Plasma non-transferrin-bound iron uptake by the small intestine leads to intestinal injury and intestinal flora dysbiosis in an iron overload mouse model and Caco-2 cells.

Iron overload often occurs during blood transfusion and iron supplementation, resulting in the presence of non-transferrin-bound iron (NTBI) in host plasma and damage to multiple organs, but effects on the intestine have rarely been reported. In this study, an iron overload mouse model with plasma NTBI was established by intraperitoneal injection of iron dextran. We found that plasma NTBI damaged intestinal morphology, caused intestinal oxidative stress injury and reactive oxygen species (ROS) accumulation, and induced intestinal epithelial cell apoptosis. In addition, plasma NTBI increased the relative abundance of Ileibacterium and Desulfovibrio in the cecum, while the relative abundance of Faecalibaculum and Romboutsia was reduced. Ileibacterium may be a potential microbial biomarker of plasma NTBI. Based on the function prediction analysis, plasma NTBI led to the weakening of intestinal microbiota function, significantly reducing the function of the extracellular structure. Further investigation into the mechanism of injury showed that iron absorption in the small intestine significantly increased in the iron group. Caco-2 cell monolayers were used as a model of the intestinal epithelium to study the mechanism of iron transport. By adding ferric ammonium citrate (FAC, plasma NTBI in physiological form) to the basolateral side, the apparent permeability coefficient (Papp) values from the basolateral to the apical side were greater than 3×10-6 cm s-1. Intracellular ferritin level and apical iron concentration significantly increased, and SLC39A8 (ZIP8) and SLC39A14 (ZIP14) were highly expressed in the FAC group. Short hairpin RNA (shRNA) was used to knock down ZIP8 and ZIP14 in Caco-2 cells. Transfection with ZIP14-specific shRNA decreased intracellular ferritin level and inhibited iron uptake. These results revealed that plasma NTBI may cause intestinal injury and intestinal flora dysbiosis due to the uptake of plasma NTBI from the basolateral side into the small intestine, which is probably mediated by ZIP14.

Effect of Long-Term and Short-Term Imbalanced Zn Manipulation on Gut Microbiota and Screening for Microbial Markers Sensitive to Zinc Status.

Zinc (Zn) imbalance is a common single-nutrient disorder worldwide, but little is known about the short-term and long-term effects of imbalanced dietary zinc in the intestinal microbiome. Here, 3-week-old C57BL/6 mice were fed diets supplemented with Zn at the doses of 0 (low Zn), 30 (control Zn), 150 (high Zn), and 600 mg/kg of body weight (excess Zn) for 4 weeks (short term) and 8 weeks (long term). The gut bacterial composition at the phyla, genus, and species levels were changed as the result of the imbalanced Zn diet (e.g., Lactobacillus reuteri and Akkermansia muciniphila). Moreover, pathways including carbohydrate, glycan, and nucleotide metabolism were decreased by a short-term low-Zn diet. Valeriate production was suppressed by a long-term low-Zn diet. Pathways such as drug resistance and infectious diseases were upregulated in high- and excess-Zn diets over 4-week and 8-week intervals. Long-term zinc fortification doses, especially at the high-Zn level, suppressed the abundance of short-chain fatty acids (SCFAs)-producing genera as well as the concentrations of metabolites. Finally, Melainabacteria (phylum) and Desulfovibrio sp. strain ABHU2SB (species) were identified to be potential markers for Zn status with high accuracy (area under the curve [AUC], >0.8). Collectively, this study identified significant changes in gut microbial composition and its metabolite concentration in altered Zn-fed mice and the relevant microbial markers for Zn status. IMPORTANCE Zn insufficiency is an essential health problem in developing countries. To prevent the occurrence of zinc deficit, zinc fortification and supplementation are widely used. However, in developed countries, the amounts of Zn consumed often exceed the tolerable upper intake limit. Our results demonstrated that dietary Zn is an essential mediator of microbial community structure and that both Zn deficiency and Zn overdose can generate a dysbiosis in the gut microbiota. Moreover, specific microbial biomarkers of Zn status were identified and correlated with serum Zn level. Our study found that a short-term low-Zn diet (0 mg/kg) and a long-term high-zinc diet (150 mg/kg) had obvious negative effects in a mouse model. Thus, these results indicate that the provision and duration of supplemental Zn should be approached with caution.

Network pharmacology-based identification of the key mechanism of quercetin acting on hemochromatosis.

Hemochromatosis is an iron overload disease, which lacks nutritional intervention strategies. This study explored the protective effect of quercetin on hemochromatosis and its possible mechanism through network pharmacology. We used Online Mendelian Inheritance in Man to screen the disease targets of hemochromatosis, and further constructed a potential protein interaction network through STITCH. The above-mentioned targets revealed by Gene enrichment analysis have played a significant role in ferroptosis, mineral absorption, basal cell carcinoma, and related signal pathways. Besides, the drug likeness of quercetin obtained by Comparative Toxicogenomics Database was evaluated by Traditional Chinese Medicine Systems Pharmacology, and potential drug targets identified by PharmMapper and similar compounds identified by PubChem were selected for further research. Moreover, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed the relationship between quercetin and glycosylation. Furthermore, we performed experiments to verify that the protective effect of quercetin on iron overload cells is to inhibit the production of reactive oxygen species, limit intracellular iron, and degrade glycosaminoglycans. Finally, iron-induced intracellular iron overload caused ferroptosis, and quercetin and fisetin were potential ferroptosis inhibitors. In conclusion, our study revealed the correlation between hemochromatosis and ferroptosis, provided the relationship between the target of quercetin and glycosylation, and verified that quercetin and its similar compounds interfere with iron overload related disease. Our research may provide novel insights for quercetin and its structurally similar compounds as a potential nutritional supplement for iron overload related diseases.

Tolerable upper intake level of iron damages the liver of weaned piglets.

Iron is one of the essential trace elements, which is often supplemented as an additive to meet the growing needs of toddlers and young animals. Recommended nutrient intake (RNI) and tolerable upper intake levels (UL) are always set when the iron is supplemented. The purpose of this study was to evaluate the subacute (28 days) toxicity of UL iron to weaned piglet liver. Thirty 23-day-old weaned piglets were divided into three groups and, respectively, supplemented with 100, 300 or 3000 (UL) mg/kg iron. UL iron caused significant weight loss in 4th week (p < 0.05). Divalent metal transporter 1(DMT1) decreased significantly, ferroportin 1 and ferritin increased significantly in the liver of UL iron group (p < 0.05). Although there was no significant effect on liver morphology, UL iron significantly increased hepatic iron, reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (p < 0.05). UL iron significantly reduced glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and total anti-oxidation capacity (T-AOC) in the liver (p < 0.05). Nuclear factor erythroid 2-related factor 2 (Nrf2) activated subunits of glutamate cysteine ligase (Gclc) and glutathione S-transferase A1 (Gsta1) upregulation in the UL iron group liver, thereby increasing resistance to oxidative stress. In conclusion, UL iron supplementation altered iron metabolism, generated free radicals, reduced antioxidant enzyme activity and activated Nrf2 signalling pathway in the weaned piglet liver.

Iron homeostasis disorder in piglet intestine.

Iron plays an essential role in preventing iron deficiency anemia and ensuring the healthy growth of animals. The special physiological condition of piglets is the main cause of iron deficiency. Iron metabolism in the intestine is the basis for understanding the effects of iron on the health of piglets. In order to scientifically evaluate dietary iron supplementation doses, it is necessary to recognize the effects of iron deficiency and iron overload on piglet intestinal health. Besides, iron as a cofactor is essential for the growth of microorganisms, and microorganisms compete with the host to absorb iron. Under the stress of iron deficiency and iron overload, various control schemes (such as precise nutrition, element balance, elimination of oxidation, etc.) are effective measures to eliminate adverse effects. In this review, we comprehensively review recent findings on the effects of iron deficiency and iron overload on intestinal health. This review will provide a rational design strategy to achieve a reasonable iron supplement, which will guide the use of iron in animal husbandry.

Tolerable upper intake level of iron damages the intestine and alters the intestinal flora in weaned piglets.

Iron supplementation has been an intervention to improve iron storage and prevent iron deficiency anemia in weaned piglets and the recommended nutrient intake (RNI) and tolerable upper intake levels (UL) of iron have been established. The purpose of this study is to investigate the potential harm of UL iron to the gut and microbes of weaned piglets. Thirty 23 day old weaned piglets were assigned to three dietary treatments: a basal diet supplemented with 100 (RNI), 300, and 3000 (UL) mg FeSO4 per kg diet for 28 days. Then, we used the intestinal porcine epithelial cell line (IPEC-1) as a cell model to study the effect of UL iron on the gut of weaned piglets. Weaned piglets showed a significant decrease in villus height after feeding on a UL iron diet (P < 0.05). The protein levels of DMT1 and Zip14 decreased, and the protein levels of ferritin increased in the duodenal mucosa (P < 0.05) of UL iron fed weaned piglets. Moreover, UL iron also increased the content of ROS and malondialdehyde and decreased the activity of superoxide dismutase in the duodenal mucosa of weaned piglets (P < 0.05). The addition of UL iron to the diet significantly reduced the expression of tight junction proteins Claudin-1, Occludin, and ZO-1 in the duodenal mucosa of weaned piglets (P < 0.05). In the IPEC-1 cell model, iron induced the production of cytosolic and mitochondrial ROS and reduced the mitochondrial membrane potential, which in turn led to cellular vacuolation and fibrosis. Furthermore, UL iron significantly altered the cecum flora of weaned piglets, and the relative abundance of Clostridiales, Faecalibacterium, and Prevotellaceae decreased significantly (P < 0.05), while the relative abundance of Desulfovibrio and Anaerovibrio increased significantly (P < 0.05). In conclusion, UL iron caused damage to the intestinal villi, induced oxidative stress, reduced iron absorption protein, damaged the intestinal barrier, and modified the intestinal microbial structure in weaned piglets.

Effects of Eucommia ulmoides leaf extracts on growth performance, antioxidant capacity and intestinal function in weaned piglets.

Eucommia ulmoides is traditional Chinese medicine, and it possesses several potential bioactivities, such as anti-inflammatory, antioxidant and immune regulatory activities. This study was conducted to determine the effects of dietary Eucommia ulmoides leaf extracts (ELE) on growth performance, antioxidant capacity and intestinal function of weaned piglets. Two hundred crossbred (Duroc × Landrace × Yorkshire) piglets with an average initial weight of 12.96 ± 0.28 kg were randomly allotted to five treatments: C0 (basal diet), C1 (basal diet + antibiotics) and basal diet supplemented with increasing levels of ELE (0.2, 0.3 or 0.4 g/kg of feed). The results showed that ELE or antibiotics supplementation remarkably decreased diarrhoea rate and 0.3 g/kg ELE increased average daily gain compared with C0 (p < .05). 0.3 g/kg ELE increased alkaline phosphatase (AKP) levels and total antioxidant capacity (T-AOC) in serum and liver, as well as increased the content of serum albumin and total protein (TP) compared with the C0 (p < .05). The lipase activity of duodenum content and trypsin activity of jejunum content were improved fed diets containing 0.3 g/kg ELE compared with C0 (p < .05). The 0.3 g/kg ELE treatments have a higher villus height of the duodenum and jejunum compared with the C0 (p < .05). These results suggested that ELE supplementation had beneficial effects on antioxidant and intestinal function in weaned piglets, which also could increase growth performance and decreased diarrhoea rate. Accordingly, ELE is a potential alternative to antibiotics.

Comparing the Influence of High Doses of Different Zinc Salts on Oxidative Stress and Energy Depletion in IPEC-J2 Cells.

The current study aimed to investigate the influence of four supplemental zinc salts (chelated: Zn glycine; non-chelated: Zn sulfate, Zn citrate, Zn gluconate) among different zinc concentrations (30-300 µM) on cell proliferation, oxidative stress, and energy depletion in intestinal porcine jejunum epithelial cells (IPEC-J2). Different zinc salts affected cell viability in a time- and dose-dependent manner, which was mainly dependent on the uptake of intracellular Zn2+. Intracellular Zn2+ of Zn sulfate has taken up almost twice as high as Zn glycine when cells were loaded with 100-200 µM zinc. After loading cells with 300 µM zinc, Zn glycine and Zn sulfate had a similar trend in accumulation of Zn2+. When the intracellular Zn2+ overloads, cells will gradually be damaged and subsequently die bearing biochemical features of necrosis or late apoptosis. Meanwhile, obviously, increased levels of intracellular ROS, mitochondrial ROS, MDA, and NO and decreased levels of GSH were observed. Excessive intracellular Zn2+ significantly decreased mitochondria membrane potential accompanied by an obvious loss of ATP and NAD+ levels. Overall, exposure to high doses of zinc salts caused cell damage, which was mainly dependent on the uptake of Zn2+. Zinc overload induced oxidative stress and energy depletion in IPEC-J2 cells, and the cell damage with non-chelated zinc addition was more serious than Zn glycine.