The dynamic shifts of IL-10-producing Th17 and IL-17-producing Treg in health and disease: a crosstalk between ancient "Yin-Yang" theory and modern immunology.

The changes in T regulatory cell (Treg) and T helper cell (Th) 17 ratios holds paramount importance in ensuring internal homeostasis and disease progression. Recently, novel subsets of Treg and Th17, namely IL-17-producing Treg and IL-10-producing Th17 have been identified. IL-17-producing Treg and IL-10-producing Th17 are widely considered as the intermediates during Treg/Th17 transformation. These "bi-functional" cells exhibit plasticity and have been demonstrated with important roles in multiple physiological functions and disease processes. Yin and Yang represent opposing aspects of phenomena according to the ancient Chinese philosophy "Yin-Yang" theory. Furthermore, Yin can transform into Yang, and vice versa, under specific conditions. This theory has been widely used to describe the contrasting functions of immune cells and molecules. Therefore, immune-activating populations (Th17, M1 macrophage, etc.) and immune overreaction (inflammation, autoimmunity) can be considered Yang, while immunosuppressive populations (Treg, M2 macrophage, etc.) and immunosuppression (tumor, immunodeficiency) can be considered Yin. However, another important connotation of "Yin-Yang" theory, the conversion between Yin and Yang, has been rarely documented in immune studies. The discovery of IL-17-producing Treg and IL-10-producing Th17 enriches the meaning of "Yin-Yang" theory and further promotes the relationship between ancient "Yin-Yang" theory and modern immunology. Besides, illustrating the functions of IL-17-producing Treg and IL-10-producing Th17 and mechanisms governing their differentiation provides valuable insights into the mechanisms underlying the dynamically changing statement of immune statement in health and diseases.

Ferritin-mediated mitochondrial iron homeostasis is essential for the survival of hematopoietic stem cells and leukemic stem cells.

Iron metabolism plays a crucial role in cell viability, but its relationship with adult stem cells and cancer stem cells is not fully understood. The ferritin complex, responsible for intracellular iron storage, is important in this process. We report that conditional deletion of ferritin heavy chain 1 (Fth1) in the hematopoietic system reduced the number and repopulation capacity of hematopoietic stem cells (HSCs). These effects were associated with a decrease in cellular iron level, leading to impaired mitochondrial function and the initiation of apoptosis. Iron supplementation, antioxidant, and apoptosis inhibitors reversed the reduced cell viability of Fth1-deleted hematopoietic stem and progenitor cells (HSPCs). Importantly, leukemic stem cells (LSCs) derived from MLL-AF9-induced acute myeloid leukemia (AML) mice exhibited reduced Fth1 expression, rendering them more susceptible to apoptosis induced by the iron chelation compared to normal HSPCs. Modulating FTH1 expression using mono-methyl fumarate increased LSCs resistance to iron chelator-induced apoptosis. Additionally, iron supplementation, antioxidant, and apoptosis inhibitors protected LSCs from iron chelator-induced cell death. Fth1 deletion also extended the survival of AML mice. These findings unveil a novel mechanism by which ferritin-mediated iron homeostasis regulates the survival of both HSCs and LSCs, suggesting potential therapeutic strategies for blood cancer with iron dysregulation.

Cellular iron depletion enhances behavioral rhythm by limiting brain Per1 expression in mice.

AIMS: Disturbances in the circadian rhythm are positively correlated with the processes of aging and related neurodegenerative diseases, which are also associated with brain iron accumulation. However, the role of brain iron in regulating the biological rhythm is poorly understood. In this study, we investigated the impact of brain iron levels on the spontaneous locomotor activity of mice with altered brain iron levels and further explored the potential mechanisms governing these effects in vitro. RESULTS: Our results revealed that conditional knockout of ferroportin 1 (Fpn1) in cerebral microvascular endothelial cells led to brain iron deficiency, subsequently resulting in enhanced locomotor activity and increased expression of clock genes, including the circadian locomotor output cycles kaput protein (Clock) and brain and muscle ARNT-like 1 (Bmal1). Concomitantly, the levels of period circadian regulator 1 (PER1), which functions as a transcriptional repressor in regulating biological rhythm, were decreased. Conversely, the elevated brain iron levels in APP/PS1 mice inhibited autonomous rhythmic activity. Additionally, our findings demonstrate a significant decrease in serum melatonin levels in Fpn1cdh5 -CKO mice compared with the Fpn1flox/flox group. In contrast, APP/PS1 mice with brain iron deposition exhibited higher serum melatonin levels than the WT group. Furthermore, in the human glioma cell line, U251, we observed reduced PER1 expression upon iron limitation by deferoxamine (DFO; iron chelator) or endogenous overexpression of FPN1. When U251 cells were made iron-replete by supplementation with ferric ammonium citrate (FAC) or increased iron import through transferrin receptor 1 (TfR1) overexpression, PER1 protein levels were increased. Additionally, we obtained similar results to U251 cells in mouse cerebellar astrocytes (MA-c), where we collected cells at different time points to investigate the rhythmic expression of core clock genes and the impact of DFO or FAC treatment on PER1 protein levels. CONCLUSION: These findings collectively suggest that altered iron levels influence the circadian rhythm by regulating PER1 expression and thereby modulating the molecular circadian clock. In conclusion, our study identifies the regulation of brain iron levels as a potential new target for treating age-related disruptions in the circadian rhythm.

Mechanisms and regulation of defensins in host defense.

As a family of cationic host defense peptides, defensins are mainly synthesized by Paneth cells, neutrophils, and epithelial cells, contributing to host defense. Their biological functions in innate immunity, as well as their structure and activity relationships, along with their mechanisms of action and therapeutic potential, have been of great interest in recent years. To highlight the key research into the role of defensins in human and animal health, we first describe their research history, structural features, evolution, and antimicrobial mechanisms. Next, we cover the role of defensins in immune homeostasis, chemotaxis, mucosal barrier function, gut microbiota regulation, intestinal development and regulation of cell death. Further, we discuss their clinical relevance and therapeutic potential in various diseases, including infectious disease, inflammatory bowel disease, diabetes and obesity, chronic inflammatory lung disease, periodontitis and cancer. Finally, we summarize the current knowledge regarding the nutrient-dependent regulation of defensins, including fatty acids, amino acids, microelements, plant extracts, and probiotics, while considering the clinical application of such regulation. Together, the review summarizes the various biological functions, mechanism of actions and potential clinical significance of defensins, along with the challenges in developing defensins-based therapy, thus providing crucial insights into their biology and potential clinical utility.

The Zinc Transporter SLC39A10 Plays an Essential Role in Embryonic Hematopoiesis.

The role of zinc in hematopoiesis is currently unclear. Here, SLC39A10 (ZIP10) is identified as a key zinc transporter in hematopoiesis. The results show that in zebrafish, Slc39a10 is a key regulator of the response to zinc deficiency. Surprisingly, both slc39a10 mutant zebrafish and hematopoietic Slc39a10-deficient mice develop a more severe form of impaired hematopoiesis than animals lacking transferrin receptor 1, a well-characterized iron gatekeeper, indicating that zinc plays a larger role than iron in hematopoiesis, at least in early hematopoietic stem cells (HSCs). Furthermore, it is shown that loss of Slc39a10 causes zinc deficiency in fetal HSCs, which in turn leads to DNA damage, apoptosis, and G1 cell cycle arrest. Notably, zinc supplementation largely restores colony formation in HSCs derived from hematopoietic Slc39a10-deficient mice. In addition, inhibiting necroptosis partially restores hematopoiesis in mouse HSCs, providing mechanistic insights into the requirement for zinc in mediating hematopoiesis. Together, these findings indicate that SLC39A10 safeguards hematopoiesis by protecting against zinc deficiency-induced necroptosis, thus providing compelling evidence that SLC39A10 and zinc homeostasis promote the development of fetal HSCs. Moreover, these results suggest that SLC39A10 may serve as a novel therapeutic target for treating anemia and zinc deficiency-related disorders.

Iron accumulation in macrophages promotes the formation of foam cells and development of atherosclerosis.

BACKGROUND: Macrophages that accumulate in atherosclerotic plaques contribute to progression of the lesions to more advanced and complex plaques. Although iron deposition was found in human atherosclerotic plaques, clinical and pre-clinical studies showed controversial results. Several epidemiological studies did not show the positive correlation between a systemic iron status and an incidence of cardiovascular diseases, suggesting that the iron involvement occurs locally, rather than systemically. RESULTS: To determine the direct in vivo effect of iron accumulation in macrophages on the progression of atherosclerosis, we generated Apoe-/- mice with a macrophage-specific ferroportin (Fpn1) deficiency (Apoe-/-Fpn1LysM/LysM). Fpn1 deficiency in macrophages dramatically accelerated the progression of atherosclerosis in mice. Pathophysiological evidence showed elevated levels of reactive oxygen species, aggravated systemic inflammation, and altered plaque-lipid composition. Moreover, Fpn1 deficiency in macrophages significantly inhibited the expression of ABC transporters (ABCA1 and ABCG1) by decreasing the expression of the transcription factor LXRα, which reduced cholesterol efflux and therefore promoted foam cell formation and enhanced plaque formation. Iron chelation relieved the symptoms moderately in vivo, but drastically ex vivo. CONCLUSIONS: Macrophage iron content in plaques is a critical factor in progression of atherosclerosis. The interaction of iron and lipid metabolism takes place in macrophage-rich atherosclerotic plaques. And we also suggest that altering intracellular iron levels in macrophages by systemic iron chelation or dietary iron restriction may be a potential supplementary strategy to limit or even regress the progression of atherosclerosis.

Auranofin mitigates systemic iron overload and induces ferroptosis via distinct mechanisms.

Iron homeostasis is essential for health; moreover, hepcidin-deficiency results in iron overload in both hereditary hemochromatosis and iron-loading anemia. Here, we identified iron modulators by functionally screening hepcidin agonists using a library of 640 FDA-approved drugs in human hepatic Huh7 cells. We validated the results in C57BL/6J mice and a mouse model of hemochromatosis (Hfe-/- mice). Our screen revealed that the anti-rheumatoid arthritis drug auranofin (AUR) potently upregulates hepcidin expression. Interestingly, we found that canonical signaling pathways that regulate iron, including the Bmp/Smad and IL-6/Jak2/Stat3 pathways, play indispensable roles in mediating AUR's effects. In addition, AUR induces IL-6 via the NF-κB pathway. In C57BL/6J mice, acute treatment with 5 mg/kg AUR activated hepatic IL-6/hepcidin signaling and decreased serum iron and transferrin saturation. Whereas chronically treating male Hfe-/- mice with 5 mg/kg AUR activated hepatic IL-6/hepcidin signaling, decreasing systemic iron overload, but less effective in females. Further analyses revealed that estrogen reduced the ability of AUR to induce IL-6/hepcidin signaling in Huh7 cells, providing a mechanistic explanation for ineffectiveness of AUR in female Hfe-/- mice. Notably, high-dose AUR (25 mg/kg) induces ferroptosis and causes lipid peroxidation through inhibition of thioredoxin reductase (TXNRD) activity. We demonstrate the ferroptosis inhibitor ferrostatin significantly protects liver toxicity induced by high-dose AUR without comprising its beneficial effect on iron metabolism. In conclusion, our findings provide compelling evidence that TXNRD is a key regulator of ferroptosis, and AUR is a novel activator of hepcidin and ferroptosis via distinct mechanisms, suggesting a promising approach for treating hemochromatosis and hepcidin-deficiency related disorders.

Dietary Intake of Homocysteine Metabolism-Related B-Vitamins and the Risk of Stroke: A Dose-Response Meta-Analysis of Prospective Studies.

Observational studies regarding the putative associations between dietary intake of homocysteine metabolism-related B-vitamins (vitamin B-6, folate, and vitamin B-12) and stroke risk have yielded inconsistent results. Thus, we conducted a systematic meta-analysis of prospective studies in order to examine the relation between the dietary (from diet and supplements) intake of these B-vitamins and the risk of stroke. PubMed and Web of Science were searched for relevant articles published through to 25 February, 2020, and RR of stroke in relation to dietary intake of vitamin B-6, folate, and vitamin B-12 were pooled using a random-effects model. Eleven publications of 12 prospective studies comprising 389,938 participants and 10,749 cases were included in the final analysis. We found that dietary intake of vitamin B-6 and folate were associated with a reduced risk of stroke, and this inverse association remained significant in studies with >10 y of follow-up periods and among participants without a pre-existing stroke event. A dose-response analysis revealed a linear inverse association between folate and vitamin B-6 intake and the risk of stroke, with a pooled RR of 0.94 (95% CI: 0.90-0.98) and 0.94 (95% CI: 0.89-0.99) for each 100 µg/d increment in folate intake and 0.5 mg/d increment in vitamin B-6 intake, respectively. In contrast, we found no significant association between dietary vitamin B-12 intake and the risk of stroke, with an RR of 1.01 (95% CI: 0.97-1.06) per 3 µg/d increase. In conclusion, our findings suggest that increased intake of vitamin B-6 and folate is associated with a reduced risk of stroke, supporting the notion that increasing habitual folate and vitamin B-6 intake may provide a small but beneficial effect with respect to stroke.

Zinc supplementation improves glycemic control for diabetes prevention and management: a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Although many studies have shown that low zinc status is associated with diabetes, the putative effects of zinc supplementation on glycemic control are inconclusive. OBJECTIVES: The aim of this meta-analysis of randomized controlled trials was to assess the effects of zinc supplementation in preventing and managing diabetes. METHODS: PubMed, Embase, and the Cochrane Library were searched for articles that were published through February 10, 2019 and contained estimates for the outcomes of interest. The pooled results were then analyzed with the use of a random-effects model. RESULTS: Thirty-two placebo-controlled interventions were extracted from 36 publications, involving a total of 1700 participants in 14 countries. Overall, compared with their respective control groups, the subjects in the zinc-supplementation group had a statistically significant reduction in fasting glucose [FG, weighted mean difference (WMD): -14.15 mg/dL; 95% CI: -17.36, -10.93 mg/dL], 2-h postprandial glucose (WMD: -36.85 mg/dL; 95% CI: -62.05, -11.65 mg/dL), fasting insulin (WMD: -1.82 mU/L; 95% CI: -3.10, -0.54 mU/L), homeostasis model assessment for insulin resistance (WMD: -0.73; 95% CI: -1.22, -0.24), glycated hemoglobin (WMD: -0.55%; 95% CI: -0.84, -0.27%), and high-sensitivity C-reactive protein (WMD: -1.31 mg/L; 95% CI: -2.05, -0.56 mg/L) concentrations. Moreover, subgroup analyses revealed that the effects of zinc supplementation on FG are significantly influenced by diabetic status and the formulation of the zinc supplement. CONCLUSIONS: Our analysis revealed that several key glycemic indicators are significantly reduced by zinc supplementation, particularly the FG in subjects with diabetes and in subjects who received an inorganic zinc supplement. Together, these findings support the notion that zinc supplementation may have clinical potential as an adjunct therapy for preventing or managing diabetes. This trial was registered at PROSPERO as CRD42018111838.

Adenine alleviates iron overload by cAMP/PKA mediated hepatic hepcidin in mice.

Hemochromatosis is prevalent and often associated with high rates of morbidity and mortality worldwide. The safe alternative iron-reducing approaches are urgently needed in order to better control iron overload. Our unbiased vitamin screen for modulators of hepcidin, a master iron regulatory hormone, identifies adenine (vitamin B4) as a potent hepcidin agonist. Adenine significantly induced hepcidin mRNA level and promoter activity activation in human cell lines, possibly through BMP/SMAD pathway. Further studies in mice validated the effect of adenine on hepcidin upregulation. Consistently, adenine dietary supplement in mice led to an increase of hepatic hepcidin expression compared with normal diet-fed mice via BMP/SMAD pathway. Notably, adenine-rich diet significantly ameliorated iron overload accompanied by the enhanced hepcidin expression in both high iron-fed mice and in Hfe-/- mice, a murine model of hereditary hemochromatosis. To further validate this finding, we selected pharmacological inhibitors against BMP (LDN193189). We found LDN193189 strongly blocked the hepcidin induction by adenine. Moreover, we uncovered an essential role of cAMP/PKA-dependent axis in triggering adenine-induced hepcidin expression in primary hepatocytes by using 8 br cAMP, a cAMP analog, and H89, a potent inhibitor for PKA signaling. These findings suggest a potential therapeutic role of adenine for hereditary hemochromatosis.

Zinc supplementation plays a crucial role in T helper 9 differentiation in allogeneic immune reactions and non-activated T cells.

T helper (Th) 9 cells play a critical role in immune-mediated diseases, including allergic airway inflammation, autoimmune diseases, and cancer development. Thus, the promotion or suppression of Th9 cell differentiation, transcriptional control, and function is very important for a healthy immune system. Interestingly, T cell maturation, differentiation and function are highly dependent on the individuals' zinc status. This is especially seen in zinc deficient individuals as in the elderly population often suffering of autoimmunity and increased incidence of infections. In this regard, this study examines the impact of zinc supplementation in pharmacological doses on Th9 differentiation in two in vitro models: 1) in mixed lymphocyte cultures (MLC) displaying allogeneic activated T cells in graft versus host disease, and 2) on non-activated resting T cells in peripheral blood mononuclear cells (PBMC). On the one hand, zinc supplementation significantly diminishes IL-4-induced Th9 differentiation in MLC thereby ameliorating this pro-inflammatory allogeneic immunoreaction. On the other hand, Th9 cells are induced in resting T cells in PBMC hence triggering the immunological defense. Thus, zinc supplementation can be considered as useful additive to dampen unwanted allogeneic immunoreactions. Moreover, the pro-inflammatory immune defense in non-reactive T cells can be strengthened, which is a frequent issue in the elderly population having a weakened immune response against invading pathogens.

Metal transporter Slc39a10 regulates susceptibility to inflammatory stimuli by controlling macrophage survival.

Zn plays a key role in controlling macrophage function during an inflammatory event. Cellular Zn homeostasis is regulated by two families of metal transporters, the SLC39A family of importers and the SLC30A family of exporters; however, the precise role of these transporters in maintaining macrophage function is poorly understood. Using macrophage-specific Slc39a10-knockout (Slc39a10fl/fl;LysM-Cre+ ) mice, we found that Slc39a10 plays an essential role in macrophage survival by mediating Zn homeostasis in response to LPS stimulation. Compared with Slc39a10fl/fl mice, Slc39a10fl/fl;LysM-Cre+ mice had significantly lower mortality following LPS stimulation as well as reduced liver damage and lower levels of circulating inflammatory cytokines. Moreover, reduced intracellular Zn concentration in Slc39a10fl/fl;LysM-Cre+ macrophages led to the stabilization of p53, which increased apoptosis upon LPS stimulation. Concomitant knockout of p53 largely rescued the phenotype of Slc39a10fl/fl;LysM-Cre+ mice. Finally, the phenotype in Slc39a10fl/fl;LysM-Cre+ mice was mimicked in wild-type mice using the Zn chelator TPEN and was reversed with Zn supplementation. Taken together, these results suggest that Slc39a10 plays a role in promoting the survival of macrophages through a Zn/p53-dependent axis in response to inflammatory stimuli.

Dietary intake of heme iron and body iron status are associated with the risk of gestational diabetes mellitus: a systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: Some potential role of iron overload in the development of diabetes mellitus have been suggested. Our study aimed to systematically assess the association between the risk of gestational diabetes mellitus (GDM) and iron intakes/body iron status. METHODS AND STUDY DESIGN: PubMed and Web of Science were searched for relevant articles. Relative risks (RR) of GDM in relation to dietary iron intakes and body iron stores were pooled with the random-effects model. Weighted mean differences of iron blood markers between GDM and non-GDM individuals were also analyzed. RESULTS: Twenty-five studies were included in the qualitative analysis, and 23 studies with 29,378 participants and 3,034 GDM patients were included in the quantitative analysis. Dietary intake of heme iron was significantly associated with GDM risk (RR=1.65, 95% CI: 1.28 to 2.12), and the pooled RR for each 1mg/day increment of heme iron intake was 1.38 (95% CI: 1.19 to 1.61). No association between GDM and the intakes of nonheme iron, total iron, or supplemental iron was detected. Body iron stores, as represented by serum ferritin level, were correlated with GDM risk (RR=1.64, 95% CI: 1.27 to 2.11). Moreover, the concentrations of both serum ferritin and serum iron were increased in GDM patients, compared with non-GDM individuals. CONCLUSIONS: Increased dietary intake of heme iron and body iron status are positively associated with the risk of GDM development in pregnant women. Future studies are warranted to better understand the role of iron in GDM development.

Zebrafish slc30a10 deficiency revealed a novel compensatory mechanism of Atp2c1 in maintaining manganese homeostasis.

Recent studies found that mutations in the human SLC30A10 gene, which encodes a manganese (Mn) efflux transporter, are associated with hypermanganesemia with dystonia, polycythemia, and cirrhosis (HMDPC). However, the relationship between Mn metabolism and HMDPC is poorly understood, and no specific treatments are available for this disorder. Here, we generated two zebrafish slc30a10 mutant lines using the CRISPR/Cas9 system. Compared to wild-type animals, mutant adult animals developed significantly higher systemic Mn levels, and Mn accumulated in the brain and liver of mutant embryos in response to exogenous Mn. Interestingly, slc30a10 mutants developed neurological deficits in adulthood, as well as environmental Mn-induced manganism in the embryonic stage; moreover, mutant animals had impaired dopaminergic and GABAergic signaling. Finally, mutant animals developed steatosis, liver fibrosis, and polycythemia accompanied by increased epo expression. This phenotype was rescued partially by EDTA- CaNa2 chelation therapy and iron supplementation. Interestingly, prior to the onset of slc30a10 expression, expressing ATP2C1 (ATPase secretory pathway Ca2+ transporting 1) protected mutant embryos from Mn exposure, suggesting a compensatory role for Atp2c1 in the absence of Slc30a10. Notably, expressing either wild-type or mutant forms of SLC30A10 was sufficient to inhibit the effect of ATP2C1 in response to Mn challenge in both zebrafish embryos and HeLa cells. These findings suggest that either activating ATP2C1 or restoring the Mn-induced trafficking of ATP2C1 can reduce Mn accumulation, providing a possible target for treating HMDPC.

Antioxidants Mediate Both Iron Homeostasis and Oxidative Stress.

Oxidative stress is a common denominator in the pathogenesis of many chronic diseases. Therefore, antioxidants are often used to protect cells and tissues and reverse oxidative damage. It is well known that iron metabolism underlies the dynamic interplay between oxidative stress and antioxidants in many pathophysiological processes. Both iron deficiency and iron overload can affect redox state, and these conditions can be restored to physiological conditions using iron supplementation and iron chelation, respectively. Similarly, the addition of antioxidants to these treatment regimens has been suggested as a viable therapeutic approach for attenuating tissue damage induced by oxidative stress. Notably, many bioactive plant-derived compounds have been shown to regulate both iron metabolism and redox state, possibly through interactive mechanisms. This review summarizes our current understanding of these mechanisms and discusses compelling preclinical evidence that bioactive plant-derived compounds can be both safe and effective for managing both iron deficiency and iron overload conditions.

Selenium Exposure and Cancer Risk: an Updated Meta-analysis and Meta-regression.

The objective of this study was to investigate the associations between selenium exposure and cancer risk. We identified 69 studies and applied meta-analysis, meta-regression and dose-response analysis to obtain available evidence. The results indicated that high selenium exposure had a protective effect on cancer risk (pooled OR = 0.78; 95%CI: 0.73-0.83). The results of linear and nonlinear dose-response analysis indicated that high serum/plasma selenium and toenail selenium had the efficacy on cancer prevention. However, we did not find a protective efficacy of selenium supplement. High selenium exposure may have different effects on specific types of cancer. It decreased the risk of breast cancer, lung cancer, esophageal cancer, gastric cancer, and prostate cancer, but it was not associated with colorectal cancer, bladder cancer, and skin cancer.

Bmp6 expression can be regulated independently of liver iron in mice.

The liver is the primary organ for storing iron and plays a central role in the regulation of body iron levels by secretion of the hormone Hamp1. Although many factors modulate Hamp1 expression, their regulatory mechanisms are poorly understood. Here, we used conditional knockout mice for the iron exporter ferroportin1 (Fpn1) to modulate tissue iron in specific tissues in combination with iron-deficient or iron-rich diets and transferrin (Tf) supplementation to investigate the mechanisms underlying Hamp1 expression. Despite liver iron overload, expression of bone morphogenetic protein 6 (Bmp6), a potent-stimulator of Hamp1 expression that is expressed under iron-loaded conditions, was decreased. We hypothesized that factors other than liver iron must play a role in controlling Bmp6 expression. Our results show that erythropoietin and Tf-bound iron do not underlie the down-regulation of Bmp6 in our mice models. Moreover, Bmp6 was down-regulated under conditions of high iron demand, irrespective of the presence of anemia. We therefore inferred that the signals were driven by high iron demand. Furthermore, we also confirmed previous suggestions that Tf-bound iron regulates Hamp1 expression via Smad1/5/8 phosphorylation without affecting Bmp6 expression, and the effect of Tf-bound iron on Hamp1 regulation appeared before a significant change in Bmp6 expression. Together, these results are consistent with novel mechanisms for regulating Bmp6 and Hamp1 expression.

Black soyabean seed coat extract regulates iron metabolism by inhibiting the expression of hepcidin.

Hepcidin, a key regulator of Fe homeostasis, is an ideal drug target for treating patients with Fe disorders such as haemochromatosis, anaemia of chronic inflammation and Fe-deficiency anaemia. However, whether (and how) traditional Chinese black foods (e.g., black soyabeans) target hepcidin and improve Fe-deficiency anaemia remains unclear. Herein, we report that black soyabean seed coat extract (BSSCE) can potently inhibit the in vitro and in vivo expression of hepcidin. In the present study, in cells treated with 200 µg/ml BSSCE, hepcidin expression was found to be reduced to only 6% of the control levels (P<0.01). An AIN-76A diet containing 2% BSSCE was fed to 8-week-old male C57BL/6 mice for 0, 1, 7, 15 or 30 d; importantly, compared with the day 0 group, the day 7 group exhibited nearly a 50% decrease in hepatic hepcidin expression (P<0.01), a 35% decrease in splenic Fe concentrations (P<0.05) and a 135% increase in serum Fe concentrations (P<0.05). Mechanistically, the effect of BSSCE on hepcidin expression was mediated via a reduction in the phosphorylation levels of mothers against decapentaplegic homolog proteins (Smad)1/5/8. Consequently, the mice in the day 30 group exhibited large increases in erythrocyte counts (111% v. day 0, P<0.01), Hb concentrations (109%, P<0.01) and haematocrit values (108%, P<0.01). In conclusion, these results indicate that black soyabean extract regulates Fe metabolism by inhibiting the expression of hepcidin. This finding can be used to optimise the intervention of patients with hepcidin-related diseases, including Fe-deficiency anaemia.

Characterization of the GufA subfamily member SLC39A11/Zip11 as a zinc transporter.

Cellular zinc influx and efflux are maintained by two major transporter families, the ZIP (SLC39A) and ZnT (SLC30A or CDF) molecules. The functions of one molecule in this class, ZIP11/SLC39A11, remain unclear. Bioinformatics analysis of the distribution and evolutionary relationships of different ZIP members in eukaryotes and prokaryotes indicated that Zip11, the sole member of gufA subfamily, is an ancient ZIP family member that might have originated in early eukaryotic ancestors. Murine Zip11 mRNA is abundantly expressed in testes and the digestive system including stomach, ileum and cecum. Analysis of cellular zinc content, metallothionein levels, and cell viability under high or low zinc conditions in cells transfected with a murine Zip11 expression plasmid, suggest that Zip11 is a zinc importer. Further, cellular zinc concentrations and metallothionein levels decreased when Zip11 was knocked down. In mice supplemented with zinc, both mRNA and protein levels of Zip11 were slightly up-regulated in several tissues. The metal response element sequences (MREs) upstream of the first exon of Zip11 responded to elevated extracellular zinc concentrations, as assessed by luciferase reporter assays. Mutagenic analysis showed that several of the MREs could regulate Zip11 promoter activity, and metal-responsive transcription factor-1 (MTF-1) was shown to be involved in this process. Collectively, these data suggest that Zip11 has unique protein sequence and structure features, it functions as a cellular zinc transporter, and its expression is at least partially regulated by zinc via hMTF-1 binding to MREs of the Zip11 promoter.