·Thirteen 8-week-old C57BL/6J male mice were randomly divided into two groups: one group of mice were fed with normal iron diet (NID group, n=5) and the other were fed with high iron diet (HID group, n=8). After 12 weeks, the murine serums and livers were collected from both groups. Liver injury, histopathological changes, hepatic fibrosis, iron deposition and peroxidation in murine liver tissues were determined by serum index test, hematoxylin-eosin (H-E) staining, Sirius red staining, Prussian blue staining and 4-hydroxynonenal (4-HNE) staining, respectively. RNA-sequencing (RNA-seq) and bioinformatics were used to analyze the effect of high iron diet on the expression of transcriptome in the murine livers and to screen new candidate genes that might regulate ferroptosis. After Hp was overexpressed or knocked down in mouse liver cells (AML-12), RAS-selective lethal small molecule 3 (RSL3) or/and extracellular regulated protein kinases 1/2 (ERK1/2) inhibitor SCH772984 was/were added to cells. The role and mechanism of Hp in hepatocyte ferroptosis were determined by cell counting, real-time PCR, C11-BODIPY^581/591 immunofluorescence and flow cytometry.

·After 12 weeks of feeding, the levels of glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) in the HID group significantly increased compared with the NID group. The high iron diet induced hepatocytes death, hepatic fibrosis, iron deposition and lipid peroxidation products accumulation. RNA-seq analysis and bioinformatics results showed that acute phase response, catalytic activity response, antioxidant activity and hemoglobin binding signaling pathways were significantly regulated in the livers of HID mice, and Hp mRNA decreased significantly. Furthermore, the mRNA and protein expression levels of Hp in liver tissues of HID mice and in AML-12 hepatocytes treated with RSL3 obviously decreased. Importantly, Hp significantly alleviated lipid peroxidation and ferroptosis induced by RSL3 in AML-12 hepatocytes. Meanwhile, Hp suppressed RSL3-induced phosphorylation of ERK1/2 in AML-12 hepatocytes. Moreover, ERK1/2 inhibitor SCH772984 totally reversed the aggravation of hepatocyte ferroptosis induced by Hp knockdown.

·Hp is a novel inhibitory molecule in hepatocytes ferroptosis, which alleviates hepatocyte ferroptosis and liver injury by inhibiting ERK1/2 signaling pathway.