甘草黄酮亚组分及甘草黄酮C对TNF-α致肠上皮屏障损伤的保护作用研究  

Protective Effect of Subcomponent GCH15 and Licoflavone C Separated from Flavones of Glycyrrhiza inflata Against TNF-α-induced Intestinal Epithelial Barrier Injury

作　　者：张娟 孙宇 徐晓琴 卿德刚 ZHANG Juan;SUN Yu;XU Xiao-qin;QING De-gang(Xinjiang Institute of Chinese Materia Medica and Ethnodrug,Urumqi 830002,China)

机构地区：[1]新疆维吾尔自治区中药民族药研究所,新疆乌鲁木齐830002

出　　处：《中国现代中药》2021年第10期1710-1716,共7页Modern Chinese Medicine

基　　金：国家自然科学基金项目(81660654);天山青年计划项目(2017Q066)。

摘　　要：目的:从甘草黄酮中筛选活性亚组分及黄酮单体,考察其对肿瘤坏死因子-α(TNF-α)诱导的肠上皮屏障损伤的保护作用。方法:以硅胶柱色谱法制备甘草黄酮亚组分;体外培养肠上皮细胞IEC-6并以TNF-α诱导炎症反应,分别以甘草黄酮亚组分(20μg·mL^(-1))提前干预,CCK-8法检测正常细胞存活率,酶联免疫吸附分析(ELISA)检测炎症细胞白细胞介素-6(IL-6)分泌水平,获得活性亚组分;高效液相色谱法(HPLC)确认活性亚组分的主要成分,进而以TNF-α诱导的Caco-2单层细胞肠道屏障损伤模型分别考察活性亚组分GCH15(5、10、20μg·mL^(-1))和甘草黄酮C(LicoC,5、10、20μmol·mL^(-1))对肠道屏障的保护作用,ELISA检测IL-6和IL-8分泌水平,电阻仪检测单层细胞跨上皮电阻(TEER),以异硫氰酸荧光素-右旋糖酐(FD-40)相对通过水平评估细胞旁通透性,免疫印迹法(Westernblot)检测闭合小环蛋白-1(ZO-1)、咬合蛋白(Occludin)和肌球蛋白轻链激酶(MLCK)表达。结果:制备得到甘草黄酮亚组分GCH1~GCH20;GCH2、GCH4、GCH8、GCH15和GCH1920μg·mL^(-1)均可显著抑制IL-6分泌(P<0.05),且以GCH15的细胞毒性最小;通过HPLC确认了GCH15的主要成分是LicoC;以TNF-α刺激Caco-2细胞,可明显促进IL-6和IL-8分泌(P<0.01),降低TEER值(P<0.01),提高FD-40相对通过水平(P<0.01),下调ZO-1和Occludin表达(P<0.01),促进MLCK表达(P<0.01)。以GCH15和LicoC提前干预则可抑制IL-6和IL-8分泌(P<0.01),增加Caco-2单层细胞TEER值(P<0.01),降低FD-40相对通过水平(P<0.01),并呈一定的剂量相关性,说明两者可以逆转TNF-α引起的炎症反应及细胞屏障功能紊乱。Westernblot结果表明,GCH1520μg·mL^(-1)和LicoC 20μmol·mL^(-1)均能促进ZO-1和Occludin表达(P<0.05,P<0.01),抑制MLCK表达(P<0.01)。结论:GCH15和LicoC均可明显缓解TNF-α引起的Caco-2细胞炎症反应及屏障功能紊乱,两者对屏障功能的调节作用可能是通过调控MLCK通路,进而促进紧密连接蛋白表�Objective:To screen the active subcomponent and monomer from flavones in Glycyrrhiza inflata Bat.and investigate the protective effect against TNF-α-induced intestinal epithelial barrier injury.Methods:IEC-6 cells were cultured in vitro and inflammatory response was induced by TNF-α.The subcomponents of flavones in G.inflata Bat.(20μg·mL^(-1)),prepared by silica gel column chromatography,were used for early intervention.The survival rate of normal cells was measured by CCK-8 assay and the level of interleukin-6(IL-6)of inflammatory cells was detected by enzyme linked immunosorbent assay(ELISA).On this basis,the active subcomponents were screened out and the major subcomponent of GCH15 was identified by high performance liquid chromatography(HPLC).Caco-2 cell monolayer injury was induced by TNF-α,and the protective effect of GCH15(5,10,20μg·mL^(-1))and licoflavone C(LicoC,5,10,20μmol·mL^(-1))was investigated,separately.The levels of IL-6 and IL-8 were detected by ELISA,and the transepithelial electrical resistance(TEER)of cell monolayer by a resistor.The flux of fluorescein isothiocyanate-dextran(FD-40)was used to evaluate the permeability of cell monolayer,and expression of zonula occluden-1(ZO-1),Occludin,and myosin light-chain kinase(MLCK)was determined by Western blot.Results:IEC-6 cells stimulated by TNF-αwere pretreated with 20 subcomponents(GCH1-GCH20).GCH2,GCH4,GCH8,GCH15,and GCH19(20μg·mL^(-1))inhibited the secretion of IL-6 induced by TNF-α(P<0.05),and GCH15 showed the least cytotoxicity.Then LicoC was identified as the main component of GCH15 by HPLC.TNF-αpromoted the secretion of IL-6 and IL-8(P<0.01),decreased the TEER value(P<0.01),increased the FD-40 flux(P<0.01),reduced the expression of ZO-1 and Occludin(P<0.01),and enhanced the expression of MLCK(P<0.01)in Caco-2 cells.Pretreatment with GCH15 and LicoC significantly reversed the inflammatory response and barrier dysfunction of Caco-2 monolayers induced by TNF-α,as evidenced by the decreased IL-6 and IL-8(P<0.01),increased TEER valu

关 键 词：甘草 黄酮 甘草黄酮C 抗炎 肠上皮屏障 紧密连接 

分 类 号：R285.5[医药卫生—中药学]