High Hepcidin Levels Promote Abnormal Iron Metabolism and Ferroptosis in Chronic Atrophic Gastritis

Abstract

Background: Chronic atrophic gastritis (CAG) is a chronic inflammatory disease and premalignant lesion of gastric cancer. As an antimicrobial peptide, hepcidin can maintain iron metabolic balance and is susceptible to inflammation.

Objectives: The objective of this study was to clarify whether hepcidin is involved in abnormal iron metabolism and ferroptosis during CAG pathogenesis.

Methods: Non-atrophic gastritis (NAG) and chronic atrophic gastritis (CAG) patient pathology slides were collected, and related protein expression was detected by immunohistochemical staining. The CAG rat model was established using MNNG combined with an irregular diet.

Results: CAG patients and rats exhibited iron deposition in gastric tissue. CAG-induced ferroptosis in the stomach was characterized by decreased GPX4 and FTH levels and increased 4-HNE levels. Hepcidin, which is mainly located in parietal cells, was elevated in CAG gastric tissue. The high gastric level of hepcidin inhibited iron absorption in the duodenum by decreasing the protein expression of DMT1 and FPN1. In addition, the IL-6/STAT3 signaling pathway induced hepcidin production in gastric tissue.

Conclusion: Our results showed that the high level of gastric hepcidin induced ferroptosis in the stomach but also inhibited iron absorption in the intestines. Inhibiting hepcidin might be a new strategy for the prevention of CAG in the future.

Keywords: IL-6/STAT3 signaling pathway; chronic atrophic gastritis; ferroptosis; hepcidin; iron.

Figure 1

Histopathological evaluation of CAG patients and rat models. (A) Endoscopic analysis of NAG and CAG patients (n = 5). (B) HE staining of NAG and CAG patient samples (Scale bar = 50 μm, n = 5). (C) HE staining of normal and CAG rat samples (Scale bar = 100 or 50 μm, n = 5). The arrows indicated local atrophy of the mucosal layer, reduced number of gastric glands, and hyperplasia of connective tissue. NAG: non-atrophic gastritis patients; CAG: chronic atrophic gastritis patients or rats; Con: the control group of rats.

Figure 2

Ferroptosis was involved in CAG injury. (A) GPX4 immunofluorescence staining in the gastric tissue of patients (Scale bar = 100 or 50 μm, n = 3). The bottom images were the larger images of the frame in the top images, and the arrows indicate the location of GPX4-positive cells. (B) The mean density of GPX4-positive cells in Panel (A). (C, D) The expression and analysis of GPX4 and 4-HNE protein levels in the gastric tissue of rats, as determined by Western blotting (n = 4). (E) TEM analysis of the gastric tissue of rats with 15,000 or 25,000 magnification (n = 3). The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs. NAG or Con groups. NAG: non-atrophic gastritis patients; CAG: chronic atrophic gastritis patients or rats; Con: the control group of rats.

Figure 3

Iron levels in CAG gastric tissue. (A) Perls’ staining of the gastric tissue of patients (n = 3). (Scale bar = 100 or 50 μm, n = 3). (B) The mean density of Fe in Panel (A). (C) Perls’ staining of the gastric tissue of rats (Scale bar = 25 μm, n = 3). (D) The mean density of Fe in Panel (C). (E) FTH immunofluorescence staining in the gastric tissue of patients (Scale bar = 100 or 50 μm, n = 3). (F) The mean density of FTH-positive cells in Panel (E). (G,H) The expression and analysis of FTH protein levels in the gastric tissue of rats, as determined by Western blotting (n = 4). The bottom images were the larger images of the frame in the top images, and the arrows indicate the location of Fe or FTH-positive cells as shown in (A,C,E). The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs. NAG or Con groups. NAG: non-atrophic gastritis patients; CAG: chronic atrophic gastritis patients or rats; Con: the control group of rats.

Figure 4

Hepcidin levels were elevated in CAG gastric tissue. (A) Hepcidin immunofluorescence staining in the gastric tissue of patients (Scale bar = 100 or 50 μm, n = 3). The bottom images were the larger images of the frame in the top images, and the arrows indicate the location of hepcidin-positive cells. (B) The mean density of hepcidin-positive cells in Panel (A). (C) Double immunofluorescence labeling of H⁺-K⁺-ATPase (green color) and hepcidin (red color) in the gastric tissue of CAG rats (Scale bar = 500 or 50 μm, n = 3). The right images (marked 1, 2) were the larger images of the frame in the Merge images. The data are presented as the mean ± SEM. ** p < 0.01 vs. NAG or Con groups. NAG: non-atrophic gastritis patients; CAG: chronic atrophic gastritis patients or rats; Con: the control group of rats.

Figure 5

The IL-6/STAT3 signaling pathways were activated in CAG gastric tissue. (A) IL-6 immunofluorescence staining in the gastric tissue of patients (Scale bar = 100 or 50 μm, n = 3). (B) The mean density of IL-6-positive cells in Panel (A). (C) p-STAT3 immunofluorescence staining of the gastric tissue of patients (Scale bar = 100 or 50 μm, n = 3). (D) The mean density of p-STAT3-positive cells in Panel (C). The bottom images were the larger images of the frame in the top images, and the arrows indicate the location of IL-6-positive cells (A) or p-STAT3-positive cells (C). (E) IL-6, TNF-α, and IL-1β immunofluorescence staining in the gastric tissue of CAG rats (n = 3). (F) The expression of IL-6 and the ratio of p-STAT3/STAT3 in the gastric tissue of CAG rats, as determined by Western blotting (n = 4). The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01 vs. NAG or Con groups. NAG: non-atrophic gastritis patients; CAG: chronic atrophic gastritis patients or rats; Con: the control group of rats.

Figure 6

Iron levels and iron-related transport proteins in the duodenum of CAG rats. (A) Perls’ staining and analysis of Fe levels in the duodenum of CAG rats. (B) DMT1 immunofluorescence staining and analysis of the duodenum of CAG rats. (C) FPN1 immunofluorescence staining and analysis of the duodenum of CAG rats. Scale bar = 25 μm, and the arrows indicate the location of Fe, DMT1-positive cells, or FPN1-positive cells. The data are presented as the mean ± SEM, n = 3. * p < 0.05 vs. Con group. Con: Control group of rats. CAG: chronic atrophic gastritis of rats.

Figure 7

A schematic showing hepcidin involvement in CAG injury. CAG can increase IL-6 expression and then activate hepcidin via the JAK2/STAT3 signaling pathway. On the one hand, high levels of hepcidin restrain FPN1, causing iron overload and ferroptosis in CAG gastric tissue, as indicated by a higher level of 4-HNE and a lower level of GPX4. On the other hand, hepcidin is expressed and released by parietal cells and flows into the duodenum. Subsequently, high levels of hepcidin inhibit FPN1 expression in the basal membrane of epithelial cells and DMT1 expression in the brush border membrane of the duodenum, resulting in iron uptake deficiency in the duodenum.