To explore the mechanism by which hedysarum polybotrys polysaccharide (HPS) improves iron death of steatotic hepatocytes through the protein kinase RNA-like endoplasmic reticulum kinase- nuclear factor-erythroid 2-related factor 2 (PERK-Nrf2) signaling pathway.

The cells were cultured with 0.6 mmol·L^-1 fatty acids to replicate the non-alcoholic fatty liver disease cell model. The LO-2 cell were divided into normal group (complete medium), model group (0.6 mmol·L^-1 fatty acid solution), positive control group (0.6 mmol·L^-1 fatty acid solution+24 μmol·L^-1 liproxstain-1) and experimental group (0.6 mmol·L^-1 fatty acid solution + 50 mg·L^-1 HPS), culture for 24 h. Stain with Oil Red O was used to analyze the positive area (%Area) of cell lipid deposition; the content of lipid peroxide(LPO), 4-hydroxynonenal(4-HNE), glutathione(GSH) and superoxide dismutase(SOD) was detected by enzyme-linked immunosorbent assay(Elisa) method; the relative expressions levels of PERK-Nrf2 signaling pathway protein in hepatocytes were detected by Western blot; immunofluorescence assay was used to detect the relative expression levels of GPX4 and SLC7A11 proteins.

The % Area of cell lipid deposition in hepatocytes of normal group, model group, control group and experimental group were 0.02±0.01, 0.26±0.08, 0.09±0.02 and 0.15±0.04; the contents of LPO were (2.62±0.17), (13.47±1.55), (6.31±1.12) and (5.71±1.50) nmol·mL^-1; the contents of 4-HNE were (8.06±0.40), (14.60±2.02), (9.40±0.81) and (12.94±0.56) ng·L^-1; the contents of GSH were (120.73±7.75), (34.60±2.98), (67.86±2.08) and (59.33±7.46) mg·L^-1; the contents of SOD were (7.25±0.61), (3.77±0.44), (5.22±0.76) and (8.12±0.78) U·mL^-1; the levels of Grp78 mRNA were 1.01±0.15, 1.47±0.11, 1.41±0.08 and 1.02±0.05; the levels of PERK mRNA were 1.00±0.09, 1.85±0.16, 1.02±0.27 and 1.16±0.01; the levels of Nrf2 mRNA were 1.01±0.12, 0.26±0.07, 0.68±0.09 and 0.66±0.05; the levels of GPX4 mRNA were 1.00±0.05, 0.79±0.02, 0.87±0.17 and 0.83±0.10; the levels of SLC7A11 mRNA were 1.01±0.17, 0.50±0.14, 0.91±0.09 and 0.98±0.05; the relative expression levels of Grp78 protein were 1.00±0.11, 2.72±0.07, 1.83±0.04 and 2.24±0.06; the relative expression levels of PERK protein were 1.00±0.06, 1.21±0.08, 1.03±0.07 and 0.95±0.06; the relative expression levels of p-PERK protein were 1.00±0.04, 2.70±0.08, 1.40±0.07 and 1.76±0.06; the relative levels of PERK protein phosphorylation were 1.00±0.09, 2.24±0.21, 1.36±0.03 and 1.85±0.09; the relative expression levels of Nrf2 protein were 1.00±0.03, 0.52±0.01, 0.73±0.02 and 0.84±0.02; the protein fluorescence intensity of GPX4 were 24.34±0.86, 13.86±1.65, 18.01±2.03 and 16.20±1.55; the protein fluorescence intensity of SLC7A11 were 69.58±4.13, 34.24±6.73, 60.75±9.35 and 45.18±6.46. Compared with the normal group, there were significant differences in the above indexes of model group (all P<0.05) ; compared with the model group, there were significant differences in the above indexes of experimental group (all P<0.05).

HPS may protect liver cells by regulating the PERK-Nrf2 signaling pathway, reducing lipid peroxidation and iron death in liver cells.