Brain iron deposition is positively correlated with cognitive impairment in patients with chronic cerebral hypoperfusion: a MRI susceptibility mapping study.

AIM: To investigate the relationship of brain iron deposition with cognitive impairment in patients with chronic cerebral hypoperfusion (CHP). MATERIALS AND METHODS: Brain iron deposition was detected using quantitative susceptibility mapping (QSM), and cognitive function by neuropsychological tests including the Mini Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), Activities of Daily Living (ADLs), and verbal fluency tests in a total of 40 participants, 23 with CHP and 17 age- and sex-matched healthy participants without CHP (controls). RESULTS: The neuropsychological tests revealed that cognitive impairment (p<0.05) and susceptibility values (p<0.05) of the bilateral hippocampus (HP) and substantia nigra (SN) in CHP patients were significantly higher than those of the controls. The susceptibility values of bilateral HP and left putamen correlated closely with the scores of neuropsychological tests in the CHP patients (p<0.05, r2>0.1). The susceptibility values in the left putamen and bilateral HP were significantly higher in CHP patients with mild cognitive impairment (MCI; n=8) than those of CHP patients without MCI (n=15; p<0.05). CONCLUSIONS: The present findings indicated that brain iron deposition in specific areas may be responsible for the cognitive impairment in CHP patients, and that QSM is a useful tool to determine brain iron, predicting cognitive impairment in CHP patients.

Association between air pollution and atopic dermatitis in Guangzhou, China: modification by age and season.

BACKGROUND: The short-term effect of ambient air pollution on atopic dermatitis (AD), along with its effect modifiers, has not been fully addressed. OBJECTIVES: To examine the short-term associations between air pollution and AD, and to identify effect modifications by age and season. METHODS: We used the generalized additive model to evaluate the short-term effect of ambient air pollution on daily hospital visits for AD, adjusting for potential confounders. Subgroup analyses were performed to identify potential effect modifications by season and age (< 18 years and ≥ 18 years). RESULTS: A total of 29 972 hospital visits for AD were recorded in Guangzhou, China, from 19 January 2013 to 31 December 2017. Among them, 72·8% were visits by children and 51·4% occurred in the cool season. Acute and delayed effects on AD hospital visits were significant for all air pollutants. Stronger effects were seen in the cool season (approximately 1·7-3·0 times higher than effects in the warm season). Stronger effects were also observed in children (approximately 1·3-1·8 times higher than effects in adults). Sensitivity analyses indicated the results were robust. CONCLUSIONS: Air pollution might be an important trigger for AD in subtropical Guangzhou, China. Children are more vulnerable than adults, and the effects are stronger in the cool season.

Role of hepcidin in murine brain iron metabolism.

Brain iron homeostasis is maintained by a balance of both iron uptake and release, and accumulating evidence has revealed that brain iron concentrations increase with aging. Hepcidin, an iron regulatory hormone produced by hepatocytes in response to inflammatory stimuli, iron, and hypoxia, has been shown to be the long-sought hormone responsible for the regulation of body iron balance and recycling in mammals. In this study, we report that hepcidin is widely expressed in the murine brain. In cerebral cortex, hippocampus and striatum, hepcidin mRNA levels increased with aging. Injection of hepcidin into the lateral cerebral ventricle resulted in decreased Fpn1 protein levels in cerebral cortex, hippocampus, and striatum. Additionally, treatment of primary cultured neurons with hepcidin caused decreased neuronal iron release and Fpn1 protein levels. Together, our data provide further evidence that hepcidin may be involved in the regulation of brain iron metabolism.

Cyclophosphamide induces a significant increase in iron content in the liver and spleen of mice.

OBJECTIVE: Oxidative stress is one of the major mechanisms of cyclophosphamide (CPX)-induced toxicities. However, it is unknown how CPX induces oxidative stress. Based on the available information, we speculated that CPX could increase iron content in the tissues and then induce oxidative stress. METHOD: We tested this hypothesis by investigating the effects of CPX on iron and ferritin contents, expression of transferrin receptor 1 (TfR1), ferroportin 1 (Fpn1), iron regulatory proteins (IRPs), hepcidin, and nuclear factor erythroid 2-related factor-2 (Nrf2) in the liver and spleen, and also on reticulocyte count, immature reticulocyte fraction, and hemoglobin (Hb) in the blood in c57/B6 mouse. RESULTS: We demonstrated that CPX could induce a significant increase in iron contents and ferritin expression in the liver and spleen, notably inhibit erythropoiesis and Hb synthesis and lead to a reduction in iron usage. The reduced expression in TfR1 and Fpn1 is a secondary effect of CPX-induced iron accumulation in the liver and spleen and also partly associated with the suppressed IRP/iron-responsive element system, upregulation of hepcidin, and downregulation of Nrf2. CONCLUSIONS: The reduced iron usage is one of the causes for iron overload in the liver and spleen and the increased tissue iron might be one of the mechanisms for CPX to induce oxidative stress and toxicities.

Simultaneous determination of nucleobases, nucleosides and saponins in Panax notoginseng using multiple columns high performance liquid chromatography.

A new multiple columns HPLC method for simultaneous determination of 16 characteristic components, 5 nucleobases and nucleosides (uracil, cytidine, uridine, guanosine and adenosine), and 11 saponins (notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, notoginsenoside R4, notoginsenoside Fa, ginsenoside Rb1, notoginsenoside R2, ginsenoside Rg2, ginsenoside Rh1, ginsenoside Rd and notoginsenoside K), in the root of Panax notoginseng, a valued traditional Chinese medicinal herb, were developed. Notoginsenoside R4, Fa and K were first quantitatively determined in P. notoginseng. The 5 nucleobases and nucleosides compounds were separated on a Zorbax SB-Aq column (150 x 4.6 mm, 5.0 microm) and 11 saponins were analyzed using a Zorbax Bonus-RP column (150 x 4.6 mm, 5.0 microm) with column switching. The column temperature was set at 30 degrees C. Mobile phase was composed of 5mM ammonium acetate aqueous (A), water (B) and acetonitrile (C) using a gradient elution. The flow rate was 1.5 mL/min and detection wavelengths were set at 260 nm for nucleobases and nucleosides, and 203 nm for saponins. The developed method had good repeatability and sensitivity for quantification of 16 analytes with overall precision (including intra- and inter-day) less than 3% (RSD), and LOD and LOQ were less than 1.33 microg/mL and 5.12 microg/mL, respectively. The method was successfully applied to the simultaneous determination of 16 analytes in 15 samples of P. notoginseng collected from different places of China, which indicated that multiple columns HPLC can be used for comprehensive quality control of P. notoginseng.

Neuroprotective role of Z-ligustilide against forebrain ischemic injury in ICR mice.

Radix Angelica sinensis, known as Danggui in Chinese, has been used to treat cardiovascular and cerebrovascular diseases in Traditional Chinese Medicine for a long time. Modern phytochemical studies showed that Z-ligustilide (LIG) is the main lipophilic component of Danggui. In this study, we examined whether LIG could protect ischemia/reperfusion-induced brain injury by minimizing oxidative stress and anti-apoptosis. Transient forebrain cerebral ischemia (FCI) was induced by the bilateral common carotid arteries occlusion for 30 min. LIG was intraperitoneally injected to ICR mice at the beginning of reperfusion. As determined via 2,3,5-triphenyl tetrazolium chloride (TTC) staining at 24 h following ischemia, the infarction volume in the FCI mice treated without LIG (22.1 +/- 2.6%) was significantly higher than that in the FCI mice treated with 5 mg/kg (11.8 +/- 5.2%) and 20 mg/kg (2.60 +/- 1.5%) LIG (P < 0.05 or P < 0.01). LIG treatment significantly decreased the level of malondialdehyde (MDA) and increased the activities of the antioxidant enzyme glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) in the ischemic brain tissues (P < 0.05 or P < 0.01 vs. FCI group). In addition, LIG provided a great increase in Bcl-2 expression as well as a significant decrease in Bax and caspase-3 immunoreactivities in the ischemic cortex. The findings demonstrated that LIG could significantly protect the brain from damage induced by transient forebrain cerebral ischemia. The antioxidant and anti-apoptotic properties of LIG may contribute to the neuroprotective potential of LIG in cerebral ischemic damage.

Changes in the uptake of transferrin-free and transferrin-bound iron during reticulocyte maturation in vivo and in vitro.

The uptake of non-transferrin-bound iron, Fe(II), transferrin-bound iron, Tf-Fe and transferrin was studied in reticulocytes from anaemic rabbits during maturation and then synchronized regeneration in vivo (following injection of actinomycin D) and while maturing during in vitro incubation. The uptake of Fe(II) and Tf-Fe decreased in parallel with each other and with the reticulocyte count and transferrin uptake during maturation in vivo and in vitro. Only during the early phase of reticulocyte regeneration in vivo was there a significant difference between the rates of Fe(II) and Tf-Fe uptake. These results suggest that a membrane carrier for iron and the transferrin receptor are lost at the same rate during reticulocyte maturation, possibly because they are associated with each other in the cell membrane. During reticulocyte maturation the rate of Fe(II) uptake into heme declined more rapidly than uptake into the total cellular cytosol. The loss of transferrin receptors and the uptake of iron from transferrin during reticulocyte maturation was not associated with a change in the affinity of the receptors for transferrin, in the relative distribution of the receptors between the outer cell membrane and intracellular sites or in the ability of the transferrin molecule to donate two iron atoms to the cell with each intracellular cycle, but the average duration of the cycle increased.

Mechanisms of iron uptake by mammalian cells.

On the basis of the discussion in this paper, the process of transferrin and iron (transferrin-bound iron and non transferrin-bound iron) uptake and transferrin release by reticulocytes are summarized diagrammatically. Although we were able to outline the pathways shown in the figure, there is still a long way to go before we achieve total understanding of the mechanisms of iron uptake. In addition, many important questions need to be answered. For example: what is the nature and properties of the iron carrier on the membrane? What is the relationship between the iron carrier and transferrin receptor? Is the iron carrier system in membranes of cells from different tissues similar or different? And how does iron cross the membrane of the endosomes after it is released from transferrin? All of these questions merit further investigation.

Effect of metabolic inhibitors on uptake of non-transferrin-bound iron by reticulocytes.

The relationship between transferrin-free iron uptake and cellular metabolism was investigated using rabbit reticulocytes in which energy metabolism was altered by incubation with metabolic inhibitors (antimycin A, 2,4-dinitrophenol, NaCN, NaN3 and rotenone) or substrates. Measurements were made of cellular ATP concentration and the rate of uptake of Fe(II) from a sucrose solution buffered at pH 6.5. There was a highly significant correlation between the rate of iron uptake into cytosolic and stromal fractions of the cells and ATP levels. Iron transport into the cytosol showed saturation kinetics. The metabolic inhibitors all reduced the Vmax but had no effect on the Km values for this process. It is concluded that the uptake of transferrin-free iron by reticulocytes is dependent on the cellular concentration of ATP and that it crosses the cell membrane by an active, carrier-mediated transport process. Additional studies were performed using transferrin-bound iron. The metabolic inhibitors also reduced the uptake of this form of iron but the inhibition could be accounted for entirely by reduction in the rate of transferrin endocytosis.

W-7 primes or inhibits the fMLP-stimulated respiratory burst in human neutrophil by concentration-dependent dual expression of the formyl peptide receptors on cell surface.

It was investigated why the fMLP-stimulated respiratory burst in human neutrophils was enhanced by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a considered calmodulin antagonist, at lower concentration but inhibited at higher concentration. Flow cytometric analysis on binding of the receptor to the fluorescence-labeled formyl peptide and the polymerization of actin in cells showed that the drug inhibited actin polymerization and promoted expression of the fMLP receptors on cell membrane at lower concentration, while promoted the actin polymerization and depressed the receptor expression at higher concentration. As intracellular Ca(2+) ([Ca(2+)](i)) is elevated, polymerization of actin decreases and the receptor expression increases. At normal physiological and two moderately high intracellular calcium levels, the dual effect of W-7 became less significant as [Ca(2+)](i) was elevated indicating that the dual effect is calcium-dependent. Under two extreme conditions that the intracellular calcium was either depleted or highly elevated, the dual effect disappeared but only an inhibitory effect on actin polymerization was observed. Colchicine and taxol study showed that disruption or stabilization of microtubules had no effect on formyl peptide receptor expression. The results suggest that W-7 primes the fMLP stimulation by direct action on actin leading to breakdown of microfilaments and more expression of formyl peptide receptors, and inhibits the stimulation by indirect action on actin through inactivation of some Ca(2+)-dependent proteins resulting in assembly of actin into microfilaments. Which action is favorable depends on the drug concentration.

Extracellular Ca2+ regulates the respiratory burst of human neutrophils.

The role of extracellular calcium in the activation of respiratory burst in human neutrophils was studied by using the receptor agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the activator of protein kinase C phorbol myristate acetate (PMA). The level of intracellular free calcium was measured by using both cell suspensions and single cells in the presence and absence of extracellular calcium. The Ca2+-ATPase inhibitor, thapsigargin, was used to activate higher Ca2+ influx, while a novel calcium channel blocker, panax notoginseng saponins (PNGS) was used to block the Ca2+ entry from extracellular space during the responding period of cells. It was found that about two-thirds of the activation of respiratory burst initiated by the receptor agonist were attributed to the Ca2+ influx under normal physiological conditions. The higher Ca2+ influx resulted in tremendous enhancement of the intensity of respiratory burst initiated by fMLP and marked acceleration of the onset of the respiratory burst stimulated by PMA. It is evident that both intra- and extracellular Ca2+ are required for full activation of the respiratory burst of human neutrophils, and the Ca2+ influx from extracellular space plays an important role either in generation of reactive oxygen metabolites or in activation of protein kinase C.

Effect of lipid peroxidation on transferrin-free iron uptake by rabbit reticulocytes.

The relationship between lipid peroxidation and uptake of transferrin- free iron, Fe(II), by reticulocytes in an experimental system for studying membrane transport of Fe(II) was investigated by using free radical scavengers: BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), superoxide dismutase, alpha-tocopherol, propyl gallate and DPPD (N,N-diphenyl-1,4-phenylenediamine), and producers: t-butyl hydroperoxide, cumene hydroperoxide, H2O2 and aluminium carbonate. Measurements were made of MDA (malondialdehyde) and the rate of Fe(II) uptake from a sucrose solution buffered at pH 6.5 by Pipes. Most scavengers and producers used could increase or decrease only slightly the rate of Fe(II) uptake and some of them had no effect on Fe(II) uptake and MDA could not be detected at iron concentration of lower than 10 microM and incubation time of 20 min. At iron concentration of higher than 100 microM and incubation time of 4 h, there was the production of MDA which increased with the increment of iron concentration of incubation medium and BHT could inhibit the production of MDA. In addition, no difference was found in the rates of Fe(II) uptake in three experimental groups whose incubation medium was buffered by Pipes, Mops and Mes respectively. The results suggested that iron could induce free radical reaction under experimental conditions, especially at high concentration of iron and longer incubation time; however, at low concentration of iron (<10 microM) and the usual incubation time (20 min) free radical reaction was very slight and the extent of the reaction was not enough to damage the integrity and function of the membrane of reticulocytes, and that Fe(II) uptake by reticulocytes was not the result of free radical reaction and lipid peroxidation. It was therefore concluded that iron could not initiate its own membrane transport in rabbit reticulocytes by free radical reaction and lipid peroxidation and that the experimental system we used for studying membrane transport of Fe(II) is valid.

Brain iron transport and neurodegeneration.

Despite years of investigation, it is still not known why iron levels are abnormally high in some regions of the brain in neurodegenerative disorders. Also, it is not clear whether iron accumulation in the brain is an initial event that causes neuronal death or is a consequence of the disease process. Here, we propose that iron and iron-induced oxidative stress constitute a common mechanism that is involved in the development of neurodegeneration. Also, we suggest that, at least in some neurodegenerative disorders, brain iron misregulation is an initial cause of neuronal death and that this misregulation might be the result of either genetic or non-genetic factors.

Intracellular free calcium regulates the onset of the respiratory burst of human neutrophils activated by phorbol myristate acetate.

Thapsigargin was used to study the regulation of different static calcium level ([Ca2+]i) on the respiratory hurst of human neutrophils stimulated with phorbol myristate acetate (PMA). The result showed that the onset time of the respiratory hurst was obviously reduced by elevation of static [Ca2+]i but is still much longer than that stimulated with N-formylmethionylleucylphenylalanine (fMLP). To find the reason, the onset times of the respiratory burst stimulated with fMLP, 1,2-dioctanoyl-sn-glycerol (DiC8), and PMA were determined at different static [Ca2+]i. It turns out that although DiC8 was unable to induce the respiratory burst at low [Ca2+], the onset time of DiC8-stimulated response at high [Ca2+]i was almost the same as that stimulated with fMLP. The study revealed that the fast onset of the fMLP-stimulated respiratory burst in comparison with PMA-stimulated response is not only due to the transient rise of [Ca2+]i, but is also due to the higher efficiency of diacylglycerol (DAG) in activating protein kinase c (PKC). The determining step in governing the onset of a respiratory burst is the activation of PKC.

Effect of iron and lipid peroxidation on development of cerebellar granule cells in vitro.

The aim of this study was to investigate the effect of chelated ferric ion on neuronal development in vitro using cultured cerebellar granule cells of the rat. The cells were exposed to ferric nitrilotriacetate at varying concentrations for seven or 14 days. In addition to morphological studies, protein content determination and malondialdehyde measurement were performed. The study showed that cell development, with the addition of a lower concentration of chelated ferric ion (5 microM), could be kept in a normal condition, no significant changes in protein content and malondialdehyde production being found as compared with those of the controls, while the addition of higher concentrations of chelated ferric ion (> or = 10 microM) to the cultures demonstrated an adverse effect on development of cerebellar granule cell in vitro. Determination of protein content showed that the neuronal population decreased significantly, and the neuronal loss was inversely proportional to the iron concentrations added. Malondialdehyde measurement demonstrated that the extent of lipid peroxidation reaction increased remarkably with increasing iron concentration. A very close and highly significant correlation (gamma=0.985) between changes of malondialdehyde production and protein content was observed. These results suggest that the neuronal loss in the cultures with higher concentrations of iron was due to lipid peroxidation reaction induced by the addition of iron, and that iron overload might accelerate the process of ageing and death of cerebellar granule cells in vitro.

Cerebellar granule cells acquire transferrin-free iron by a carrier-mediated process.

In this study, the mechanism of transferrin-free iron uptake by brain neuronal cells was investigated using the cultured cerebellar granule cells. Effects of incubation time, iron concentration, temperature and other divalent metals on the cellular uptake were determined. After five days of plating, the cells were incubated with different concentrations of transferrin-free iron in isotonic sucrose solution at different temperatures for a certain time. The cellular transferrin-free iron uptake was analysed by measuring the cellular radioactivity with a gamma-counter. The result showed that the cultured cerebellar granule cells had the capacity to acquire transferrin-free iron at pH 6.5, at which it was demonstrated that transferrin binds iron very poorly and only very little transferrin can be internalized by reticulocytes and HeLa cells. The iron uptake by cells increased with incubation time in a linear manner at a rate of 0.1076 pmol/microg protein/min within the first 10 min. The uptake was time- and temperature-dependent, iron concentration saturable, and inhibited by several divalent metal ions, such as Co2+, Zn2+, Mn2+ and Ni2+. These characteristics of transferrin-free iron uptake by the cultured cerebellar granule cells observed in this study, similar to those obtained from cells outside of the brain, implied that a carrier-mediated iron transport system might be present on the membrane of this type of brain neuronal cells. In addition, no significant difference in malondialdehyde measurement was found when the cells were incubated without or with the lower concentrations of iron (< 4 microM) for 20 min at 37 degrees C, demonstrating that this system was valid for studying membrane iron transport in this type of brain neuronal cell.

Effect of lead on the transport of transferrin-free and transferrin-bound iron into rabbit reticulocytes.

The effects of Pb on iron transport into rabbit reticulocytes was investigated using two sources of iron, non-transferrin-bound ferrous iron, Fe(II), and transferrin-bound iron, and fractionating the cells into haem, cytosolic and stromal fractions. Uptake of Fe(II) into all three fractions was inhibited by low concentrations of Pb, 50% inhibition of uptake to the cytosol (IC50) occurring at 1 microM Pb. Fe(II) uptake could be divided into saturable and non-saturable components. The saturable component was inhibited at lower concentrations of Pb than the non-saturable component. Pb reduced the Vmax and increased the Km values for saturable Fe(II) transport. The effects of Pb on Fe(II) transport were reversible and were observed with PbCl2 and Pb (NO3)2 as well as with lead acetate. Pb also inhibited the uptake of transferrin-bound iron but at higher concentrations (IC50, 4 microM) and the inhibition was less readily reversible. The effect was attributable to inhibition of transferrin endocytosis which resulted in a redistribution of transferrin receptors from intracellular to cell surface sites. These results show that Pb can inhibit transferrin endocytosis and iron transport across the cell membrane of reticulocytes and raise the possibility that these effects may contribute to the hypochromic anaemia associated with Pb poisoning, in addition to the previously established inhibition of enzymes of the haem synthesis pathway.

Increased expression of transferrin receptor on membrane of erythroblasts in strenuously exercised rats.

This study investigated the effects of strenuous exercise on transferrin (Tf)-receptor (TfR) expression and Tf-bound iron (Tf-Fe) uptake in erythroblasts of rat bone marrow. Female Sprague-Dawley rats were randomly assigned to either an exercise or sedentary group. Animals in the exercise group swam 2 h/day for 3 mo in a glass swimming basin. Both groups received the same amount of handling. At the end of 3 mo, the bone marrow erythroblasts were freshly isolated for Tf-binding assay and determination of Tf-Fe uptake in vitro. Tissue nonheme iron and hematological iron indexes were measured. The number of Tf-binding sites found in erythroblasts was approximately 674,500 +/- 132,766 and 1,270,011 +/- 235,321 molecules/cell in control and exercised rats, respectively (P < 0. 05). Total Fe and Tf uptake by the cells was also significantly increased in the exercised rats after 30 min of incubation. Rates of cellular Fe accumulation were 5.68 and 2.58 fmol. 10(6) cells(-1). min(-1) in the exercised and control rats, respectively (P < 0.05). Tf recycling time and TfR affinity were not different in exercised and control rats. Increased cellular Fe was mainly located in the stromal fraction, suggesting that most of accumulated Fe was transported to the mitochondria for heme synthesis. The findings demonstrated that the increased cellular Fe uptake in exercised rats was a consequence of the increased TfR expression rather than the changes in TfR affinity and Tf recycling time. The increase in TfR expression and cellular Fe accumulation, as well as the decreased serum Fe concentration and nonheme Fe in the liver and the spleen induced by exercise, probably represented the early signs of Fe deficiency.

Transferrin-bound iron uptake by the cultured cerebellar granule cells.

Excessive brain iron has been found in several neurodegenerative diseases. However, little information is available about mechanism of iron uptake by different types of brain cells including neurons. In this study, transferrin-bound iron (Tf-Fe) accumulation in the cultured cerebellar granule cell was investigated in vitro. After 5 days of culture, the cells were incubated with 1 microM of double-labelled transferrin (1251-Tf-59Fe) at 37 degrees C for 60 min. The cellular Tf-Fe and transferrin (Tf) uptake was analysed. The result showed (1) Tf uptake by the cells increased rapidly at the first 5 min, reaching its maximum after about 20 min of incubation; (2) Tf-Fe uptake kept increasing in a linear manner during the whole period of incubation; (3) the addition of either NH4Cl or CH3NH2, the blockers of Tf-Fe uptake via inhibiting iron release from Tf within endosomes, decreased the cellular Tf-Fe uptake but had no significant effect on Tf uptake; (4) trypsin and unlabelled Tf-Fe inhibited the uptake rate of Tf-Fe as well as Tf. The results suggested that Tf-Fe transport across the membrane of this type of neuron, much like other mammalian cells, was mediated by Tf-TfR endocytosis. Dysfunction of Tf or TfR would possibly lead to iron irregulation in the brain and consequently cause damage to neuronal functions.