辣木多肽对LPS所致Caco-2细胞间高通透性的保护作用

Protection of LPS-induced Intercellular Hyperpermeability in Caco-2 Cells by Polypeptide from Moringa oleifera

摘要:

探讨了辣木多肽(MOPP)对脂多糖(LPS，2 μg/mL)诱发Caco-2细胞高通透性的保护作用。MTT法测定细胞生存率，细胞乳酸脱氢酶(LDH)水平依说明用试剂盒测定。酶联法(ELISA)检测白介素-1β (IL-1β)、IL-8和肿瘤坏死因子(TNF-α)的分泌水平。跨上皮细胞电阻(TEER)值和异硫氰酸荧光素-右旋糖酐(FD40)透过度评估细胞通透的改变。实时定量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(MLCK)的mRNA表达。与模型细胞相比，高浓度(150 μg/mL)的MOPP处理能显著提高受损细胞生存率至83.8%，并有效抑制50.3%的LDH溢出。MOPP有效降低模型细胞中IL-1β (19.8%)，IL-8 (43.7%)和TNF-α (37.9%)的分泌并抑制其mRNA转录(IL-1β: 44.3%，IL-8: 35.0%，TNF-α: 33.5%)。此外，MOPP可增强细胞紧密连接因子(Occludin、claudin-1、ZO-1)的mRNA转录，并抑制31.3%的MLCK的mRNA转录。结果意味着，MOPP具有较强的抗炎活性，可由上调细胞内紧密连接相关因子的mRNA转录显著来改善LPS所致Caco-2细胞间高通透性的发生。

Abstract:

The protective effect of polypeptide from Moringa oleifera (MOPP) on lipopolysaccharide (LPS, 2 μg/mL) induced hypepermeability in Caco-2 cells was investigated. 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 Interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α. The transepithelialelectricalresistance (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). A high concentration of MOPP (150 μg/mL) treatment significantly increased the cell survival rate (to 83.8%) and inhibited the spillover of LDH (50.3%) compared with that in model cells. MOPP treatment effectively inhibited the secretion of inflammatory cytokines (IL-1β: 19.8%, IL-8: 43.7% and TNF-α: 37.9%) and reduced their mRNA transcription (IL-1β: 44.3%，IL-8: 35.0% and TNF-α: 33.5%)in model cells. In addition, MOPP also significantly increased the mRNA levels of Occludin, claudin-1, ZO-1, and inhibit the 31.3% of MLCK mRNA transcription to improve the intercellular permeability in LPS treated Caco-2 cells. These results suggest that the MOPP exhibited 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.