Influence of cigarette smoking on crocidolite-induced ferritin release by human alveolar macrophages

Alveolar macrophages (AMs) mobilize iron from the surface of iron-containing minerals such as asbestos and synthesize ferritin for intracellular iron storage or secretion. Although the synthesis of iron-free ferritin (apoferritin) provides antioxidant protection, the secretion of iron-containing ferritin by AMs could increase the availability of catalytic iron in the lungs. Cigarette smoking may promote the secretion of ferritin by AMs after iron acquisition from mineral sources, because smokers' AMs are iron loaded. The first objective of this study was to determine whether ferritin secretion/release by AMs after in vitro exposure to crocidolite asbestos is enhanced by cigarette smoking. The second objective was to assess whether exogenous ferritin-bound iron could enhance the toxicity of crocidolite to lung cells in vitro. AMs recovered from nonsmokers (n = 8) or smokers (n = 8) were exposed to crocidolite or titanium dioxide (TiO2)(1 x 10(6) AMs, 50 to 200 microg/mL) for up to 18 hours. AMs exposed to crocidolite but not TiO2 showed increased cell content of iron and ferritin and increased cell supernatant ferritin concentrations. Increases in iron and ferritin content were similar for AMs recovered from smokers and those recovered from nonsmokers; however, increases in supernatant ferritin were >7-fold greater for smokers' AMs than for nonsmokers' AMs (P < .001). Exposure of A549 cells, a lung cancer-derived cell line, to crocidolite (50 to 200 microg/mL, 18 hours) caused dose-dependent cell death as indicated by lactate dehydrogenase release. The addition of ferritin (> or = 500 mg/mL) but not apoferritin to culture media enhanced crocidolite-induced LDH release (P < .01). These findings suggest that cigarette smoking and crocidolite exposure have synergistic effects that promote ferritin release by AMs, which could catalyze oxidative injury to other alveolar cells.     

Nitric oxide acts on the mitochondria and protects human endothelial cells from apoptosis

Proliferation of small blood vessels in synovial tissues is one of the pathologic features of rheumatoid arthritis. In this study we tested the hypothesis that nitric oxide (NO) protects endothelial cells (ECs) against apoptogenic agents in vitro. Human umbilical-vein endothelial cells (HUVECs) were cultured with and without NO donor S -nitro- N -acetylpenicillamine (SNAP) and further incubated in the presence or absence of Z-leucine-leucine-leucine-aldehyde (LLL-CHO), etoposide, or C2-ceramide. After cultivation, apoptosis of HUVECs was quantified on the basis of disruption of mitochondrial transmembrane potential (DeltaPsim), activation of caspases, and the presence of hypodiploid DNA-positive cells. Treatment of HUVECs with LLL-CHO, etoposide, or C2-ceramide induced DeltaPsim, activation of caspase-3, caspase-8, and caspase-9 and the appearance of hypodiploid DNA-positive cells. NO production in HUVECs was clearly increased by SNAP. Apoptotic cell death in HUVECs induced by LLL-CHO, etoposide, and C2-ceramide was significantly suppressed by SNAP treatment. HUVECs in vitro expressed Bcl-2, Bcl-xL, and Bax; however, expression was not changed by SNAP treatment in the presence or absence of LLL-CHO, etoposide, or C2-ceramide. Although the molecule(s) responsible for the protective effects of NO remains to be identified, our data imply that NO protects HUVECs against mitochondrial perturbation caused by apoptogenic agents. These results suggest that NO promotes endothelial-cell proliferation and angiogenesis in the synovial tissues of patients with rheumatoid arthritis and that NO may be a therapeutic target for rheumatoid arthritis.     

Effect of hyperoxia on reduced glutathione in alveolar macrophages and polymorphonuclear leukocytes

Reduced glutathione (GSH) protects alveolar macrophages (AMs) and polymorphonuclear leukocytes (PMNs) against oxidative damage. To obtain further knowledge of the oxygen toxicity we determined GSH in AMs and PMNs of guinea pigs exposed to an oxygen concentration of 85% for up to 90 h. AMs and PMNs from control animals contained 17.93 and 11.67 nmol GSH/mg protein, respectively. During the exposure to a FIO2 of 85% we observed a significant continuous increase of GSH in AMs. By 90 h of oxygen exposure, AMs contained 51.22 nmol GSH/mg protein. In addition, the protein content of AMs decreased during hyperoxia. In contrast, no change of the GSH amount and protein content was detectable in PMNs. The increase of GSH in AMs could serve as an adaptation of the cells to hyperoxia. The lack of the GSH increase in PMNs could be due to the different oxygen concentrations between the lung and the peritoneal cavity. The greater GSH content in AMs may account for the difference between these cells in their susceptibility to oxidant injury.     

Hypocapnic alkalosis enhances oxidant-induced apoptosis of human alveolar epithelial type II cells

Apoptosis of alveolar epithelial type II (AEC-II) cells induced by reactive oxygen species (ROS) contributes to extensive alveolar damage during acute lung injury. Hypercapnic acidosis and hypocapnic alkalosis are known to modulate ROS-mediated lung damage. This study assessed the effects of acid-base balance disturbances on hydrogen peroxide (H2O2)-induced apoptosis of the AEC-II-like human cell line A549, which was cultured under different conditions of pH and CO2 tension (normal pH and CO2, hypercapnic acidosis, metabolic acidosis, hypocapnic alkalosis and metabolic alkalosis). H2O2-induced apoptosis was assessed by a dye-uptake bioassay and induction of caspase activity, which were quantified using analytical digital photomicroscopy. Acidosis or alkalosis of the culture medium alone did not induce A549 cell apoptosis. Hypocapnic alkalosis significantly increased H2O2-induced apoptosis and caspase activation of A549 cells. Metabolic alkalosis non-significantly increased H2O2-induced A549 cell apoptosis and caspase activation. These data suggest that hypocapnic alkalosis intensifies oxidative-induced apoptosis of alveolar epithelial cells.     

Cigarette smoke and asbestos activate poly-ADP-ribose polymerase in alveolar epithelial cells.

BACKGROUND: Cigarette smoke augments asbestos-induced bronchogenic carcinoma in a synergistic manner by mechanisms that are not established. One important mechanism may involve alveolar epithelial cell (AEC) injury resulting from oxidant-induced DNA damage that subsequently activates poly (ADP-ribose) polymerase (PARP), an enzyme involved in DNA repair that can deplete cellular energy stores. We previously showed that whole aqueous cigarette smoke extracts (CSE) augment amosite asbestos-induced DNA damage and cytotoxicity to cultured AEC in part by generating iron-induced free radicals. We hypothesized that CSE increase asbestos-induced AEC injury by triggering PARP activation resulting from DNA damage caused by iron-induced free radicals. METHODS: Aqueous CSE were prepared fresh on the day of each experiment. PARP activity in WI-26 (a type I-like cell line) and A549 (a type II-like cell line) cells was assessed by the uptake of labeled NAD over 4 hours and confirmed on the basis of the reduction of PARP levels in the presence of a PARP inhibitor, 3-aminobenzamide (3-ABA). Cell survival was assessed by trypan blue dye exclusion. RESULTS: Hydrogen peroxide (H2O2; 1-250 microM), CSE (0.4-10 vol%), and amosite asbestos (5-250 micrograms/cm2) each caused PARP activation in WI-26 and A549 cells. The combination of asbestos (5 micrograms/cm2) and CSE (0.04-10%) induced WI-26 and A549 cell PARP activation without evidence of synergism. 3-ABA significantly attenuated WI-26 and A549 cell PARP activity and cell death after exposure to H2O2, CSE, and asbestos. Phytic acid, an iron chelator, catalase, and superoxide dismutase each decreased WI-26 cell PARP activation caused by asbestos and CSE. CONCLUSIONS: CSE and asbestos induced PARP activation in cultured AEC in a nonsynergistic manner. These data provide further support that asbestos and cigarette smoke are genotoxic to relevant lung target cells and that iron-induced free radicals in part cause these effects.     

Neutrophil Elastase Increases Airway Epithelial Nonheme Iron Levels

Alpha‐1‐antitrypsin (A1AT) deficiency is characterized by increased neutrophil elastase (NE) activity and oxidative stress in the lung. We hypothesized that NE exposure generates reactive oxygen species by increasing lung nonheme iron. To test this hypothesis, we measured bronchoalveolar lavage (BAL) iron and ferritin levels, using inductively coupled plasma (ICP) optical emission spectroscopy and an ELISA, respectively, in A1AT‐deficient patients and healthy subjects. To confirm the role of NE in regulating lung iron homeostasis, we administered intratracheally NE or control buffer to rats and measured BAL and lung iron and ferritin. Our results demonstrated that A1AT‐deficient patients and rats postelastase exposure have elevated levels of iron and ferritin in the BAL. To investigate the mechanism of NE‐induced increased iron levels, we exposed normal human airway epithelial cells to either NE or control vehicle in the presence or absence of ferritin, and quantified intracellular iron uptake using calcein fluorescence and ICP mass spectroscopy. We also tested whether NE degraded ferritin in vitro using ELISA and western analysis. We demonstrated in vitro that NE increased intracellular nonheme iron levels and degraded ferritin. Our results suggest that NE digests ferritin increasing the extracellular iron pool available for cellular uptake.     

Iron binding, internalization, and fate in human alveolar macrophages

Chronic inflammation in such diseases as rheumatoid arthritis has been associated with the accumulation of iron in mononuclear phagocytes. Cigarette smoking, which also produces chronic pulmonary inflammation, may be associated with iron accumulation in alveolar macrophages (AM). We have examined the total iron content in human AM and found it to be 43.0 +/- 7.7 (mean +/- SEM) and 12.8 +/- 1.3 nmol/1 X 10(6) cells (P less than 0.01) from smokers and nonsmokers, respectively. Because the higher iron content in smokers' macrophages may reflect increased internalization, the binding and uptake of iron-saturated transferrin was examined in cells from smokers and nonsmokers. However, no significant differences were found between the two groups. The smoking-related alteration in iron content may instead reflect differences in the fate of internalized iron. Iron internalized by AM as iron 59 initially bound to transferrin was distributed to a cytoplasmic, largely ferritin-associated, pool more slowly in smokers than in nonsmokers, during a 24-hour incubation in vitro. Significantly less newly internalized iron was returned to the culture medium by AM from smokers, which by 24 hours had released 11.0% +/- 3.7% of the initially internalized 59Fe compared with 36.0% +/- 2.3% for nonsmokers (P less than 0.01). The increased accumulation of iron by AM in the alveolar space of smokers may modulate hydroxyl radical production in the microenvironment of these cells.     

Oxygen toxicity and iron accumulation in the lungs of mice lacking heme oxygenase-2.

Heme oxygenase (HO) activity leads to accumulation of the antioxidant bilirubin, and degradation of the prooxidant heme. Moderate overexpression of the inducible form, HO-1, is associated with protection against oxidative injury. However, the role of HO-2 in oxidative stress has not been explored. We evaluated survival, indices of oxidative injury, and lung and HO expression in HO-2 null mutant mice exposed to > 95% O2 compared with wild-type controls. Similar basal levels of major lung antioxidants were observed, except that the knockouts had a twofold increase in total glutathione content. Despite increased HO-1 expression from HO-1 induction, knockout animals were sensitized to hyperoxia-induced oxidative injury and mortality, and also had significantly increased markers of oxidative injury before hyperoxic exposure. Furthermore, during hyperoxia, lung hemoproteins and iron content were significantly increased without increased ferritin, suggesting accumulation of available redox-active iron. These results demonstrate that the absence of HO-2 is associated with induction of HO-1 and increased oxygen toxicity in vivo, apparently due to accumulation of lung iron. These results suggest that HO-2 functions to augment the turnover of lung iron during oxidative stress, and that this function does not appear to be compensated for by induction of HO-1 in the knockouts.     

Modulation of alveolar macrophage leukotriene B4 released by complement component C5

The release of neutrophil chemotactic activity by the guinea pig alveolar macrophage (AM) is dependent on the fifth component of complement (C5) on the cell surface. Because one potent chemotactic factor released by AMs is leukotriene B4 (LTB4), we hypothesized that cell surface C5 may modulate LTB4 release. To test this hypothesis, human AMs obtained by bronchoalveolar lavage from 12 subjects were cultured for 4 hours in the presence of anti-C5 Fab' antibodies with stimuli. The cultures were harvested and evaluated for LTB4 by radioimmunoassay. The LTB4 levels in supernatants obtained from AMs cultured in media alone were variable (447 +/- 63 pg/ml), but the levels were increased when AMs were cultured with the stimuli-opsonized zymosan, immune complexes, or lipopolysaccharide (233%, 49%, and 114% increase, respectively, compared with macrophages cultured in media alone, p less than 0.05). Culturing the AMs with anti-C5 Fab' antibodies inhibited the release of LTB4 induced by opsonized zymosan, immune complexes, or lipopolysaccharide (78%, 41%, and 82% inhibition, respectively, p less than 0.05). Consistent with these observations, anti-C5 Fab' antibodies also decreased the neutrophil chemotactic activity of culture supernatants obtained from AMs stimulated with the same stimuli (p less than 0.001). These data suggest that AM release of LTB4 may be C5-dependent.     

Anti-oxidant and Anti-hypercholesterolemic Activities of Wasabia japonica

The effects of Wasabia japonica (WJ) were investigated in vitro and in vivo for their anti-oxidant and anti-hypercholesterolemic activities. It was found that the aqueous extracts of WJ leaves (WJL) had strong scavenging activities towards 1,1-Diphenyl-2-picryhydrazyl (DPPH) and nitric oxide (NO) free radicals in cell free systems. WJL also inhibited NO production and the expressions of inducible NO synthase (iNOS) mRNA and enzyme protein, determined by Griess reactions, RT-PCR or Western blotting respectively in Lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages cells. The anti-hypercholesterolemic effects of WJ diet were investigated in hypercholesterolemia rats. Sprague-Dawley rats were divided into four groups and were fed with either normal diet (Group 1), or diet containing 1%(w/w) cholesterol (Groups 2, 3 and 4). After 4 weeks, Group 2 was changed to normal diet, Groups 3 and 4 were changed to the diet containing 5% WJ leaf and or 5% WJ root, respectively. 3 weeks after WJ diets, Serum HDL-cholesterol levels were significantly increased in WJ diet groups compared with the normal diet hypercholesterolemia rats. In contrast, the serum LDL-cholesterol levels and liver xanthine oxidase (XO) activity in WJ diet groups were significantly decreased. The results indicate that the WJ extracts have significant anti-oxidant activities, and the WJ diet exhibited anti-hypercholesterolemic action in high cholesterol diet rats, which was companied with modulations of cholesterol metabolism and decrease in liver XO activity.     

TPA诱导K562细胞分化过程中线粒体铁蛋白表达情况研究

为了研究K562白血病细胞在十四烷酰佛波醇-乙酯（TPA）诱导分化过程中线粒体铁蛋白（MtF）、运铁蛋白受体Ⅰ（TfR1）和铁蛋白（Fn）mRNA表达水平的变化情况,并探讨MtF在白血病细胞增殖和细胞铁代谢方面的作用,采用TPA（终浓度16nmol/L）诱导K562白血病细胞向单核细胞定向分化,并于诱导分化后第1、3和5天收集细胞,用瑞氏染色法观察各时点细胞形态,流式细胞术检测细胞表面分化抗原CD64的表达水平,使用半定量RT-PCR方法检测各时点细胞MtF、TfR1和Fn mRNA表达水平,管家基因β-actin用作对照。结果表明,TPA（16nmol/L）可诱导K562细胞向单核细胞分化,细胞呈现单核细胞典型的形态学特征,在诱导培养第5天诱导分化率可达95%,细胞表面CD64表达水平显著增加。诱导分化前K562细胞具有一定水平MtF表达,但随着TPA诱导细胞分化的增强,MtF和TfR1 mRNA表达水平进行性下调,诱导分化第5天的表达水平分别为诱导分化前的50.3%和68.2%,而Fn mRNA表达水平则逐渐上调,诱导分化第5天表达水平为诱导分化前的1.97倍。结论：TPA诱导K562细胞向单核细胞分化过程中MtF和TfR1 mRNA表达下调,而Fn mRNA表达上调;这种协调变化有助于降低细胞通过TfR1介导的铁摄取,从而抑制或影响细胞增殖能力。     

Stimulation of growth of neuroblastoma cells by ferritin in vitro.

Among children with advanced neuroblastoma, serum concentrations of the iron storage protein ferritin correlate inversely with prognosis. To determine whether ferritin stimulates tumor cell growth, the effects of graded concentrations on cell number were studied for each of three neuroblastoma cell lines (CHP-126, CHP-100, IMR-32) plated in serum-free tissue culture medium. Ferritin extracted from human liver, spleen, or CHP-126 cells (150 ng/ml, final concentration) but not from human heart (150-300 ng/ml) resulted in 1.4-fold +/- 0.2-fold increases in cell numbers over 72 hours as measured spectrophotometrically after reduction of a tetrazolium dye. Higher concentrations of isoferritins (up to 1000 ng/ml) did not further increase cell number, but stimulation was abrogated by rabbit immunoglobulin G antiferritin. Although specific receptors for iodine 125-labeled ferritin could not be demonstrated on the two cell lines tested, deoxyribonucleic acid (DNA) synthesis, measured by incorporation of 3H-thymidine, also increased after addition of ferritin, by approximately 25%. We conclude that ferritin has mitogenic activity for human neuroblastoma cells in vitro which may explain the clinical correlation between levels of that protein and prognosis. Possible implications for therapy are discussed.     

Asbestos causes apoptosis in alveolar epithelial cells: role of iron-induced free radicals

Asbestos causes asbestosis and malignancies by mechanisms that are not fully understood. Alveolar epithelial cell (AEC) injury by iron-induced reactive oxygen species (ROS) is one important mechanism. To determine whether asbestos causes apoptosis in AECs, we exposed WI-26 (human type I-like cells), A549 (human type II-like cells), and rat alveolar type II cells to amosite asbestos and assessed apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate-biotin nick end labeling (TUNEL) staining, nuclear morphology, annexin V staining, DNA nucleosome formation, and caspase 3 activation. In contrast to control medium and TiO2, amosite asbestos and H2O2 each caused AEC apoptosis. A role for iron-catalyzed ROS was suggested by the finding that asbestos-induced AEC apoptosis and caspase 3 activation were each attenuated by either an iron chelator (phytic acid and deferoxamine) or a.OH scavenger (dimethyl-thiourea, salicylate, and sodium benzoate) but not by iron-loaded phytic acid. To determine whether asbestos causes apoptosis in vivo, rats received a single intratracheal instillation of amosite (5 mg) or normal saline solution, and apoptosis in epithelial cells in the bronchoalveolar duct regions was assessed by TUNEL staining. One week after exposure, amosite asbestos caused a 3-fold increase in the percentage of apoptotic cells in the bronchoalveolar duct regions as compared with control (control, 2.1% +/- 0.35%; asbestos, 7.61% +/- 0.15%; n = 3). However, by 4 weeks the number of apoptotic cells was similar to control. We conclude that asbestos-induced pulmonary toxicity may partly be caused by apoptosis in the lung epithelium that is mediated by iron-catalyzed ROS and caspase 3 activation.     

丹参酮ⅡA磺酸钠对海水浸泡人肺上皮A549细胞水通道蛋白5的影响

目的 探讨水通道蛋白5(AQP5)在海水浸泡致细胞损伤中的变化,同时了解丹参酮Ⅱ A可能的作用机制.方法 体外传代培养肺腺癌细胞株A549细胞,接种于培养皿中,按不同海水含量分为空白对照组及15%、25%、50%、75%、100%海水组;以25%海水浸泡不同时间分为空白对照组及海水1、4、8 h组;按给予不同剂量丹参酮ⅡA干预分为空白对照组、25%海水组及25、50、75、100μg/ml丹参酮ⅡA干预4 h组.用蛋白质免疫印迹法(Western blotting)检测AQP5蛋白表达;用免疫组化法检测AQP5阳性表达.结果 Western blotting结果显示,25%与50%海水组8 h时A549细胞AQP5蛋白表达均较空白对照组明显增高(1.053±0.231、1.116±0.316比0.101±0.081,均P＜0.05);海水1 h组AQP5表达较空白对照组稍有增加(0.306±0.125比0.288±0.098,P＞0.05),4 h组(1.423±0.377)明显增加(P＜0.01),8 h组AQP5表达(1.507±0.461)较4 h组略有增加,但差异无统计学意义;25μg/ml与50 μg/ml丹参酮Ⅱ A组4 h时AQP5蛋白表达较25%海水组明显减少(0.580±0.186、0.499±0.172比1.013±0.287,均P＜0.05).免疫组化显示,25%海水4 h组AQP5阳性表达较空白对照组明显增多(7.21±0.78比0.41±0.07,P＜0.01),染色变深;25μg/ml丹参酮ⅡA干预4 h组AQP5阳性表达(3.02±0.23)较25%海水4 h组明显减少(P＜0.05).结论 丹参酮Ⅱ A在25μg/ml浓度时毒副作用最小,对海水浸泡A549细胞的保护作用最佳,其机制可能与抑制AQP5的过度表达有关.

Abstract：

Objective To explore the effects of tanshinone Ⅱ A on the activity of aquaporin-5 (AQP5)in human alveolar epithelial cells (A549) after seawater exposure and its possible mechanism. Methods Routinely cultured A549 cells were divided into different groups according to different content of seawater:blank control group, 15%, 25%, 50%, 75%, 100% seawater groups; they were divided into different groups according to the duration of exposure to 25 % seawater : blank control group, 1, 4, 8 hours groups ;they were also divided into different groups according to concentration of tanshinone ⅡA and exposed to seawater for 4 hours: blank control group, 25% seawater group, 25, 50, 75, 100 μg/ml tanshinone ⅡA intervention groups. The expressions of AQP5 were respectively assayed by Western blotting and immunohistochemistry. Results The results of Western blotting showed that the expressions of AQP5 were remarkably higher at 8 hours of exposure to seawater in 25% and 50% seawater groups than those in blank control group (1. 053±0. 231, 1. 116±0. 316 vs. 0. 101±0. 081, both P＜0. 05); the expression of AQP5 in 1-hour group showed a slight increase compared with blank control group (0. 306±0. 125 vs. 0. 288±0. 098,P＞0. 05), that in 4-hour group was increased significantly (1. 423±0. 377, P＜0. 01), and in 8-hour group (1. 507± 0. 461 ) it was slightly higher than that in 4-hour group without statistical significance. The AQP5 expression was significantly lower in tanshinone ⅡA 25 μg/ml and 50μg/ml intervention groups than that in 25% seawater group (0. 580 ± 0. 186, 0. 499 ± 0. 172 vs. 1.013 ± 0. 287, both P ＜ 0. 05). Immunohistochemistry showed that the expression of AQP5 was markedly up-regulated after A549 cells were stimulated with 25% seawater for 4 hours as compared with blank control group (7.21±0. 78 vs. 0. 41 ±0.07, P＜0.01), but intervention of tanshinone ⅡA significantly inhibited the up-regulation of AQP5 expression (3.02±0.23) induced by 25% seawater (P＜0.05). Conclusion The experimental results showed that tanshinone ⅡA is innocuous to A549 at a dosage of 25 μg/ml, and it can decrease the overexpression of AQP5 induced by seawater.     

Methylation-dependent gene silencing induced by interleukin 1beta via nitric oxide production

Interleukin (IL)-1beta is a pleiotropic cytokine implicated in a variety of activities, including damage of insulin-producing cells, brain injury, or neuromodulatory responses. Many of these effects are mediated by nitric oxide (NO) produced by the induction of NO synthase (iNOS) expression. We report here that IL-1beta provokes a marked repression of genes, such as fragile X mental retardation 1 (FMR1) and hypoxanthine phosphoribosyltransferase (HPRT), having a CpG island in their promoter region. This effect can be fully prevented by iNOS inhibitors and is dependent on DNA methylation. NO donors also cause FMR1 and HPRT gene silencing. NO-induced methylation of FMR1 CpG island can be reverted by demethylating agents which, in turn, produce the recovery of gene expression. The effects of IL-1beta and NO appear to be exerted through activation of DNA methyltransferase (DNA MeTase). Although exposure of the cells to NO does not increase DNA MeTase gene expression, the activity of the enzyme selectively increases when NO is applied directly on a nuclear protein extract. These findings reveal a previously unknown effect of IL-1beta and NO on gene expression, and demonstrate a novel pathway for gene silencing based on activation of DNA MeTase by NO and acute modification of CpG island methylation.     

Impact of parturition on iron status in nonanaemic iron deficiency

BACKGROUND: Iron-deficient nonanaemic parturients risk underdiagnosis as a result of the reliance on postpartum ferritin and haemoglobin as markers of iron status. Ferritin is an acute-phase protein whose levels increase during the inflammatory response, as occurs after delivery. Our aims were to evaluate the impact of parturition on iron status, erythropoiesis and the inflammatory response, and identify the optimal parameters and timing for diagnosing iron deficiency in the presence of postpartum inflammation. MATERIALS AND METHODS: Conventional parameters of iron status, erythropoiesis and the inflammatory response (serum ferritin, serum iron, transferrin saturation, C-reactive protein) were compared with more recent parameters [soluble transferrin receptors (sTfR), hypochromic red cells, reticulocyte indices] within 48 h either side of delivery in 64 iron-deficient nonanaemic women (defined by a prepartum serum ferritin < or =15 microg L(-1), and a pre- and postpartum haemoglobin of > or =11.0 g dL(-1) and > or =10.0 g dL(-1), respectively). RESULTS: Mean sTfR decreased pre to postpartum from 7.3 to 5.8 microg mL(-1) (P<0.01), while mean serum ferritin increased from 9.7 to 16.9 microg L(-1) (P<0.01). Serum ferritin did not correlate with haemoglobin pre or postpartum (r=0.04, P=0.7; r=0.2, P=0.09), but a correlation persisted postpartum between hypochromic red blood cells and haemoglobin (r=-0.26; P<0.05). The percentage of hypochromic red cells remained virtually unchanged pre- and postpartum (4.0% vs. 3.8%; NS). Postpartum mean reticulocyte haemoglobin content (CHr) was 27.1 +/- 1.6 pg. CONCLUSION: Iron status should be tested prepartum, in the absence of an inflammatory response, rather than in the early postpartum. A valuable additional parameter, where available, might be the hypochromic red cell percentage, which is virtually uninfluenced by the inflammatory response. Furthermore, hypochromic red cell percentage, CHr and sTfR can be helpful to differentiate between functional iron deficiency and depleted iron stores.     

高氧所致氧化应激状态下肺泡上皮细胞Toll样受体的表达及其功能研究

目的 观察高氧暴露后肺泡上皮细胞内活性氧(ROS)水平与Toll样受体(TLRs)基因、蛋白表达变化及其与信号通路功能之间的关系,探讨高氧所致肺损伤炎症反应的可能机制.方法 将体外培养的人肺腺癌A549细胞株分为空气对照组、高氧组、N-乙酰半胱氨酸(NAC)预处理组;高氧组细胞在＞90％O2的高氧环境中暴露2、6、12、24及48 h,NAC预处理组细胞予以ROS清除剂NAC预处理后高氧暴露6h.于相应时间点用流式细胞术检测细胞内ROS含量以及TLR2/4蛋白在细胞中的分布和表达;用逆转录-聚合酶链反应(RT-PCR)法检测TLR2/4 mRNA表达;用酶联免疫吸附法(ELISA)检测细胞上清液中白细胞介素(IL-6、IL-8)的含量.结果 A549细胞有TLR2/4表达,并且以细胞质内表达为主.与空气对照组比较,高氧组暴露2h细胞内ROS含量(荧光强度)即明显增高(11.820±3.123比7.223±1.170,P＜0.01),随高氧暴露时间延长呈进行性增高,于48 h达高峰(113.837±5.247,P＜0.01);高氧暴露2 h TLR2/4 mRNA表达至高峰(TLR2 mRNA:1.820±0.056比1.263±0.023;TLR4 mRNA:2.080±0.220比1.317±0.107,均P＜0.01),随高氧暴露时间延长,TLR2/4 mRNA表达虽仍有增多,但无显著变化;高氧暴露后TLR2/4蛋白表达显著增高,6h表达至高峰(TLR2蛋白:8.370±1.548比3.930±0.277;TLR4蛋白:25.803±5.783比8.867±2.230,均P＜0.01),以细胞质内表达为主;随高氧暴露时间延长,细胞上清液中IL-6(ng/L)、IL-8(ng/L)水平呈进行性增高,于48 h达高峰(IL-6:2 213.41±69.99比9.76±1.47;IL-8:11 520.38±429.93比159.56±20.80,均P＜0.01).在NAC预处理情况下,高氧刺激后,细胞内ROS水平明显降低(14.050±1.257比31.180±2.336,P＜0.01),细胞TLR2/4 mRNA和蛋白表达显著降低(TLR2 mRNA:1.270±0.061比1.683±0.025; TLR4 mRNA:1.507±0.058比1.650±0.139;TLR2蛋白:3.458±0.258比8.370±1.548;TLR4蛋白:11.611±3.403比25.803±5.783,均P＜0.05),细胞上清液中IL-6、IL-8水平显著下降(IL-6:8.42±0.70比73.51±16.70;IL-8:134.94±5.19比772.82±96.05,均P＜0.05),与空气对照组比较差异均无统计学意义.结论 A549细胞在高氧暴露后,细胞内ROS能够激活人肺泡上皮细胞TLR2/4的表达,导致前炎症细胞因子IL-6和IL-8的大量释放.     

Nitric oxide promotes the internalization and passage of viable bacteria through cultured Caco-2 intestinal epithelial cells

Nitric oxide (NO) may play an important role in the pathophysiology of intestinal barrier disruption. Our purpose was to investigate the effects of NO donors on the internalization and passage of bacteria through cultured intestinal epithelial cells. Human intestinal epithelial cell line Caco-2 cells were grown on microtiter plastic plates. The cells were incubated with Escherichia coli and sodium nitroprusside (SNP) or S-nitroso-N-acetyl-penicillamine (SNAP), as NO donors, at several concentrations. The numbers of viable bacteria internalized into the epithelial cells were measured. Caco-2 cells were also grown to confluency on membranes of bicameral systems. The cells were incubated with E. coli and SNP. The numbers of viable bacteria passed through the epithelial layer were determined. Viability of the bacteria and the intestinal epithelial cells after culture with SNP or SNAP were also determined. Both SNP and SNAP at .1 or 1 mmol/L increased the number of viable bacteria internalized into the enterocytes. Both 1 or 10 mmol/L SNP promoted bacterial passage through the intestinal epithelial layer. However, 10 mmol/L SNP decreased the number of viable Caco-2 cells and failed to increase the bacterial internalization into Caco-2 cells. Incubation of E. coli with SNAP at 10 mmol/L slightly decreased the number of viable bacteria and failed to increase the bacterial internalization into Caco-2 cells. We conclude that NO donors promote both the viable bacterial uptake and passage through the intestinal epithelial layer.     

Nitric oxide directly impairs intestinal barrier function

Excess production of nitric oxide (NO) has been implicated in endotoxin-induced loss of gut barrier function in vivo. Thus, we tested the direct effect of NO on the barrier function of intestinal mucosal membranes suspended ex vivo in Ussing chambers and on IEC-6 enterocyte monolayers. In these experiments, ex vivo-mounted ileal membranes or IEC-6 cell enterocyte monolayers were exposed to the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) over a dose range (10 microm to 2 mM) or medium. SNAP at concentrations of 1 or 2 mM, but not 10 or 100 microM, increased the rates of bacterial translocation (BT) across both the ileal membranes and the IEC-6 monolayers by >1 log (P < 0.05), as well as the permeability of the IEC-6 monolayers to phenol red (P < 0.05). The ileal membranes exposed to 1 or 2 mM SNAP for 3 h manifested histologic evidence of mucosal injury and decreases in electrical resistance and potential difference values (P < 0.05), while the IEC-6 cells exposed to SNAP for 18 h had increased levels of cell death (P < 0.05). Since NO produced locally by stimulated enterocytes could contribute to barrier dysfunction, NO production, iNOS mRNA levels, and monolayer permeability were measured in enterocytes (IEC-6 and Caco-2) exposed to medium, endotoxin (lipopolysaccharide [25 microg/mL]) or a cytokine mixture (IL-1beta 10 ng/mL, TNF-alpha 10 ng/mL, and INF-gamma 250 U/mL) for 6 or 24 h. Endotoxin increased NO production, iNOS mRNA expression, and monolayer permeability in the IEC-6, but not the Caco-2 cells, while exposure to the cytokine mixture increased both NO production, iNOS mRNA expression, and monolayer permeability in both the IEC-6 and Caco-2 cell lines. Based on the results of these studies it appears that NO can directly increase ileal mucosal membrane and enterocyte monolayer permeability and BT and that increased NO production and iNOS mRNA expression is associated with endotoxin- and/or cytokine-induced loss of enterocyte monolayer barrier function.