柚叶总多酚对LPS所致Caco-2细胞间高通透性的保护作用

Protective Effect of Total Polyphenolic Extracts from Pomelo Levels on LPS-induced Intercellular Hyperpermeability in Caco-2 Cells

摘要:

本文探讨了柚叶总多酚对脂多糖(LPS，2 μg/mL)致Caco-2细胞高通透性的保护作用。MTT法测定细胞生存率，细胞内乳酸脱氢酶(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，IL)-1β、IL-8和肿瘤坏死因子(tumor necrosis factor，TNF)-α分泌水平。跨上皮细胞电阻(trans epithelial electrieal resistanee，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)、ZO-1和肌球蛋白轻链激酶(myosin light chain kinase，MLCK)的mRNA表达。柚叶总多酚能显著提高受损细胞生存率，抑制LDH溢出，有效抑制受损细胞炎性细胞因子(IL-1β、IL-8、TNF-α)的分泌及mRNA转录。柚叶总多酚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转录从而改善细胞间高通透性。结果提示，柚叶总多酚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。

Abstract:

To investigate the protective effect of total polyphenolic extracts from pomelo levels (PLTPE) on lipopolysaccharide (LPS, 2 g/mL) induced hypepermeability in Caco-2 cells in this paper. The 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 (IL)-1β, IL-8, and tumor necrosis factor (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). PLTPE treatment significantly increased the cell survival rate and inhibited the spillover of LDH in Caco-2 cells. PLTPE treatment effectively inhibited the secretion of inflammatory cytokines (IL-1β, IL-8, TNF-α) and reduced their mRNA transcription in Caco-2 cells. At the same time, PLTPE also significantly increased the mRNA levels of occludin, claudin-1, ZO-1, and inhibit the MLCK mRNA transcription to improve the intercellular permeability in LPS treated Caco-2 cells. These results suggest that the PLTPE showed a strong anti-inflammatory activity, and can improve the LPS induced high permeability of Caco-2 cells may associate with regulating the mRNA transcription of tight junction related factors.