞的炎症性损伤。
Objective: To investigate the protective effect of glycyrrhizic acid( GA) on lipopolysaccharide( LPS) induced inflammation of the intestinal epithelial cells( IEC-6) and the potential mechanism. Methods: The cultured IEC-6 cells were observed. Four groups including Control,LPS model,LPS+GA and GA groups were set up in this study. Western blot was used to detect the protein expression levels of TLR4,CD14,MyD88,IκBα,p-IκBα,NF-κB p65 and its downstream genes; RT-qPCR was used to measure the expression of mRNA. Results: Compared with the Control group,LPS significantly augmented the expression of TLR4,CD14,MyD88,phosphorylation of IκBα and the nuclear translocaton of NF-κB p65 in the intestinal epithelial cells( P<0. 05); It also up-regulated the expression of ICAM-1,COX-2,i NOS and IL-6,meanwhile down-regulated the expression of IL-10( P<0. 05).Glycyrrhizic acid could significantly reverse the above changes,the differences were statistically significant( P < 0. 05). Conclusion: Glycyrrhizic acid can inhibit LPS-activated TLR4/NF-κB signaling pathway and down-regulate pro-inflammatory cytokines,reducing the inflammatory injury in intestinal epithelium cells.
Objective: To investigate the protective effect of glycyrrhizic acid( GA) on lipopolysaccharide( LPS) induced inflammation of the intestinal epithelial cells( IEC-6) and the potential mechanism. Methods: The cultured IEC-6 cells were observed. Four groups including Control,LPS model,LPS+GA and GA groups were set up in this study. Western blot was used to detect the protein expression levels of TLR4,CD14,MyD88,IκBα,p-IκBα,NF-κB p65 and its downstream genes; RT-qPCR was used to measure the expression of mRNA. Results: Compared with the Control group,LPS significantly augmented the expression of TLR4,CD14,MyD88,phosphorylation of IκBα and the nuclear translocaton of NF-κB p65 in the intestinal epithelial cells( P<0. 05); It also up-regulated the expression of ICAM-1,COX-2,i NOS and IL-6,meanwhile down-regulated the expression of IL-10( P<0. 05).Glycyrrhizic acid could significantly reverse the above changes,the differences were statistically significant( P < 0. 05). Conclusion: Glycyrrhizic acid can inhibit LPS-activated TLR4/NF-κB signaling pathway and down-regulate pro-inflammatory cytokines,reducing the inflammatory injury in intestinal epithelium cells.
引文
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[13] Wang XR,Hao HG,Lan C. Glycyrrhizin inhibits LPS-induced inflammatory mediator production in endometrial epithelial cells[J].Microb Pathog,2017,109(8):110-113.
[14] Wang CY,Kao TC,Lo WH,et al. Glycyrrhizic acid and 18β-glycyrrhetinic acid modulate lipopolysaccharide-induced inflammatory response by suppression of NF-κB through PI3K p110δand p110γinhibitions[J]. J Agric Food Chem,2011,59(14):7726-7733.
[15] Wu CX,He LX,Guo H,et al. Inhibition effect of glycyrrhizin in lipopolysaccharide-induced high-mobility group box 1 releasing and expression from RAW264. 7 cells[J]. Shock,2015,43(4):412-421.
[16] Barakat W,Safwet N,El-Maraghy NN,et al. Candesartan and glycyrrhizin ameliorate ischemic brain damage through downregulation of the TLR signaling cascade[J]. Eur J Pharmacol,2014,724(3):43-50.
[2] Honda H,Nagai Y,Matsunaga T,et al. Glycyrrhizin and isoliquiritigenin suppress the LPS sensor Toll-like receptor 4/MD-2 complex signaling in a different manner[J]. J Leukoc Biol,2012,91(6):967-976.
[3] Schr felbauer B,Raffetseder J,Hauner M,et al. Glycyrrhizin,the main active compound in liquorice,attenuates pro-inflammatory responses by interfering with membrane-dependent receptor signalling[J].Biochem J,2009,421(3):473-482.
[4]张娴,黄羽,曾星.甘草酸对LPS刺激的小肠上皮细胞炎症因子表达的影响[J].医药前沿,2012,2(16):100-101.Zhang X,Huang Y,Zeng X. Effect of glycyrrhizic acid on inflammatory factors in LPS-stimulated IEC-6 cells[J]. Med Frontier,2012,2(16):100-101.
[5] Nagai Y,Akashi S,Nagafuku M,et al. Essential role of MD-2 in LPS responsiveness and TLR4 distribution[J]. Nature Immunol,2002,3(7):667-672.
[6] Póciennikowska A,Hromada-Judycka A,Borz cka K,et al. Co-operation of TLR4 and raft proteins in LPS-induced pro-inflammatory signaling[J].Cell Mol Life Sci,2015,72(3):557-881.
[7] Chaturvedi MM,Sung B,Yadav VR,et al. NF-κB addiction and its role in cancer:"one size does not fit all"[J]. Oncogene,2010,30(14):1615-1630.
[8] Wang YM,Du GQ. Glycyrrhizic acid prevents enteritis through reduction of NF-κB p65 and p38MAPK expression in rat[J]. Mol Med Rep,2016,13(4):3639-3646.
[9] Jobin C,Hellerbrand C,Licato LL,et al. Mediation by NF-kappa B of cytokine induced expression of intercellular adhesion molecule 1(ICAM-1)in an intestinal epithelial cell line,a process blocked by proteasome inhibitors[J].Gut,1998,42(6):779-787.
[10] Wang ZH,Hsieh CH,Liu WH,et al. Glycyrrhizic acid attenuated glycative stress in kidney of diabetic mice through enhancing glyoxalase pathway[J]. Mol Nutr Food Res,2014,58(7):1426-1435.
[11] Bendjelloul F,Mal P,Mandys V,et al. Intercellular adhesion molecule-1(ICAM-1)deficiency protects mice against severe forms of experimentally induced colitis[J]. Clin Exp Immunol,2000,119(1):57-63.
[12] Zhao H,Zhao M,Wang Y,et al. Glycyrrhizic acid attenuates sepsis-induced acute kidney injury by inhibiting NF-κB signaling pathway[J].Evid Based Complement Alternat Med,2016,2016(1):8219287.
[13] Wang XR,Hao HG,Lan C. Glycyrrhizin inhibits LPS-induced inflammatory mediator production in endometrial epithelial cells[J].Microb Pathog,2017,109(8):110-113.
[14] Wang CY,Kao TC,Lo WH,et al. Glycyrrhizic acid and 18β-glycyrrhetinic acid modulate lipopolysaccharide-induced inflammatory response by suppression of NF-κB through PI3K p110δand p110γinhibitions[J]. J Agric Food Chem,2011,59(14):7726-7733.
[15] Wu CX,He LX,Guo H,et al. Inhibition effect of glycyrrhizin in lipopolysaccharide-induced high-mobility group box 1 releasing and expression from RAW264. 7 cells[J]. Shock,2015,43(4):412-421.
[16] Barakat W,Safwet N,El-Maraghy NN,et al. Candesartan and glycyrrhizin ameliorate ischemic brain damage through downregulation of the TLR signaling cascade[J]. Eur J Pharmacol,2014,724(3):43-50.
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