Melittin Regulates Iron Homeostasis as a Key Mediator of Macrophage Polarization in Rat Lumbar Spinal Stenosis

BackgroundLumbar spinal stenosis (LSS) is defined as the narrowing of the spinal canal, which compresses the nerves traveling through the lower back into the legs. Inflammation is the most common cause of LSS. Chronic pain induced by nerve damage results from chronic inflammation, and the inflammation response worsens with elevated iron stores. Furthermore, macrophage polarization to the M1 (inflammatory) or M2 (anti-inflammatory) type is essential for controlling host defense or repairing tissues. However, the precise function of macrophage polarization in iron release or retention in LSS pathophysiology is not well-understood. Here, we introduce melittin to modulate macrophage polarization related to iron metabolism for LSS treatment.MethodsPrimary peritoneal macrophage were cultured in 200 or 500 ng/mL of melittin and FeSO4-containing medium for 24 h. Macrophage polarization was assessed by Immunofluorescence staining to CD86 or Arg1 antibodies. In an in vivo rat model of LSS, melittin were administered at 100 and 250 µg/kg, and in vivo effects of melittin on iron deposition-induced macrophage polarization was evaluated by immunochemistry, real time-PCR, western blot, and flow-cytometry. The locomotor functions were assessed by BBB, ladder scoring, and Von Frey test for up to 3 weeks. ResultsIn vitro experiments demonstrated that macrophages can be polarized toward an M2 phenotype after melittin treatment in iron-insulted primary macrophages. Treatment with 100 and 250 μg/kg melittin in a rat LSS model increased the proportion of M2 macrophages in the damaged spinal cord. Moreover, we found that melittin attenuated iron overload-induced M1 polarization via regulating iron metabolism-related genes in LSS rats. As a result, melittin improved locomotor recovery and stimulated axonal growth following LSS.ConclusionsMelittin can promote functional recovery in LSS models by activating M2 macrophages via controlling macrophage iron metabolism, suggesting the potential applications of melittin for treating LSS.

X-ray Characterization of Conformational Changes of Human Apo- and Holo-Transferrin

Human serum transferrin (Tf) is a bilobed glycoprotein whose function is to transport iron through receptor-mediated endocytosis. The mechanism for iron release is pH-dependent and involves conformational changes in the protein, thus making it an attractive system for possible biomedical applications. In this contribution, two powerful X-ray techniques, namely Macromolecular X-ray Crystallography (MX) and Small Angle X-ray Scattering (SAXS), were used to study the conformational changes of iron-free (apo) and iron-loaded (holo) transferrin in crystal and solution states, respectively, at three different pH values of physiological relevance. A crystallographic model of glycosylated apo-Tf was obtained at 3.0 Å resolution, which did not resolve further despite many efforts to improve crystal quality. In the solution, apo-Tf remained mostly globular in all the pH conditions tested; however, the co-existence of closed, partially open, and open conformations was observed for holo-Tf, which showed a more elongated and flexible shape overall.

Berberine alleviates liver fibrosis through inducing ferrous redox to activate ROS-mediated hepatic stellate cells ferroptosis

Berberine (BBR) has been explored as a potential anti-liver fibrosis agent, but the underlying mechanisms are unknown. In the current study, we aimed to investigate the molecular mechanisms underlying the effect of BBR against liver fibrogenesis in thioacetamide (TAA) and carbon tetrachloride (CCl4) induced mouse liver fibrosis. In addition to i.p. injection with TAA or CCl4, mice in the treatment group received BBR intragastrically. Concurrently, combined with TAA and BBR treatment, mice in the inhibitor group were injected i.p. with ferrostatin-1 (Fer-1). Hepatic stellate cells (HSCs) were also used in the study. Our results showed that BBR obviously alleviated mouse liver fibrosis and restored mouse liver function; however, the pharmacological effects of BBR against liver fibrosis were significantly diminished by Fer-1 treatment. Mechanically, BBR impaired the autophagy–lysosome pathway (ALP) and increased cell reactive oxygen species (ROS) production in HSCs. ROS accelerated the breakdown of the iron-storage protein ferritin and sped up iron release from ferritin, which resulted in redox-active iron accumulation in HSCs. Lipid peroxidation and glutathione (GSH) depletion triggered by the Fenton reaction promoted ferroptosis and attenuated liver fibrosis. Furthermore, impaired autophagy enhanced BBR-mediated ferritin proteolysis to increase cellular ferrous overload via the ubiquitin–proteasome pathway (UPS) in HSCs and triggered HSC ferroptosis. Collectively, BBR alleviated liver fibrosis by inducing ferrous redox to activate ROS-mediated HSC ferroptosis. Our findings may be exploited clinically to provide a potential novel therapeutic strategy for liver fibrosis.

Iron Encapsulation by Deacetylated Glucomannan as an Excipient Using the Gelation Method: Characteristics and Controlled Release

Research background. Deacetylation and the use of CaCl2 as a gelation agent improve the performance of glucomannan as iron encapsulant using the gelation method. This study was conducted to investigate the effects of deacetylation using NaOH and pH gelation on the characteristics of encapsulated iron using the CaCl2 gelation method. Experimental approach. Glucomannan was deacetylated at various NaOH concentrations and was subsequently utilized as an iron excipient using the pipette-dropped gelation method in CaCl2 solution to directly investigate the gelation process of encapsulation. The pH of the gelation solution was also changed. The beads were subsequently vacuum-dried. Results and conclusions. Deacetylation led to lower endothermic peak temperature of the glucomannan than that of the native one. The deacetylation degree (DD) and gelation pH did not significantly affect the diameter of the beads but influenced their appearance and physical characteristics. The backbone of glucomannan was not changed by either the deacetylation degree or the pH of the gelation treatment. The highest encapsulation efficiency (73.27 %) was observed in the encapsulated iron using the glucomannan matrix of the highest deacetylation degree (82.56 %) and gelated in pH=10 solution. The highest deacetylation degree of glucomannan caused the beads to have the highest swelling, which led to the release of a higher amount of iron. Glucomannan deacetylation improved the pH sensitivity of iron encapsulation, in which more iron was released at a pH=6.8 than of pH=1.2. The Weibull model was the best-fitted model to represent the profile of iron release from the deacetylated glucomannan matrix using the gelation method (R2 > 0.93) at pH=6.8 and pH=1.2 solutions. Novelty and scientific contribution. This result supports the application of deacetylated glucomannan using NaOH as a pH-sensitive matrix on iron encapsulation using CaCl2 solution as gelation agent. A higher deacetylation degree leads to the release of a higher amount of iron from the matrix. The encapsulation is not only protecting the iron but also delivering it to the absorption site and controlling the iron release which are useful in supplement formulation. or food fortifications. The results show that the deacetylated glucomannan as the matrix holds more iron in encapsulation process.

Antibacterial Activity and Iron Release of Organic-Inorganic Hybrid Biomaterials Synthesized via the Sol-Gel Route

The aim of this work was the synthesis of hybrid materials of iron (II)-based therapeutic systems via the sol-gel method. Increasing amounts of polyethylene glycol (PEG 6, 12, 24, 50 wt%) were added to SiO2/Fe20 wt% to modulate the release kinetics of the drug from the systems. Fourier-transform infrared (FTIR) spectroscopy was used to study the interactions between different components in the hybrid materials. The release kinetics in a simulated body fluid (SBF) were investigated, and the amount of Fe2+ released was detected via ultraviolet-visible spectroscopy (UV-Vis) after reaction with ortho-phenanthroline. Furthermore, biological characterization was carried out. The bioactivity of the synthesized hybrid materials was evaluated via the formation of a layer of hydroxyapatite on the surface of samples soaked in SBF using spectroscopy. Finally, the potential antibacterial properties of seven different materials against two different bacteria—E. coli and S. aureus—were investigated.

Role of Hepcidin as a biomarker for iron status in patients on regular hemodialysis

Background The etiology of anemia in End Stage Renal Disease is multifactorial. Importantly, ESRD patients also have several abnormalities in systemic homeostasis of iron, an essential component in the production of red blood cells. Aim of the Work to assess hepcidin level in negative virology End Stage Renal Disease patients & its relation to iron level and erythropoiesis. Patients and Methods This study was conducted on 45 patients who are stage V chronic kidney disease on regular haemodialysis. Ten age and sex matched controls were included in the study. The study included 29 (64.4%) males and 16 (35.6%) females; their mean age was 53.40± 11.56 years. The prevalence of diabetes among the studied cases was 17.8%, while that of hypertensive was 42.2%. Mean of serum iron level was 64.23±19.53. Mean of TIBC was 409.96±67.85. Mean of Ferritin level 394.55±139.23 and mean of Hepcidin level was 218.51±127. Results Significant negative correlation between Hepcidin level and the Hemoglobin level, and highly significant positive correlation between Hepcidin level and serum Ferritin. Hepcidin up-regulation in the setting of CKD, with subsequent increased serum levels, results in impaired iron absorption from the intestine and decreased iron release from body storage sites. Ultimately, in the setting of such elevated levels, a state of functional iron deficiency may develop and lead to anemia due to iron-restricted erythropoiesis. Conclusion Based on current evidence, it seems likely that hepcidin represents a potentially modifiable mediator of anemia of CKD and is thus a potential target for future anemia therapy.

Towards Bioleaching of a Vanadium Containing Magnetite for Metal Recovery

Vanadium – a transition metal – is found in the ferrous-ferric mineral, magnetite. Vanadium has many industrial applications, such as in the production of high-strength low-alloy steels, and its increasing global industrial consumption requires new primary sources. Bioleaching is a biotechnological process for microbially catalyzed dissolution of minerals and wastes for metal recovery such as biogenic organic acid dissolution of bauxite residues. In this study, 16S rRNA gene amplicon sequencing was used to identify microorganisms in Nordic mining environments influenced by vanadium containing sources. These data identified gene sequences that aligned to the Gluconobacter genus that produce gluconic acid. Several strategies for magnetite dissolution were tested including oxidative and reductive bioleaching by acidophilic microbes along with dissimilatory reduction by Shewanella spp. that did not yield significant metal release. In addition, abiotic dissolution of the magnetite was tested with gluconic and oxalic acids, and yielded 3.99 and 81.31% iron release as a proxy for vanadium release, respectively. As a proof of principle, leaching via gluconic acid production by Gluconobacter oxydans resulted in a maximum yield of 9.8% of the available iron and 3.3% of the vanadium. Addition of an increased concentration of glucose as electron donor for gluconic acid production alone, or in combination with calcium carbonate to buffer the pH, increased the rate of iron dissolution and final vanadium recoveries. These data suggest a strategy of biogenic organic acid mediated vanadium recovery from magnetite and point the way to testing additional microbial species to optimize the recovery.

Decreased ferroportin in hepatocytes promotes macrophages polarize towards an M2-like phenotype and liver fibrosis

Iron release from macrophages is closely regulated by the interaction of hepcidin, a peptide hormone produced by hepatocytes, with the macrophage iron exporter ferroportin (FPN1). However, the functions of FPN1 in hepatocyte secretion and macrophage polarization remain unknown. CD68 immunohistochemical staining and double immunofluorescence staining for F4/80 and Ki67 in transgenic mouse livers showed that the number of macrophages in FPN1−/+ and FPN1−/− mouse livers was significantly increased compared to that in WT (FPN+/+) mice. FPN1 downregulation in hepatic cells increased the levels of the M2 markers CD206, TGF- β, VEGF, MMP-9, Laminin, Collagen, IL-4 and IL-10. Furthermore, the expression of CD16/32 and iNOS, as M1 markers, exhibited the opposite trend. Meanwhile, α-SMA immunohistochemistry and Sirius red staining showed that the trend of liver fibrosis in FPN1−/− mice was more significant than that in control mice. Similarly, in vitro FPN1 knockdown in L02-Sh/L02-SCR liver cell lines yielded similar results. Taken together, we demonstrated that downregulated FPN1 expression in hepatocytes can promote the proliferation and polarization of macrophages, leading to hepatic fibrosis. Above all, the FPN1 axis might provide a potential target for hepatic fibrosis.

Intestinal iron absorption is appropriately modulated to match physiological demand for iron in wild-type and iron-loaded Hamp (hepcidin) knockout rats during acute colitis

Mucosal damage, barrier breach, inflammation, and iron-deficiency anemia (IDA) typify ulcerative colitis (UC) in humans. The anemia in UC appears to mainly relate to systemic inflammation. The pathogenesis of this ‘anemia of inflammation’ (AI) involves cytokine-mediated transactivation of hepatic Hamp (encoding the iron-regulatory hormone, hepcidin). In AI, high hepcidin represses iron absorption (and iron release from stores), thus lowering serum iron, and restricting iron for erythropoiesis (causing anemia). In less-severe disease states, inflammation may be limited to the intestine, but whether this perturbs iron homeostasis is uncertain. We hypothesized that localized gut inflammation will increase overall iron demand (to support the immune response and tissue repair), and that hepatic Hamp expression will decrease in response, thus derepressing (i.e., enhancing) iron absorption. Accordingly, we developed a rat model of mild, acute colitis, and studied iron absorption and homeostasis. Rats exposed (orally) to DSS (4%) for 7 days had intestinal (but not systemic) inflammation, and biomarker analyses demonstrated that iron utilization was elevated. Iron absorption was enhanced (by 2-3-fold) in DSS-treated, WT rats of both sexes, but unexpectedly, hepatic Hamp expression was not suppressed. Therefore, to gain a better understanding of regulation of iron absorption during acute colitis, Hamp KO rats were used for further experimentation. The severity of DSS-colitis was similar in Hamp KOs as in WT controls. In the KOs, increased iron requirements associated with the physiological response to colitis were satisfied by mobilizing hepatic storage iron, rather than by increasing absorption of enteral iron (as occurred in WT rats). In conclusion then, in both sexes and genotypes of rats, iron absorption was appropriately modulated to match physiological demand for dietary iron during acute intestinal inflammation, but regulatory mechanisms may not involve hepcidin.

Synthesis and Characterization of CSH/CS/n-HA Composite Scaffold for Bone Tissue Engineering

In this paper, chitosan/hydroxyapatite (CS/n-HA) were synthesized by ultrasound-assisted precipitation combined with inverse crosslinking-emulsion method. In order to obtain a scaffold material with excellent properties, Calcium sulfate hemihydrate (CSH) were combined with CS-HA obtained CSH/CS/n-HA composite scaffold via setting citric acid as solidifying liquid, which possessed better biodegradability, bioactivity, mechanical properties. The physicochemical, morphological properties of scaffolds were characterized by FTIR, XRD and TFSEM. In addition, explored were the mechanical, degradable, biocompatibility and iron release properties. The mechanical strength study indicated that the compressive strength of the porous composite scaffold was influenced by adding an appropriate amount of CS/n-HA composite microspheres. It was proved that the composite scaffold with 6% CS/n-HA content obtained the highest mechanical strength (17.46±1.29 MPa). The results illustrated that the composite scaffold possessed biodegradability and can form hydroxyapatite by dynamic balance of Ca and P elements. The hemolysis tests demonstrate that materials are non-hemolytic and have good blood compatibility. Therefore, the developed composite scaffolds are safe medical inorganic materials, which can potentially be applied in bone tissue engineering.