石蒜碱可能通过调控NF-κB通路抑制TNF-α诱导的肠上皮细胞损伤

doi:10.3969/j.issn.1673-8640.2026.07.010

摘要/Abstract

摘要：

目的探讨石蒜碱（Lyc）调控核因子-κB（NF-κB）通路抑制肿瘤坏死因子-α（TNF-α）诱导肠上皮细胞损伤的机制。方法选取人结直肠腺癌细胞系Caco2和人正常肠上皮细胞系NCM460，根据处理方式分为对照组（不做任何处理）、Lyc组（仅用Lyc处理）、TNF-α组（仅用TNF-α处理）和TNF-α+Lyc组（先用TNF-α处理，再用Lyc处理）。采用CCK-8试验评估不同剂量TNF-α和Lyc对Caco2细胞和NCM460细胞活力的影响，以确定适宜实验浓度。采用实时荧光定量聚合酶链反应检测各组白细胞介素（IL）-6、诱导型-氮化氮合酶（iNOS）、环氧合酶-2（COX-2）和IL-1β基因表达。采用免疫荧光法和免疫印迹法检测各组闭锁小带蛋白1（ZO-1）、闭合蛋白（OCLN）和紧密连接蛋白1（CLDN-1）表达。采用Calcein/PI染色检测细胞凋亡情况。采用免疫印迹法检测各组凋亡调控蛋白（Bax、Bcl2）和NF-κB通路蛋白[核因子κB抑制蛋白α（IκBα）、磷酸化核因子κB抑制蛋白α（p-IκBα）、核因子-κB p65和磷酸化核因子-κB p65（p-NF-κB p65）]的表达。结果在Caco2细胞和NCM460细胞中，与对照组和Lyc组比较，TNF-α组IL-6 mRNA、iNOS mRNA、COX2 mRNA和IL-1β mRNA相对表达量均显著升高（P<0.05），ZO-1、OCLN和CLDN-1蛋白表达量均显著降低（P<0.05）；与TNF-α组比较，TNF-α+Lyc组IL-6 mRNA、iNOS mRNA、COX2 mRNA和IL-1β mRNA相对表达量均显著降低（P<0.05），ZO-1、OCLN和CLDN-1蛋白表达量均显著升高（P<0.05）。TNF-α组PI阳性细胞数显著多于对照组（P<0.05）；TNF-α+Lyc组PI阳性细胞数显著少于TNF-α组（P<0.05），但仍多于对照组（P<0.05）。TNF-α组Bax蛋白表达显著高于对照组和Lyc组（P<0.05），Bcl2蛋白表达显著低于对照组和Lyc组（P<0.05）；TNF-α+Lyc组Bax蛋白表达低于TNF-α组（P<0.05），Bcl2蛋白表达高于TNF-α组（P<0.05）。TNF-α组p-IκBα/IκBα比值和p-NF-κB/NF-κB比值均显著高于对照组（P<0.05）；TNF-α+Lyc组p-IκBα/IκBα比值和p-NF-κB/NF-κB比值均显著低于TNF-α组（P<0.05），但仍高于对照组（P<0.05）。结论 Lyc可能通过调控NF-κB通路抑制TNF-α诱导的肠上皮细胞损伤。

关键词: 石蒜碱, 肿瘤坏死因子-ɑ, Caco2细胞, NCM460细胞, 炎症性肠病

Abstract:

Objective To investigate the mechanism by which lycorine（Lyc） regulates nuclear factor-kappa B（NF-κB） pathway and inhibits tumor necrosis factor-alpha（TNF-α）-induced intestinal epithelial cell damage. Methods Human colorectal adenocarcinoma cell line Caco2 and human normal intestinal epithelial cell line NCM460 were selected. They were classified into control group（no treatment），Lyc group（treated with Lyc only），TNF-α group（treated with TNF-α only） and TNF-α+Lyc group（treated with TNF-α first and then Lyc）. The effects of different concentrations of TNF-α and Lyc on the viability of Caco2 cells and NCM460 cells were evaluated by CCK-8 assay to determine the appropriate experimental concentrations. The expressions of interleukin（IL）-6，inducible nitric oxide synthase（iNOS），cyclooxygenase-2（COX-2）and IL-1β in each group were determined by real-time fluorescence quantitative polymerase chain reaction. The expressions of zonula occludens-1（ZO-1），occludin（OCLN） and claudin-1（CLDN-1） in each group were determined by immunofluorescence and immunoblotting. The apoptosis of cells was determined by Calcein/PI staining. The expressions of Bax protein，Bcl2 protein and inhibitor of kappa B alpha（IκBα），phosphorylated inhibitor of kappa B alpha（p-IκBα），phosphorylated nuclear factor-kappa B p65（p-NF-κB p65）in each group were determined by immunoblotting. Results Compared with control group and Lyc group，the relative expression levels of IL-6 mRNA，iNOS mRNA，COX2 mRNA and IL-1β mRNA in TNF-α group were increased（P<0.05），and the protein expression levels of ZO-1，OCLN and CLDN-1 were decreased（P<0.05）. Compared with TNF-α group，the relative expression levels of IL-6 mRNA，iNOS mRNA，COX2 mRNA and IL-1β mRNA in TNF-α+Lyc group were decreased（P<0.05），and the protein expression levels of ZO-1，OCLN and CLDN-1 were increased（P<0.05）. The number of PI-positive cells in TNF-α group was higher than that in control group（P<0.05），and the number of PI-positive cells in TNF-α+Lyc group was lower than that in TNF-α group（P<0.05），but still higher than that in control group（P<0.05）. The expression of Bax protein in TNF-α group was higher than those in control group and Lyc group（P<0.05），and the expression of Bcl2 protein was lower than those in control and Lyc group（P<0.05）. The expression of Bax protein in TNF-α+Lyc group was lower than that in TNF-α group（P<0.05），and the expression of Bcl2 protein was higher than that in TNF-α group（P<0.05）. The p-IκBα/IκBα ratio and p-NF-κB/NF-κB ratio in TNF-α group were higher than those in control group（P<0.05）. The ratios of p-IκBα/IκBα and p-NF-κB/NF-κB in TNF-α+Lyc group were lower than those in TNF-α group（P<0.05），but still higher than those in control group（P<0.05）. Conclusions Lyc may inhibit TNF-α-induced intestinal epithelial cell damage by regulating NF-κB pathway.

Key words: Lycorine, Tumor necrosis factor-alpha, Caco2 cell, NCM460 cell, Inflammatory bowel disease

中图分类号:

Q946.88

程阳, 冯一鸣, 金姝, 蔡骁垚, 胡奕雯, 闫佩毅. 石蒜碱可能通过调控NF-κB通路抑制TNF-α诱导的肠上皮细胞损伤[J]. 检验医学, 2026, 41(7): 676-684.

CHENG Yang, FENG Yiming, JIN Shu, CAI Xiaoyao, HU Yiwen, YAN Peiyi. Role of lycorine in regulating NF-κB pathway to inhibit TNF-α-induced intestinal epithelial cell damage possibly[J]. Laboratory Medicine, 2026, 41(7): 676-684.

图/表 8

表1 引物序列

基因名称	正向引物（5'~3'）	反向引物（5'~3'）
IL-6	ACTCACCTCTTCAGAACGAATTG	CCATCTTTGGAAGGTTCAGGTTG
iNOS	TTCAGTATCACAACCTCAGCAAG	TGGACCTGCAAGTTAAAATCCC
COX-2	CTGGCGCTCAGCCATACAG	CGCACTTATACTGGTCAAATCCC
IL-1β	ATGATGGCTTATTACAGTGGCAA	GTCGGAGATTCGTAGCTGGA
β-actin	GTGACGTTGACATCCGTAAAGA	GCCGGACTCATCGTACTCC

图1 不同TNF-α浓度处理24、48、72 h对Caco2细胞和NCM460细胞存活率的影响注：（a）Caco2细胞；（b）NCM460细胞；处理24 h，处理48 h，处理72 h。

图2 不同浓度Lyc处理24 h对Caco2细胞和NCM460细胞存活率的影响注：（a）Caco2细胞；（b）NCM460细胞；与0 μmol·L-1比较，*P<0.05。

图3 4个组IL-6 mRNA、iNOS mRNA、COX2 mRNA和IL-1β mRNA相对表达量比较注：（a）Caco2细胞；（b）NCM460细胞；对照组， Lyc组， TNF-α组， TNF-α+Lyc组；与对照组比较，*P<0.05；与 TNF-α组比较，#P<0.05。

图4 Caco2细胞各组ZO-1、OCLN和CLDN-1表达比较注：（a）免疫荧光法检测Caco2细胞ZO-1的表达；（b）免疫荧光法检测Caco2细胞OCLN的表达；（c）免疫荧光法检测Caco2细胞CLDN-1的表达；（d）各组ZO-1、OCLN和CLDN-1表达的相对MFI值；（e）ZO-1、OCLN和CLDN-1表达的免疫印迹图；（f）各组ZO-1、OCLN和CLDN-1的蛋白相对表达量；对照组， Lyc组， TNF-α组， TNF-α+Lyc组；与对照组比较，*P<0.05；与TNF-α组比较，#P<0.05。

图5 NCM460细胞各组ZO-1、OCLN和CLDN-1表达比较注：（a）免疫荧光法检测NCM460细胞ZO-1的表达；（b）免疫荧光法检测NCM460细胞OCLN的表达；（c）免疫荧光法检测NCM460细胞CLDN-1的表达；（d）各组ZO-1、OCLN和CLDN-1表达的相对MFI值；（e）ZO-1、OCLN和CLDN-1表达的免疫印迹图；（f）各组ZO-1、OCLN和CLDN-1的蛋白相对表达量；对照组， Lyc组， TNF-α组， TNF-α+Lyc组；与对照组比较，*P<0.05；与 TNF-α组比较，#P<0.05。

图6 Lyc对TNF-α诱导的细胞凋亡的影响注：（a）Caco2细胞和NCM460细胞的Calcein/PI染色；（b）Caco2细胞各组PI阳性细胞比例比较；（c）NCM460细胞各组PI阳性细胞比例比较；（d）Caco2细胞和NCM460细胞Bax和Bcl2蛋白表达的免疫印迹图；（e）Caco2细胞各组Bax和Bcl2蛋白相对表达量比较；（f）NCM460细胞各组Bax和Bcl2蛋白相对表达量比较；对照组， Lyc组， TNF-α组， TNF-α+Lyc组；与对照组比较，*P<0.05；与 TNF-α组比较，#P<0.05。

图7 Caco2细胞和NCM460细胞各组NF-κB通路蛋白表达比较注：（a）Caco2细胞和NCM460细胞各组p-IκBα、IκBα、p-NF-κB p65和NF-κB p65蛋白表达的免疫印迹图；（b）Caco2细胞各组p-IκBα/IκBα比值、p-NF-κB p65/NF-κB p65比值比较；（c）Caco2细胞各组p-IκBα/IκBα比值、p-NF-κB p65/NF-κB p65比值比较；与对照组比较，*P<0.05；与TNF-α组比较，#P<0.05。

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