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

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.