Ferrous Ion Alleviates Lipid Deposition and Inflammatory Responses Caused by a High Cottonseed Meal Diet by Modulating Hepatic Iron Transport Homeostasis and Controlling Ferroptosis in Juvenile Ctenopharyngodon idellus

Abstract

To investigate the mechanisms through which ferrous ion (Fe²⁺) addition improves the utilization of a cottonseed meal (CSM) diet, two experimental diets with equal nitrogen and energy content (low-cottonseed meal (LCM) and high-cottonseed meal (HCM) diets, respectively) containing 16.31% and 38.46% CSM were prepared. Additionally, the HCM diet was supplemented with graded levels of FeSO₄·7H₂O to establish two different Fe²⁺ supplementation groups (HCM + 0.2%Fe²⁺ and HCM + 0.4%Fe²⁺). Juvenile Ctenopharyngodon idellus (grass carps) (5.0 ± 0.5 g) were fed one of these four diets (HCM, LCM, HCM + 0.2%Fe²⁺ and HCM + 0.4%Fe²⁺ diets) for eight weeks. Our findings revealed that the HCM diet significantly increased lipid peroxide (LPO) concentration and the expression of lipogenic genes, e.g., sterol regulatory element binding transcription factor 1 (srebp1) and stearoyl-CoA desaturase (scd), leading to excessive lipid droplet deposition in the liver (p < 0.05). However, these effects were significantly reduced in the HCM + 0.2%Fe²⁺ and HCM + 0.4%Fe²⁺ groups (p < 0.05). Plasma high-density lipoprotein (HDL) concentration was also significantly lower in the HCM and HCM + 0.2%Fe²⁺ groups compared to the LCM group (p < 0.05), whereas low-density lipoprotein (LDL) concentration was significantly higher in the HCM + 0.2%Fe²⁺ and HCM + 0.4%Fe²⁺ groups than in the LCM group (p < 0.05). Furthermore, the plasma levels of liver functional indices, including alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose (GLU), were significantly lower in the HCM + 0.4%Fe²⁺ group (p < 0.05). Regarding the expression of genes related to iron transport regulation, transferrin 2 (tfr2) expression in the HCM group and Fe²⁺ supplementation groups were significantly suppressed compared to the LCM group (p < 0.05). The addition of 0.4% Fe²⁺ in the HCM diet activated hepcidin expression and suppressed ferroportin-1 (fpn1) expression (p < 0.05). Compared to the LCM group, the expression of genes associated with ferroptosis and inflammation, including acyl-CoA synthetase long-chain family member 4b (acsl4b), lysophosphatidylcholine acyltransferase 3 (lpcat3), cyclooxygenase (cox), interleukin 1β (il-1β), and nuclear factor kappa b (nfκb), were significantly increased in the HCM group (p < 0.05), whereas Fe²⁺ supplementation in the HCM diet significantly inhibited their expression (p < 0.05) and significantly suppressed lipoxygenase (lox) expression (p < 0.05). Compared with the HCM group without Fe²⁺ supplementation, Fe²⁺ supplementation in the HCM diet significantly upregulated the expression of genes associated with ferroptosis, such as heat shock protein beta-associated protein1 (hspbap1), glutamate cysteine ligase (gcl), and glutathione peroxidase 4a (gpx4a) (p < 0.05), and significantly decreased the expression of the inflammation-related genes interleukin 15/10 (il-15/il-10) (p < 0.05). In conclusion, FeSO₄·7H₂O supplementation in the HCM diet maintained iron transport and homeostasis in the liver of juvenile grass carps, thus reducing the occurrence of ferroptosis and alleviating hepatic lipid deposition and inflammatory responses caused by high dietary CSM contents.

Keywords: herbivorous fish; inflammation; ion transporter; lipid metabolism; plant protein source.

Figure 1

Effect of Fe²⁺ supplementation in the HCM diet on the plasma biochemical parameters of grass carp. (A) ALP, alkaline phosphatase; (B) AST, aspartate aminotransferase; (C) ALT, alanine aminotransferase; (D) GLU, glucose; (E) TP, total protein; (F) ALB, albumin. All data are expressed as mean ± standard error (SE). Different small letters above the bars indicate significant differences among groups (Duncan’s test, p < 0.05).

Figure 2

Effects of Fe²⁺ supplementation in HCM diet on hepatic and plasma lipid metabolism of grass carp. (A) Morphology of hepatic adipocytes using Oil Red O staining. a–d: Microscopic magnification 200× hepatic adipocyte morphology; e–h: Microscopic magnification 400× hepatic adipocyte morphology. The lipid droplets are stained in red, whereas the nuclei are stained in blue. (B) Quantification of the relative area of lipid droplets; (C) LPO, lipid peroxide; (D) HDL, high-density lipoprotein; (E) LDL, low-density lipoprotein; (F) TC, total cholesterol; (G) srebp1, sterol regulatory element binding transcription factor 1; (H) scd, stearoyl-CoA desaturase; (I) cpt, carnitine palmitoyl-transferase; (J) cyp7b1, cytochrome p450 family 7 subfamily B member 1. All data are expressed as mean ± standard error (SE). Different small letters above the bars indicate significant differences among groups (Duncan’s test, p < 0.05).

Figure 3

Effect of Fe²⁺ supplementation in HCM diet on gene expressions related to iron transportation in grass carp liver. (A) tfr2, transferrin 2; (B) fpn1, ferroportin-1; (C) hepcidin; (D) ferritin. All data are expressed as mean ± standard error (SE). Different small letters above the bars indicate significant differences among groups (Duncan’s test, p < 0.05).

Figure 4

Effect of Fe²⁺ supplementation in HCM diet on the expression of genes related to ferroptosis in grass carp liver. (A) acsl4b, acyl-CoA synthetase long chain family member 4b; (B) lpcat3, lysophosphatidylcholine acyltransferase 3; (C) lox, lipoxygenase; (D) cox, cyclooxygenase; (E) hspbap1, heat shock protein beta associated protein1; (F) gcl, glutamate cysteine ligase; (G) gpx4a, glutathione peroxidase 4a. All data are expressed as mean ± standard error (SE). Different small letters above the bars indicate significant differences among groups (Duncan’s test, p < 0.05).

Figure 5

Effect of Fe²⁺ supplementation in HCM diet on the expression of genes related to inflammation in grass carp liver. (A) nfκb, nuclear factor kappa b; (B–D) il-1β/10/15, interleukin-1β/10/15. All data are expressed as mean ± standard error (SE). Different small letters above the bars indicate significant differences among groups (Duncan’s test, p < 0.05).

Figure 6

Regulation mechanisms of Fe²⁺ supplementation on HCM diet-induced lipid metabolism disorders and ferroptosis. Recombinant solute carrier family 7, member 11 (SLC7A11); lipid peroxide (LPO); sterol regulatory element binding protein (srebp1); stearoyl-CoA desaturase (scd); glutamate cysteine ligase (gcl); glutathione (GSH); glutathione peroxidase 4a(gpx4a); acyl-CoA synthetase long-chain family member 4b (acsl4b); lysophosphatidylcholine acyltransferase 3 (lpcat3); lipoxygenase (lox); transferrin 2 (tfr2); ferroportin-1 (fpn1); hepcidin; cyclooxygenase (cox); nuclear factor kappa b (nfκb); Interleukin-1β/10/15 (il-1β/10/15).