摘要:
探讨柚叶总黄酮(PLTFE)对脂多糖(LPS,2 μg/mL)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、150 μg/mL)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,qRT-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mRNA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mRNA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mRNA转录,抑制MLCK的mRNA转录,改善细胞间高通透性qRT-PCR法检测细胞中相关因子的mRNA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mRNA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)。
Abstract:
To investigate the protective effect of total flavonoid extracts from pomelo levels (PLTFE) on lipopolysaccharide (LPS, 2 g/mL) induced hypepermeability in Caco-2 cells. The model cells were treated with different concentrations (0、10、50、100、150 μg/mL) 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 mRNA transcription in LPS treated Caco-2 cells (p<0.05). At the same time, it could effectively inhibit the secretion of inflammatory cytokines (IL-1β, IL-8, TNF-α) and mRNA transcription in damaged cells. In addition, PLTFE also significantly increased the mRNA levels of Occludin, claudin-1, ZO-1, and inhibited the MLCK mRNA 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 mRNA transcription of tight junction related factors.
百度学术
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