青蒿素减轻LPS诱导的肠上皮细胞屏障功能损伤的实验研究

摘要

[ ABSTRACT] AIM:To investigate the effect of artemisinin on lipopolysaccharide ( LPS)-induced intestinal epi-thelial barrier damage in IEC-6 cells and its molecular mechanism.METHODS:Cultured IEC-6 cells were divided to 5 groups:control group, LPS (100 mg/L) group and LPS +Artemisinin (30, 50 and 100μmol/L) groups.The cytotoxici-ty was detected by MTT assay.The releases of TNF-α, IL-1βand IL-6 in the IEC-6 cells were measured by ELISA.The transepithelial electrical resistance ( TER) was detected by electrical resistance tester, and the horseradish peroxidase (HRP) flux permeability were analyzed by a microplate reader.The expression of tight junction proteins, ZO-1, claudin-1 and occludin, and the expression of TLR4/MyD88/NF-κB at mRNA and protein levels were determined by RT-qPCR and Western blot.RESULTS:Artemisinin alone (up to 100 μmol/L) or in combination with LPS (100 mg/L) was not toxic to IEC-6 cells.Compared with control group, the releases of TNF-α, IL-1βand IL-6 in the culture supernatant of IEC-6 cells significantly increased after treatment with LPS.The expression of TLR4/MyD88/NF-κB was activated by LPS.LPS down-regulated the protein expression of ZO-1, claudin-1 and occludin.However, artemisinin treatment decreased the re-leases of TNF-α, IL-1βand IL-6 in the culture supernatant of IEC-6 cells.The expression of TLR4/MyD88/NF-κB at mR-NA and protein levels was gradually reduced after treatment with artemisinin.In addition, artemisinin upregulated the pro-tein expression of ZO-1, claudin-1 and occludin significantly (P<0.01) in a dose-dependent manner.CONCLUSION:Artemisinin attenuates LPS-induced intestinal epithelial barrier damage by inhibiting TLR4/MyD88/NF-κB activation in the IEC-6 cells.%目的：探究青蒿素对脂多糖（ lipopolysaccharide， LPS）诱导的大鼠肠上皮IEC－6细胞屏障功能损伤的影响。方法：体外培养IEC－6细胞，随机分为5组：对照组、LPS （100 mg／L ）组和LPS＋青蒿素（30、50和100μmol／L）组，MTT法检测各组细胞毒性变化，ELISA检测各组细胞分泌炎性因子TNF－α、IL－1β和IL－6水平的变化，电阻仪检测肠上皮细胞跨上皮电阻（TER），酶标仪检测单层细胞对辣根过氧化物酶（HRP）的通透性，RT－qPCR和Western blot检测各组细胞紧密连接蛋白（ ZO－1、claudin－1和occludin）以及TLR4／MyD88／NF－κB mRNA和蛋白表达的变化。结果：LPS与青蒿素在本实验浓度范围对IEC－6细胞均无毒性。与对照组相比， LPS处理下，细胞分泌TNF－α、IL－1β和IL－6水平以及TLR4／MyD88／NF－κB的mRNA和蛋白表达明显增加，ZO－1、claudin－1和occludin的mRNA和蛋白表达降低。而青蒿素干预下，细胞分泌TNF－α、IL－1β和IL－6水平以及TLR4／MyD88／NF－κB mRNA和蛋白表达明显降低，ZO－1、claudin－1和occludin的mRNA和蛋白表达升高（P＜0．05），均呈现浓度依赖性。结论：青蒿素可能通过抑制TLR4／MyD88／NF－κB通路减轻LPS诱导的肠上皮细胞屏障功能损伤。