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 / 中国实验方剂学杂志
Chinese Journal of Experimental Traditional Medical Formulae
; (24): 144-153, 2023.
Article
in Zh
| WPRIM
| ID: wpr-978460
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WPRO
ABSTRACT
ObjectiveTo 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. MethodCaco2 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 Fe2+ 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-1β (IL-1β), 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. ResultCompared with the normal group, the model group showed mitochondrial contraction, increased mitochondrial membrane thickness, and smaller mitochondrial morphology, increased Fe2+ 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 Fe2+ 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). ConclusionGR-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.
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Index:
WPRIM
Language:
Zh
Journal:
Chinese Journal of Experimental Traditional Medical Formulae
Year:
2023
Type:
Article