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Effect of Glycyrrhizae Radix et Rhizoma-containing Serum on LPS-induced Inflammation in Caco2 Cells Based on Inhibition of Ferroptosis by Nrf2/HO-1 Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 16, Pages: 144-153(2023)
- 作者机构：
安徽中医药大学 药学院，中药复方安徽省重点实验室，合肥 230012
- 作者简介：
- 基金信息：
DOI：10.13422/j.cnki.syfjx.20230601
CLC： R2-0;R22;R285.5;R289;R33Published：20 August 2023，
Published Online：14 February 2023，
Received：31 October 2022，
- 稿件说明：
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孔金融,施高翔,侯静等.基于Nrf2/HO-1通路抑制铁死亡探究甘草含药血清对LPS诱导的Caco2细胞炎症的影响[J].中国实验方剂学杂志,2023,29(16):144-153.
KONG Jinrong,SHI Gaoxiang,HOU Jing,et al.Effect of Glycyrrhizae Radix et Rhizoma-containing Serum on LPS-induced Inflammation in Caco2 Cells Based on Inhibition of Ferroptosis by Nrf2/HO-1 Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(16):144-153.
孔金融,施高翔,侯静等.基于Nrf2/HO-1通路抑制铁死亡探究甘草含药血清对LPS诱导的Caco2细胞炎症的影响[J].中国实验方剂学杂志,2023,29(16):144-153. DOI： 10.13422/j.cnki.syfjx.20230601.
KONG Jinrong,SHI Gaoxiang,HOU Jing,et al.Effect of Glycyrrhizae Radix et Rhizoma-containing Serum on LPS-induced Inflammation in Caco2 Cells Based on Inhibition of Ferroptosis by Nrf2/HO-1 Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(16):144-153. DOI： 10.13422/j.cnki.syfjx.20230601.
摘要
目的
2
探讨甘草含药血清激活核因子E
2
相关因子2（Nrf2）/血红素加氧酶-1（HO-1）通路抑制铁死亡对脂多糖（LPS）诱导的人结肠上皮腺癌细胞（Caco2）炎症的影响。
方法
2
将Caco2细胞分为正常组、模型组（LPS，200 μg·L
-1
）、甘草含药血清低、中、高浓度组（5%、10%、20%）、铁死亡抑制剂组（3-氨基-4-环己基氨基苯甲酸乙酯，Fer-1，10 μmol·L
-1
）。正常组细胞正常培养，其他组建立炎症模型，甘草含药血清低、中、高浓度组分别加入5%、10%、20%含药血清处理24 h，铁死亡抑制剂组给予Fer-1处理24 h。透射电镜观察各组线粒体形态；流式细胞术检测细胞内Fe
2+
水平；微板法检测超氧化物歧化酶（SOD）活性、丙二醛（MDA）和谷胱甘肽过氧化物酶（GSH-Px）水平，酶联免疫吸附测定法（ELISA）检测白细胞介素-1
β
（IL-1
β
）、IL-6、IL-10、肿瘤坏死因子-
α
（TNF-
α
）含量；蛋白免疫印迹法（Western blot）检测Nrf2、HO-1、铁蛋白重链1（FTH1）、谷胱甘肽过氧化物酶4（GSH-Px4）蛋白的表达水平。运用小干扰核糖核酸（siRNA）的方式来观察Nrf2在铁死亡调控中的作用。将干扰后细胞分为空载体组（NC）、Si-Nrf2组、含药血清（20%）+Si-Nrf2组、含药血清（20%）+NC组。微板法检测MDA、SOD、GSH-Px水平；Western blot检测Nrf2、HO-1、FTH1、GSH-Px4蛋白表达水平。
结果
2
与正常组比较，模型组线粒体固缩，线粒体膜增厚，线粒体形态变小；模型组Fe
2+
含量显著增加（
P
<
0.01）；SOD活性显著降低（
P
<
0.01），GSH-Px表达显著下降（
P
<
0.01），MDA含量显著增高（
P
<
0.01）；Nrf2、HO-1表达降低（
P
<
0.05），FTH1表达显著性下降（
P
<
0.01），GSH-Px4表达显著降低（
P
<
0.01）。甘草含药血清处理组，中、高浓度组Fe
2+
含量显著降低（
P
<
0.01），SOD活性和GSH-Px活性显著升高（
P
<
0.01），MDA水平显著降低（
P
<
0.01）；高浓度组Nrf2表达明显升高（
P
<
0.05），中浓度组HO-1与GSH-Px4蛋白的表达有所增高（
P
<
0.05），低、中、高浓度组FTH1水平皆显著增加（
P
<
0.01）。机制研究发现，与NC组比较，转染Nrf2 siRNA的细胞中MDA含量增加（
P
<
0.01），SOD活性下降（
P
<
0.01），GSH-Px活性减少（
P
<
0.01）；Nrf2、HO-1表达下降（
P
<
0.01），FTH1和GSH-Px4蛋白含量降低（
P
<
0.01）；与Si-Nrf2组比较，在甘草含药血清干预后，细胞内MDA含量降低（
P
<
0.01），SOD活性提高（
P
<
0.01），GSH-Px活性上升（
P
<
0.01）；Nrf2、FTH1蛋白表达增加（
P
<
0.05），HO-1和GSH-Px4蛋白表达水平明显升高（
P
<
0.01）。
结论
2
甘草含药血清可降低LPS诱导的Caco2细胞炎症因子及氧化应激水平，其机制与促进Nrf2/HO-1信号通路的表达，减轻细胞内脂质过氧化从而抑制铁死亡的发生有关。
Abstract
Objective
2
To investigate the effect of Glycyrrhizae Radix et Rhizoma （GR）-containing serum on lipopolysaccharide （LPS）-induced inflammation in human colon epithelial adenocarcinoma cells （Caco2） based on inhibition of ferroptosis by the nuclear factor erythroid 2-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1） pathway.
Method
2
Caco2 cells were divided into a normal group， a model group （LPS， 200 μg·L
-1
）， low-， medium-， and high-dose GR-containing serum groups （5%， 10%， 20%）， and a ferroptosis inhibitor group （3-amino-4-cyclohexylamino-benzoic acid ethyl ester， Fer-1， 10 μmol·L
-1
）. The cells in the normal group were cultured normally， while those in other groups underwent the induction of an inflammation model. The cells in the low-， medium-， and high-dose GR-containing serum groups were treated with 5%， 10%， and 20% GR-containing serum for 24 hours， respectively， and the cells in the ferroptosis inhibitor group were treated with Fer-1 for 24 hours. Transmission electron microscopy was used to observe mitochondrial morphology in each group. Flow cytometry was used to detect intracellular Fe
2+
levels. Microplate assays were performed to measure superoxide dismutase （SOD） activity， malondialdehyde （MDA） and glutathione peroxidase （GSH-Px） levels. Enzyme-linked immunosorbent assay （ELISA） was used to measure interleukin-1
β
（IL-1
β
）， IL-6， IL-10， and tumor necrosis factor-
α
（TNF-
α
） levels. Western blot was used to measure the expression levels of Nrf2， HO-1， ferritin heavy chain 1 （FTH1）， and glutathione peroxidase 4 （GSH-Px4） proteins. Small interfering RNA （siRNA） was used to investigate the role of Nrf2 in ferroptosis regulation. The cells after interference were divided into a negative control （NC） group， a Si-Nrf2 group， a GR-containing serum （20%） + Si-Nrf2 group， and a GR-containing serum （20%） + NC group. Microplate assays were performed to measure MDA， SOD， and GSH-Px levels， and Western blot was used to measure the expression levels of Nrf2， HO-1， FTH1， and GSH-Px4 proteins.
Result
2
Compared with the normal group， the model group showed mitochondrial contraction， increased mitochondrial membrane thickness， and smaller mitochondrial morphology， increased Fe
2+
content （
P
<
0.01）， blunted SOD activity （
P
<
0.01）， decreased GSH-Px expression （
P
<
0.01）， increased MDA content （
P
<
0.01）， reduced expression levels of Nrf2 and HO-1 （
P
<
0.05）， reduced FTH1 expression （
P
<
0.01）， and down-regulated GSH-Px4 expression （
P
<
0.01）. In the GR-containing serum groups， the medium- and high-dose groups showed a significant decrease in Fe
2+
content （
P
<
0.01）， potentiated SOD and GSH-Px activities （
P
<
0.01）， and decreased MDA levels （
P
<
0.01）. The high-dose group showed a significant increase in Nrf2 expression （
P
<
0.05）， and the medium-dose group showed increased expression of HO-1 and GSH-Px4 proteins （
P
<
0.05）. The expression levels of FTH1 significantly increased in the low-， medium-， and high-dose groups （
P
<
0.01）. The study on mechanism revealed that compared with the NC group， the cells transfected with Nrf2 siRNA showed increased MDA content （
P
<
0.01）， blunted SOD activity （
P
<
0.01）， decreased GSH-Px activity （
P
<
0.01）， decreased expression of Nrf2 and HO-1 （
P
<
0.01）， and reduced levels of FTH1 and GSH-Px4 proteins （
P
<
0.01）. Compared with the Si-Nrf2 group， the cells treated with GR-containing serum showed a decrease in MDA content （
P
<
0.01）， an increase in SOD activity （
P
<
0.01）， an increase in GSH-Px activity （
P
<
0.01）， increased expression of Nrf2 and FTH1 proteins （
P
<
0.05）， and higher expression levels of HO-1 and GSH-Px4 proteins （
P
<
0.01）.
Conclusion
2
GR-containing serum can reduce the inflammatory cytokines and oxidative stress levels in LPS-induced Caco2 cells. Its mechanism is related to the promotion of Nrf2/HO-1 signaling pathway expression， alleviating intracellular lipid peroxidation and inhibiting ferroptosis.
关键词
甘草溃疡性结肠炎核因子E2相关因子2（Nrf2）血红素加氧酶-1（HO-1）铁死亡
Keywords
Glycyrrhizae Radix et Rhizomaulcerative colitisnuclear factor erythroid 2-related factor 2 （Nrf2）heme oxygenase-1 （HO-1）ferroptosis
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