The hypoferremic response to acute inflammation is maintained in thalassemia mice even under parenteral iron loading.

BACKGROUND: Hepcidin controls iron homeostasis by inducing the degradation of the iron efflux protein, ferroportin (FPN1), and subsequently reducing serum iron levels. Hepcidin expression is influenced by multiple factors, including iron stores, ineffective erythropoiesis, and inflammation. However, the interactions between these factors under thalassemic condition remain unclear. This study aimed to determine the hypoferremic and transcriptional responses of iron homeostasis to acute inflammatory induction by lipopolysaccharide (LPS) in thalassemic (Hbbth3 /+) mice with/without parenteral iron loading with iron dextran. METHODS: Wild type and Hbbth3 /+ mice were intramuscularly injected with 5 mg of iron dextran once daily for two consecutive days. After a 2-week equilibration, acute inflammation was induced by an intraperitoneal injection of a single dose of 1 µg/g body weight of LPS. Control groups for both iron loading and acute inflammation received equal volume(s) of saline solution. Blood and tissue samples were collected at 6 hours after LPS (or saline) injection. Iron parameters and mRNA expression of hepcidin as well as genes involved in iron transport and metabolism in wild type and Hbbth3 /+ mice were analyzed and compared by Kruskal-Wallis test with pairwise Mann-Whitney U test. RESULTS: We found the inductive effects of LPS on liver IL-6 mRNA expression to be more pronounced under parenteral iron loading. Upon LPS administration, splenic erythroferrone (ERFE) mRNA levels were reduced only in iron-treated mice, whereas, liver bone morphogenetic protein 6 (BMP6) mRNA levels were decreased under both control and parenteral iron loading conditions. Despite the altered expression of the aforementioned hepcidin regulators, the stimulatory effect of LPS on hepcidin mRNA expression was blunt in iron-treated Hbbth3 /+ mice. Contrary to the blunted hepcidin response, LPS treatment suppressed FPN1 mRNA expression in the liver, spleen, and duodenum, as well as reduced serum iron levels of Hbbth3 /+ mice with parenteral iron loading. CONCLUSION: Our study suggests that a hypoferremic response to LPS-induced acute inflammation is maintained in thalassemic mice with parenteral iron loading in a hepcidin-independent manner.

PLGA nanoparticles containing α-fetoprotein siRNA induce apoptosis and enhance the cytotoxic effects of doxorubicin in human liver cancer cell line.

Hepatocellular carcinoma (HCC) is one of the most common cancers and is a leading cause of death. Delivery of therapeutic molecules, e.g., siRNA, to HCC cells could potentially be an alternative treatment for HCC. In this study, the siRNA targeting α-fetoprotein (AFP) mRNA was found to specifically induce apoptosis and significant cell death in HepG2 cells. It also enhanced the cytotoxic effects of doxorubicin by about two-fold, making it the candidate therapeutic molecule for HCC treatment. To deliver the siRNAs into HCC cells, the AFP siRNAs were loaded into the nanoparticles based on poly (lactic-co-glycolic) acid (PLGA). These nanoparticles induced apoptosis in HepG2 cells and synergistically increased the cytotoxicity of doxorubicin. In summary, the delivery of the AFP siRNA-loaded PLGA nanoparticles in combination with doxorubicin could be a very promising approach for the treatment of HCC.

Curcuminoids supplementation ameliorates iron overload, oxidative stress, hypercoagulability, and inflammation in non-transfusion-dependent ß-thalassemia/Hb E patients.

Curcuminoids, polyphenol compounds in turmeric, possess several pharmacological properties including antioxidant, iron-chelating, and anti-inflammatory activities. Effects of curcuminoids in thalassemia patients have been explored in a limited number of studies using different doses of curcuminoids. The present study aims to evaluate the effects of 24-week curcuminoids supplementation at the dosage of 500 and 1000 mg/day on iron overload, oxidative stress, hypercoagulability, and inflammation in non-transfused ß-thalassemia/Hb E patients. In general, both curcuminoids dosages significantly lowered the levels of oxidative stress, hypercoagulability, and inflammatory markers in the patients. In contrast, reductions in iron parameter levels were more remarkable in the 1000 mg/day group. Subgroup analysis revealed that a marker of hypercoagulability was significantly decreased only in patients with baseline ferritin ≤ 1000 ng/ml independently of curcuminoids dosage. Moreover, the alleviation of iron loading parameters was more remarkable in patients with baseline ferritin > 1000 ng/ml who receive 1000 mg/day curcuminoids. On the other hand, the responses of oxidative stress markers were higher with 500 mg/day curcuminoids regardless of baseline ferritin levels. Our study suggests that baseline ferritin levels should be considered in the supplementation of curcuminoids and the appropriate curcuminoids dosage might differ according to the required therapeutic effect. Thai Clinical Trials Registry (TCTR): TCTR20200731003; July 31, 2020 "retrospectively registered".

Iron homeostasis in a mouse model of thalassemia intermedia is altered between adolescence and adulthood.

BACKGROUND: Iron overload is one of common complications of ß-thalassemia. Systemic iron homeostasis is regulated by iron-regulatory hormone, hepcidin, which inhibits intestinal iron absorption and iron recycling by reticuloendothelial system. In addition, body iron status and requirement can be altered with age. In adolescence, iron requirement is increased due to blood volume expansion and growth spurt. Heterozygous ß-globin knockout mice (Hbbth3 /+; BKO) is a mouse model of thalassemia widely used to study iron homeostasis under this pathological condition. However, effects of age on iron homeostasis, particularly the expression of genes involved in hemoglobin metabolism as well as erythroid regulators in the spleen, during adolescence have not been explored in this mouse model. METHODS: Iron parameters as well as the mRNA expression of hepcidin and genes involved in iron transport and metabolism in wildtype (WT) and BKO mice during adolescence (6-7 weeks old) and adulthood (16-20 weeks old) were analyzed and compared by 2-way ANOVA. RESULTS: The transition of adolescence to adulthood was associated with reductions in duodenal iron transporter mRNA expression and serum iron levels of both WT and BKO mice. Erythrocyte parameters in BKO mice remained abnormal in both age groups despite persistent induction of genes involved in hemoglobin metabolism in the spleen and progressively increased extramedullary erythropiesis. In BKO mice, adulthood was associated with increased liver hepcidin and ferroportin mRNA expression along with splenic erythroferrone mRNA suppression compared to adolescence. CONCLUSION: Our results demonstrate that iron homeostasis in a mouse model of thalassemia intermedia is altered between adolescence and adulthood. The present study underscores the importance of the age of thalassemic mice in the study of molecular or pathophysiological changes under thalassemic condition.

Role of stratifin (14-3-3 sigma) in adenocarcinoma of gallbladder: A novel prognostic biomarker.

BACKGROUND: Gallbladder cancer (GBC) is a rare and fatal biliary tract malignancy. Genetic derangements are one of many factors that determine the prognosis of GBC. In this study, the expression of the stratifin (SFN) gene encoding 14-3-3 sigma protein, which is reported to be associated with the metastatic property of cholangiocarcinoma cells, was investigated in GBC. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded cancer (n = 37) and non-cancer control tissues (n = 14) of gallbladders from patients who underwent surgical resection from January 2006 to May 2015 were retrieved. The expression of SFN normalized with that of ACTB was determined using RT-qPCR. Multivariate analysis of factors affecting disease-free survival (DFS) and overall survival (OS) including the type of SFN expression was performed. RESULT: The average expression level of SFN in cancer was higher than that in control tissues (p = 0.002). The relative SFN expression in cancer tissue was classified as overexpression (n = 14) and control level expression (n = 23) according to the receiver operating characteristic (ROC) curves for discriminating early GBC recurrence or metastasis after surgery. The SFN overexpression group was associated with lower rates of distant metastasis and early tumor recurrence following resection. The univariate analysis demonstrated factors affecting DFS, including resection margin (p < 0.001), lymphovascular invasion (p = 0.040), perineural invasion (p = 0.046), and SFN expression (p < 0.001). The multivariate analysis revealed that the resection margin (p = 0.019) and SFN expression (P = 0.040) were independent prognostic factors of DFS. CONCLUSION: To achieve the longest survival, margin-free resection is recommended. The overexpression of SFN in GBC is associated with better prognosis, lower rates of early cancer recurrence, and distant metastasis following resection. SFN expression might be a novel prognostic biomarker in GBC treatment. Further studies to elucidate the role of SFN might unveil its clinical benefit in cancer treatment regimens.

Differentially expressed plasma proteins of ß-thalassemia/hemoglobin E patients in response to curcuminoids/vitamin E antioxidant cocktails.

OBJECTIVE: Iron overload and oxidative stress are the major causes of serious complications and mortality in thalassemic patients. Our previous work supports the synergistic effects of antioxidant cocktails (curcuminoids or vitamin E, N-acetylcysteine, and deferiprone) in treatment of ß-thalassemia/Hb E patients. This further 2-DE-based proteomic study aimed to identify the plasma proteins that expressed differentially in response to antioxidant cocktails. METHODS: Frozen plasma samples of ten normal subjects and ten ß-thalassemia/Hb E patients at three-time points (baseline, month 6, and month 12) were reduced the dynamic range of proteome using ProteoMiner kit and separated proteins by two-dimensional gel electrophoresis. Differentially expressed proteins were identified using tandem mass spectrometry. Several plasma proteins were validated by ELISA and Western blot analysis. RESULTS: Thirteen and 11 proteins were identified with altered expression levels in the curcuminoids- and vitamin E cocktail groups, respectively. The associations between vitronectin (VTN) expression and total bilirubin levels, as well as between serum paraoxonase/arylesterase 1 (PON1) expression and blood reactive oxygen species were observed. Validation results were consistent with proteomics results. DISCUSSION AND CONCLUSIONS: These plasma proteins may provide better understanding of the mechanisms underlying the therapeutic effects of antioxidant cocktails in thalassemic patients.

Expression of ABCG2 (BCRP) in mouse models with enhanced erythropoiesis.

Haem is a structural component of numerous cellular proteins which contributes significantly to iron metabolic processes in mammals but its toxicity demands that cellular levels must be tightly regulated. Breast Cancer Resistance Protein (BCRP/ABCG2), an ATP Binding Cassette G-member protein has been shown to possess porphyrin/haem efflux function. The current study evaluated the expression and regulation of Abcg2 mRNA and protein levels in mouse tissues involved in erythropoiesis. Abcg2 mRNA expression was enhanced in bone marrow hemopoietic progenitor cells from mice that were treated with phenylhydrazine (PHZ). Abcg2 mRNA expression was increased particularly in the extramedullary haematopoietic tissues from all the mice models with enhanced erythropoiesis. Haem oxygenase (ho1) levels tended to increase in the liver of mice with enhanced erythropoiesis and gene expression patterns differed from those observed in the spleen. Efflux of haem biosynthetic metabolites might be dependent on the relative abundance of Abcg2 or ho1 during erythropoiesis. Abcg2 appears to act principally as a safety valve regulating porphyrin levels during the early stages of erythropoiesis and its role in systemic haem metabolism and erythrophagocytosis, in particular, awaits further clarification.

The transcription factor ATOH8 is regulated by erythropoietic activity and regulates HAMP transcription and cellular pSMAD1,5,8 levels.

ATOH8 has previously been shown to be an iron-regulated transcription factor, however its role in iron metabolism is not known. ATOH8 expression in HEK293 cells resulted in increased endogenous HAMP mRNA levels as well as HAMP promoter activity. Mutation of the E-box or SMAD response elements within the HAMP promoter significantly reduced the effects of ATOH8, indicating that ATOH8 activates HAMP transcription directly as well as through bone morphogenic protein (BMP) signalling. In support of the former, Chromatin immunoprecipitation assays provided evidence that ATOH8 binds to E-box regions within the HAMP promoter while the latter was supported by the finding that ATOH8 expression in HEK293 cells led to increased phosphorylated SMAD1,5,8 levels. Liver Atoh8 levels were reduced in mice under conditions associated with increased erythropoietic activity such as hypoxia, haemolytic anaemia, hypotransferrinaemia and erythropoietin treatment and increased by inhibitors of erythropoiesis. Hepatic Atoh8 mRNA levels increased in mice treated with holo transferrin, suggesting that Atoh8 responds to changes in plasma iron. ATOH8 is therefore a novel transcriptional regulator of HAMP, which is responsive to changes in plasma iron and erythroid activity and could explain how changes in erythroid activity lead to regulation of HAMP.

Protective effects of mangosteen extract on H2O2-induced cytotoxicity in SK-N-SH cells and scopolamine-induced memory impairment in mice.

Mangosteen extracts (ME) contain high levels of polyphenolic compounds and antioxidant activity. Protective effects of ME against ß-amyloid peptide (Aß), induced cytotoxicity have been reported. Here, we further studied the protective effects of ME against oxidative stress induced by hydrogen peroxide (H2O2) and polychlorinated biphenyls (PCBs), and demonstrated the protection against memory impairment in mice. The cytoprotective effects of ME were measured as cell viability and the reduction in ROS activity. In SK-N-SH cell cultures, 200 µg/ml ME could partially antagonize the effects of 150 or 300 µM H2O2 on cell viability, ROS level and caspase-3 activity. At 200, 400 or 800 µg/ml, ME reduced AChE activity of SK-N-SH cells to about 60% of the control. In vivo study, Morris water maze and passive avoidance tests were used to assess the memory of the animals. ME, especially at 100 mg/kg body weight, could improve the animal's memory and also antagonize the effect of scopolamine on memory. The increase in ROS level and caspase-3 activity in the brain of scopolamine-treated mice were antagonized by the ME treatment. The study demonstrated cytoprotective effects of ME against H2O2 and PCB-52 toxicity and having AChE inhibitory effect in cell culture. ME treatment in mice could attenuate scopolamine-induced memory deficit and oxidative stress in brain.

Regulation of iron metabolism in Hamp (-/-) mice in response to iron-deficient diet.

BACKGROUND: Hepcidin, the liver-secreted iron regulatory peptide, maintains systemic iron homeostasis in response to several stimuli including dietary iron levels and body iron status. In addition, iron metabolism is controlled by several local regulatory mechanisms including IRP and Hif-2α activities independently of hepcidin. However, the roles of these mechanisms and their interaction particularly in hepcidin-deficient individuals are not yet fully understood. We, therefore, aimed to explore whether Hamp disruption affects iron homeostatic responses to dietary iron deficiency. METHODS: Hepcidin1 knockout (Hamp (-/-)) mice and heterozygous littermates were fed with control or iron-deficient diet for 2 weeks. The expression of iron-related genes and proteins were determined by quantitative PCR and Western blot, respectively. RESULTS: Two-week iron-deficient diet feeding in Hamp (-/-) mice did not alter serum iron but significantly reduced liver non-heme iron levels. This was also associated with increased ferroportin protein expression in the duodenum and spleen, whereas decreased expression was found in the liver. In addition, significant inductive effects of iron-deficient diet on Dcytb and DMT1 mRNA expression in the duodenum were noted with more pronounced effects in Hamp (-/-) mice compared with controls. CONCLUSIONS: Hamp (-/-) mice exhibited a more dramatic increase in the expression of iron transport machinery, which may be responsible for the unaltered serum iron levels upon iron-deficient diet feeding in these mice. Despite the lack of hepcidin, Hamp (-/-) mice can maintain a degree of iron homeostasis in response to altered dietary iron through several hepcidin-independent mechanisms.

Duodenal reductase activity and spleen iron stores are reduced and erythropoiesis is abnormal in Dcytb knockout mice exposed to hypoxic conditions.

Duodenal cytochrome b (Dcytb, Cybrd1) is a ferric reductase localized in the duodenum that is highly upregulated in circumstances of increased iron absorption. To address the contribution of Dcytb to total duodenal ferric reductase activity as well as its wider role in iron metabolism, we first measured duodenal ferric reductase activity in wild-type (WT) and Dcytb knockout (Dcytb(-/-)) mice under 3 conditions known to induce gut ferric reductase: dietary iron deficiency, hypoxia, and pregnancy. Dcytb(-/-) and WT mice were randomly assigned to control (iron deficiency experiment, 48 mg/kg dietary iron; hypoxia experiment, normal atmospheric pressure; pregnancy experiment, nonpregnant animals) or treatment (iron deficiency experiment, 2-3 mg/kg dietary iron; hypoxia experiment, 53.3 kPa pressure; pregnancy experiment, d 20 of pregnancy) groups and duodenal reductase activity measured. We found no induction of ferric reductase activity in Dcytb(-/-) mice under any of these conditions, indicating there are no other inducible ferric reductases present in the duodenum. To test whether Dcytb was required for iron absorption in conditions with increased erythropoietic demand, we also measured tissue nonheme iron levels and hematological indices in WT and Dcytb(-/-) mice exposed to hypoxia. There was no evidence of gross alterations in iron absorption, hemoglobin, or total liver nonheme iron in Dcytb(-/-) mice exposed to hypoxia compared with WT mice. However, spleen nonheme iron was significantly less (6.7 ± 1.0 vs. 12.7 ± 0.9 nmol · mg tissue(-1); P < 0.01, n = 7-8) in hypoxic Dcytb(-/-) compared with hypoxic WT mice and there was evidence of impaired reticulocyte hemoglobinization with a lower reticulocyte mean corpuscular hemoglobin (276 ± 1 vs. 283 ± 2 g · L(-1); P < 0.05, n = 7-8) in normoxic Dcytb(-/-) compared with normoxic WT mice. We therefore conclude that DCYTB is the primary iron-regulated duodenal ferric reductase in the gut and that Dcytb is necessary for optimal iron metabolism.

Iron metabolism in hepcidin1 knockout mice in response to phenylhydrazine-induced hemolysis.

Hepcidin, an iron regulatory peptide, plays a central role in the maintenance of systemic iron homeostasis by inducing the internalization and degradation of the iron exporter, ferroportin. Hepcidin expression in the liver is regulated in response to several stimuli including iron status, erythropoietic activity, hypoxia and inflammation. Hepcidin expression has been shown to be reduced in phenylhydrazine-treated mice, a mouse model of acute hemolysis. In this mouse model, hepcidin suppression was associated with increased expression of molecules involved in iron transport and recycling. The present study aims to explore whether the response to phenylhydrazine treatment is affected by hepcidin deficiency and/or the subsequently altered iron metabolism. Hepcidin1 knockout (Hamp(-/-)) and wild type mice were treated with phenylhydrazine or saline and parameters of iron homeostasis were determined 3 days after the treatment. In wild type mice, phenylhydrazine administration resulted in significantly reduced serum iron, increased tissue non-heme iron levels and suppressed hepcidin expression. The treatment was also associated with increases in membrane ferroportin protein levels and spleen heme oxygenase 1 mRNA expression. In addition, trends toward increased mRNA expression of duodenal iron transporters were also observed. In contrast, serum iron and tissue non-heme iron levels in Hamp(-/-) mice were unaffected by the treatment. Moreover, the effects of phenylhydrazine on the expression of ferroportin and duodenal iron transporters were not observed in Hamp(-/-) mice. Interestingly, mRNA levels of molecules involved in splenic heme uptake and degradation were significantly induced by Hamp disruption. In summary, our study demonstrates that the response to phenylhydrazine-induced hemolysis differs between wild type and Hamp(-/-) mice. This observation may be caused by the absence of hepcidin per se or the altered iron homeostasis induced by the lack of hepcidin in these mice.

BMPER protein is a negative regulator of hepcidin and is up-regulated in hypotransferrinemic mice.

The BMP/SMAD4 pathway has major effects on liver hepcidin levels. Bone morphogenetic protein-binding endothelial cell precursor-derived regulator (Bmper), a known regulator of BMP signaling, was found to be overexpressed at the mRNA and protein levels in liver of genetically hypotransferrinemic mice (Trf(hpx/hpx)). Soluble BMPER peptide inhibited BMP2- and BMP6-dependent hepcidin promoter activity in both HepG2 and HuH7 cells. These effects correlated with reduced cellular levels of pSMAD1/5/8. Addition of BMPER peptide to primary human hepatocytes abolished the BMP2-dependent increase in hepcidin mRNA, whereas injection of Bmper peptide into mice resulted in reduced liver hepcidin and increased serum iron levels. Thus Bmper may play an important role in suppressing hepcidin production in hypotransferrinemic mice.

Iron absorption in hepcidin1 knockout mice.

Hepcidin, the Fe-regulatory peptide, has been shown to inhibit Fe absorption and reticuloendothelial Fe recycling. The present study was conducted to explore the mechanism of in vivo Fe regulation through genetic disruption of hepcidin1 and acute effects of hepcidin treatment in hepcidin1 knockout (Hepc1-/-) and heterozygous mice. Hepcidin1 disruption resulted in significantly increased intestinal Fe uptake. Hepcidin injection inhibited Fe absorption in both genotypes, but the effects were more evident in the knockout mice. Hepcidin administration was also associated with decreased membrane localisation of ferroportin in the duodenum, liver and, most significantly, in the spleen of Hepc1-/- mice. Hypoferraemia was induced in heterozygous mice by hepcidin treatment, but not in Hepc1-/- mice, 4 h after injection. Interestingly, Fe absorption and serum Fe levels in Hepc1-/- and heterozygous mice fed a low-Fe diet were not affected by hepcidin injection. The present study demonstrates that hepcidin deficiency causes increased Fe absorption. The effects of hepcidin were abolished by dietary Fe deficiency, indicating that the response to hepcidin may be influenced by dietary Fe level or Fe status.