柚叶总黄酮对LPS所致Caco-2结肠上皮细胞间高通透性的保护作用
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摘要
探讨柚叶总黄酮(PLTFE)对脂多糖(LPS,2μg/m L)诱发Caco-2高通透性细胞模型的保护作用。Caco-2模型细胞以不同浓度的PLTFE(0、10、50、100、150μg/m L)处理培养24 h进行后续实验。MTT法测定细胞生存率,细胞乳酸脱氢酶(lactate dehydrogenase,LDH)水平依说明书使用试剂盒测定。酶联法(enzyme linked immunosorbent assay,ELISA)测定白介素(interlukin-1β,IL-1β)、IL-8和肿瘤坏死因子(tumor necrosis factor-α,TNF-α)分泌水平。跨上皮细胞电阻(trans epithelial electrical resistance,TEER)值和异硫氰酸荧光素-右旋糖酐(FD40)透过度用于评估细胞通透性水平。实时定量PCR(Quantitative real-time PCR,q RT-PCR)检测细胞IL-1β、IL-8、TNF-α、闭锁蛋白(Occludin)、紧密连接蛋白-1(claudin-1)、封闭小带蛋白(ZO-1)和肌球蛋白轻链激酶(myosin light chain kinase,MLCK)的m RNA表达。结果表明,柚叶总黄酮能显著提高受损细胞生存率至86.1%,抑制受损细胞中LDH的溢出(p <0.05)。同时还能有效抑制受损细胞中炎性细胞因子(IL-1β、IL-8、TNF-α)的分泌及m RNA转录。此外,柚叶总黄酮可增强细胞紧密连接因子(Occludin、claudin-1、ZO-1)的m RNA转录,抑制MLCK的m RNA转录,改善细胞间高通透性q RT-PCR法检测细胞中相关因子的m RNA转录水平。结果提示,柚叶总黄酮具有较强的抗炎活性,能通过上调细胞内细胞紧密连接相关因子的m RNA转录显著改善LPS造成的Caco-2细胞间高通透性的发生(p <0.05)。
To investigate the protective effect of total flavonoid extracts from pomelo levels( PLTFE) on lipopolysaccharide( LPS,2 g/m L) induced hypepermeability in Caco-2 cells.The model cells were treated with different concentrations( 0、10、50、100、150 μg/m L) of PLTFE for 24 h.Following this treatment,cell survival rate was measured by MTT assay.The cellular level of lactate dehydrogenase( LDH) was determined by kit.Enzyme linked immunosorbent assay( ELISA) was used to determine the level of Interlukin-1β( IL-1β),IL-8,and tumor necrosis factor-α( TNF-α). The transepithelial cell resistance( TEER) and fluorescein dextran( FD40) permeability were used to evaluate the permeability of cells. Quantitative real-time PCR( qRT-PCR) was used to detect the expression of IL-1β,IL-8,TNF-α,Occludin,claudin-1,ZO-1,and myosin light chain kinase( MLCK).PLTFE treatment significantly increased the cell survival rate( to 86.1%) and inhibited the spillover of LDH in LPS treated Caco-2 cells( p < 0.05).PLTFE treatment effectively inhibited the secretion of inflammatory cytokines( IL-1β,IL-8,TNF-α) and reduced their m RNA transcription in LPS treated Caco-2 cells( p < 0.05).At the same time,it could effectivelyinhibit the secretion of inflammatory cytokines( IL-1β,IL-8,TNF-α) and m RNA transcription in damaged cells.In addition,PLTFE also significantly increased the m RNA levels of Occludin,claudin-1,ZO-1,and inhibited the MLCK m RNA transcription to improve the intercellular permeability in LPS treated Caco-2 cells( p < 0.05). These results suggest that the PLTFE showed a strong anti-inflammatory activity,and could improve the LPS induced high permeability of Caco-2 cells associated with regulating the m RNA transcription of tight junction related factors.
To investigate the protective effect of total flavonoid extracts from pomelo levels( PLTFE) on lipopolysaccharide( LPS,2 g/m L) induced hypepermeability in Caco-2 cells.The model cells were treated with different concentrations( 0、10、50、100、150 μg/m L) of PLTFE for 24 h.Following this treatment,cell survival rate was measured by MTT assay.The cellular level of lactate dehydrogenase( LDH) was determined by kit.Enzyme linked immunosorbent assay( ELISA) was used to determine the level of Interlukin-1β( IL-1β),IL-8,and tumor necrosis factor-α( TNF-α). The transepithelial cell resistance( TEER) and fluorescein dextran( FD40) permeability were used to evaluate the permeability of cells. Quantitative real-time PCR( qRT-PCR) was used to detect the expression of IL-1β,IL-8,TNF-α,Occludin,claudin-1,ZO-1,and myosin light chain kinase( MLCK).PLTFE treatment significantly increased the cell survival rate( to 86.1%) and inhibited the spillover of LDH in LPS treated Caco-2 cells( p < 0.05).PLTFE treatment effectively inhibited the secretion of inflammatory cytokines( IL-1β,IL-8,TNF-α) and reduced their m RNA transcription in LPS treated Caco-2 cells( p < 0.05).At the same time,it could effectivelyinhibit the secretion of inflammatory cytokines( IL-1β,IL-8,TNF-α) and m RNA transcription in damaged cells.In addition,PLTFE also significantly increased the m RNA levels of Occludin,claudin-1,ZO-1,and inhibited the MLCK m RNA transcription to improve the intercellular permeability in LPS treated Caco-2 cells( p < 0.05). These results suggest that the PLTFE showed a strong anti-inflammatory activity,and could improve the LPS induced high permeability of Caco-2 cells associated with regulating the m RNA transcription of tight junction related factors.
引文
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[19]胡玥,吕宾.应激对肠上皮紧密连接功能的影响[J].生理科学进展,2017,48(2):113-116.
[20]马军宏,于向阳,张楠,等.紧密连接蛋白与肠黏膜屏障损伤研究进展[J].中国中西医结合外科杂志,2015,21(1):104-105.
[21]Shen Y,Zhou M,Yan J,et al.mi R-200b inhibits TNFα-induced IL-8 secretion and tight junction disruption of intestinal epithelial cells in vitro[J]. American Journal of Physiology Gastrointestinal and Liver Physiology,2016,312(2):G123-G132.
[22]丁少桢,丁浩,梅俏,等.同型半胱氨酸调控MEK-ERKMLCK通路影响结肠炎大鼠肠黏膜通透性的实验研究[J].中国药理学通报,2016,32(4):498-502.
[2]尤新.植物多酚黄酮抗氧化剂与人体健康[J].食品与生物技术学报,2011,30(4):481-488.
[3]郑亚美,任娇艳,史传超.蜜柚叶黄酮的提取及其抗氧化与降尿酸活性研究[J].食品工业科技,2017,38(8):262-266,271.
[4]黄友秋,赵雪荔,陈席,等.HPLC-DAD法测定通贤柚叶中两种活性黄酮的含量[J].食品工业科技,2015,36(7):286-289.
[5]Kaplan G G,Ng S C.Understanding and preventing the global increase of inflammatory bowel disease[J]. Gastroenterology,2017,152(2):313-321.
[6]马志杰,王爱民.胃肠道疾病与紧密连接蛋白关系的研究进展[J].中华实验外科杂志,2017,34(2):358-360.
[7]Liu T C,Stappenbeck T S. Genetics and pathogenesis of inflammatory bowel disease[J].Annual Reviewa Pathology.2016,11(1):127-148.
[8]Greeenhill C. IBD:Glucocorticoids revealed to augment intestinal epithelial barrier function[J]. Nature Reviews Gastroenterology&Hepatology,2014,11(2):75.
[9]Vlantis K,Polykratis A,Welz P S,et al.Original article:TLR-independent anti-inflammatory function of intestinal epithelial TRAF6 signalling prevents DSS-induced colitis in mice[J].Gut,2016,65(6):935-943.
[10]Li B,Zani A,Martin Z,et al.Intestinal epithelial cell injury is rescued by hydrogen sulfide[J]. Journal of Pediatric Surgery,2016,51(5):775-778.
[11]孙钦娟,李琴,宛东,等.细胞因子在炎症性肠病患者肠黏膜中表达的分析[J].胃肠病学,2018,23(1):13-17.
[12]唐齐林,周国华,王为.TNF-α、IL-6在炎症性肠病发病机制中的研究进展[J].医学综述,2014,20(7):1174-1176.
[13]王艳萍,姬林松,倪猛,等.不同严重程度溃疡性结肠炎患者血清TNF-α、IL-6及IL-8的表达及意义[J].中国老年学杂志,2015,35(14):3940-3941.
[14]周勇.检测血清IL-1β、IL-6、IL-8水平对于评估溃疡性结肠炎患者病情的应用价值分析[J].中国中西医结合消化杂志,2015,23(4):286-287,290.
[15]Perinelli D R,Vllasaliu D,Bonacucina G,et al.Rhamnolipids as epithelial permeability enhancers for macromolecular therapeutics[J]. European Journal of Pharmaceutics and Biopharmaceutics,2017,119:419-425.
[16]Seike S,Takehara M,Takagishi T,et al. Delta-toxin from Clostridium perfringens perturbs intestinal epithelial barrier function in Caco-2 cell monolayers[J]. Biochim Biophys Acta,2018,1860(2):428-433.
[17]Chopyk D M,Kuma P,Raeman R,et al. Dysregulation of junctional adhesion molecule-A contributes to ethanol-induced barrier disruption in intestinal epithelial cell monolayers.[J].Physiological Reports,2017,5(23):e13541.
[18]Xu P,Becker H,Elizalde M,et al.Intestinal organoid culture model is a valuable system to study epithelial barrier function in IBD[J].Gut,2017:gutjnl-2017-315685.
[19]胡玥,吕宾.应激对肠上皮紧密连接功能的影响[J].生理科学进展,2017,48(2):113-116.
[20]马军宏,于向阳,张楠,等.紧密连接蛋白与肠黏膜屏障损伤研究进展[J].中国中西医结合外科杂志,2015,21(1):104-105.
[21]Shen Y,Zhou M,Yan J,et al.mi R-200b inhibits TNFα-induced IL-8 secretion and tight junction disruption of intestinal epithelial cells in vitro[J]. American Journal of Physiology Gastrointestinal and Liver Physiology,2016,312(2):G123-G132.
[22]丁少桢,丁浩,梅俏,等.同型半胱氨酸调控MEK-ERKMLCK通路影响结肠炎大鼠肠黏膜通透性的实验研究[J].中国药理学通报,2016,32(4):498-502.