Effect of thymoquinone on cytosolic pH and Na+/H+ exchanger activity in mouse dendritic cells

The anti-inflammatory Nigella sativa component thymoquinone compromises the function of dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. DC function is regulated by the Na(+)/H(+) exchanger (NHE), which is stimulated by lipopolysaccharides (LPS) and required for LPS-induced cell swelling, reactive oxygen species (ROS) production, TNF-α release and migration. Here we explored, whether thymoquinone influences NHE activity in DCs. To this end, bone marrow derived mouse DCs were treated with LPS in the absence and presence of thymoquinone (10 μM). Cytosolic pH (pH(i)) was determined from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNF-α production utilizing ELISA and DC migration with transwell migration assays. As a result, exposure of DCs to LPS (1 μg/ml) led within 4 hours to transient increase of NHE activity. Thymoquinone did not significantly modify cytosolic pH or cellular NHE activity in the absence of LPS, but abrogated the effect of LPS on NHE activity. Accordingly, in the presence of thymoquinone LPS-treatment resulted in cytosolic acidification. LPS further increased forward scatter and ROS formation, effects similarly abrogated by thymoquinone. Again, in the absence of LPS, thymoquinone did not significantly modify ROS formation and cell volume. LPS further triggered TNF-α release and migration, effects again blunted in the presence of thymoquinone. NHE1 inhibitor cariporide (10 μM) blunted LPS induced TNF-α release and migration. The effects of thymoquinone on NHE activity and migration were reversed upon treatment of the cells with t-butyl hydroperoxide (TBOOH, 5 μM). In conclusion, thymoquinone blunts LPS induced NHE activity, cell swelling, oxidative burst, cytokine release and migration of bone marrow derived murine dendritic cells. NHE inhibition may thus contribute to the antiinflammatory action of thymoquinone.     

Effect of azathioprine on Na(+)/H(+) exchanger activity in dendritic cells

Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na(+)/H(+) exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na(+)/H(+) exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) was estimated utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 μg/ml) led to a transient increase of Na(+)/H(+) exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 μM) did not significantly alter the Na(+)/H(+) exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na(+)/H(+) exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na(+)/H(+) exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug.     

OSR1-sensitive regulation of Na+/H+ exchanger activity in dendritic cells

The oxidative stress-responsive kinase 1 (OSR1) is activated by WNK (with no K kinases) and in turn stimulates the thiazide-sensitive Na-Cl cotransporter (NCC) and the furosemide-sensitive Na-K-2Cl cotransporter (NKCC), thus contributing to transport and cell volume regulation. Little is known about extrarenal functions of OSR1. The present study analyzed the impact of decreased OSR1 activity on the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs were cultured from bone marrow of heterozygous WNK-resistant OSR1 knockin mice (osr(KI)) and wild-type mice (osr(WT)). Cell volume was estimated from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescence, and Na(+)/H(+) exchanger activity from Na(+)-dependent realkalinization following ammonium pulse and migration utilizing transwell chambers. DCs expressed WNK1, WNK3, NCC, NKCC1, and OSR1. Phosphorylated NKCC1 was reduced in osr(KI) DCs. Cell volume and pH(i) were similar in osr(KI) and osr(WT) DCs, but Na(+)/H(+) exchanger activity and ROS production were higher in osr(KI) than in osr(WT) DCs. Before LPS treatment, migration was similar in osr(KI) and osr(WT) DCs. LPS (1 μg/ml), however, increased migration of osr(WT) DCs but not of osr(KI) DCs. Na(+)/H(+) exchanger 1 inhibitor cariporide (10 μM) decreased cell volume, intracellular reactive oxygen species (ROS) formation, Na(+)/H(+) exchanger activity, and pH(i) to a greater extent in osr(KI) than in osr(WT) DCs. LPS increased cell volume, Na(+)/H(+) exchanger activity, and ROS formation in osr(WT) DCs but not in osr(KI) DCs and blunted the difference between osr(KI) and osr(WT) DCs. Na(+)/H(+) exchanger activity in osr(WT) DCs was increased by the NKCC1 inhibitor furosemide (100 nM) to values similar to those in osr(KI) DCs. Oxidative stress (10 μM tert-butyl-hydroperoxide) increased Na(+)/H(+) exchanger activity in osr(WT) DCs but not in osr(KI) DCs and reversed the difference between genotypes. Cariporide virtually abrogated Na(+)/H(+) exchanger activity in both genotypes and blunted LPS-induced cell swelling and ROS formation in osr(WT) mice. In conclusion, partial OSR1 deficiency influences Na(+)/H(+) exchanger activity, ROS formation, and migration of dendritic cells.     

Lipopolysaccharide-sensitive H+ current in dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells equipped to transport antigens from the periphery to lymphoid tissues and to present them to T cells. Ligation of Toll-like receptor 4 (TLR4), expressed on the DC surface, by lipopolysaccharides (LPS), elements of the Gram-negative bacteria outer wall, induces DC maturation. Initial steps of maturation include stimulation of antigen endocytosis and enhanced reactive oxygen species (ROS) production with eventual downregulation of endocytic capacity in fully matured DCs. ROS production depends on NADPH oxidase (NOX2), the activity of which requires continuous pH and charge compensation. The present study demonstrates, for the first time, the functional expression of voltage-gated proton (Hv1) channels in mouse bone marrow-derived DCs. In whole cell patch-clamp experiments, we recorded Zn(2+) (50 μM)-sensitive outwardly rectifying currents activated upon depolarization, which were highly selective for H(+), with the reversal potential shift of 38 mV per pH unit. The threshold voltage of activation (V(threshold)) was dependent on the pH gradient and was close to the empirically predicted V(threshold) for the Hv1 currents. LPS (1 μg/ml) had bimodal effects on Hv1 channels: acute LPS treatment increased Hv1 channel activity, whereas 24 h of LPS incubation significantly inhibited Hv1 currents and decreased ROS production. Activation of H(+) currents by acute application of LPS was abolished by PKC inhibitor GFX (10 nM). According to electron current measurements, acute LPS application was associated with increased NOX2 activity.     

Intracellular pH regulation by Na⁺/H⁺ exchanger-1 (NHE1) is required for growth factor-induced mammary branching morphogenesis

Regulation of intracellular pH (pHi) and protection against cytosolic acidification is primarily a function of the ubiquitous plasma membrane Na+/H+exchanger-1 (NHE1), which uses a highly conserved process to transfer cytosolic hydrogen ions (H+) across plasma membranes in exchange for extracellular sodium ions (Na+). Growth factors, which are essential regulators of morphogenesis, have also been found to be key activators of NHE1 exchanger activity; however, the crosstalk between both has not been fully evaluated during organ development. Here we report that mammary branching morphogenesis induced by transforming growth factor-alpha (TGFα) requires PI3K-dependent NHE1-activation and subsequent pHi alkalization. Inhibiting NHE1 activity after TGFα stimulation with 10 μM of the NHE1-specific inhibitor N-Methyl-N-isobutyl Amiloride (MIA) dramatically disrupted branching morphogenesis, induced extensive proliferation, ectopic expression of the epithelial hyper-proliferative marker Keratin-6 and sustained activation of MAPK. Together these findings indicate a novel developmental signaling cascade involving TGFα>PI3K>NHE1>pHi alkalization, which leads to a permissible environment for MAPK negative feedback inhibition and thus regulated mammary branching morphogenesis.     

Protein kinase CK1α regulates erythrocyte survival

Protein kinase CK1 (casein kinase 1) isoforms are involved in the regulation of various physiological functions including apoptosis. The specific CK1 inhibitor D4476 may either inhibit or foster apoptosis. Similar to apoptosis of nucleated cells, eryptosis, the suicidal death of erythrocytes, is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity following energy depletion (removal of glucose) or oxidative stress (exposure to the oxidant tert-butyl hydroperoxide [TBOOH]). Western blotting was utilized to verify that erythrocytes express the protein kinase CK1α, and FACS analysis to determine whether the CK1 inhibitor D4476 and CK1α activator pyrvinium pamoate modify forward scatter (reflecting cell volume), annexin V binding (reflecting phosphatidylserine exposure), and Fluo3 fluorescence (reflecting cytosolic Ca(2+) activity). As a result, both, human and murine erythrocytes express CK1 isoform α. Glucose depletion (48 hours) and exposure to 0.3 mM TBOOH (30 minutes) both decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence. CK1 inhibitor D4476 (10 μM) significantly blunted the decrease in forward scatter, the increase in annexin V binding and the increase in Fluo 3 fluorescence. (R)-DRF053, another CK1 inhibitor, similarly blunted the increase in annexin V binding upon glucose depletion. The CK1α specific activator pyrvinium pamoate (10 μM) significantly enhanced the increase in annexin V binding and Fluo3 fluorescence upon glucose depletion and TBOOH exposure. In the presence of glucose, pyrvinium pamoate slightly but significantly increased Fluo3 fluorescence. In conclusion, CK1 isoform α participates in the regulation of erythrocyte programmed cell death by modulating cytosolic Ca(2+) activity.     

Nitric oxide inhibits LPS-induced tumor necrosis factor synthesis in vitro and in vivo

The effect of nitric oxide (NO) on LPS-stimulated TNF-α synthesis has been studied in vitro and in vivo. The synthesis of TNF-α in J774 macrophages stimulated with LPS (0.l μg/ml) was increased in concentration-related fashion by NO synthase inhibitor L-NMMA (3-30-300 μM) and reduced by either L-arginine (3-30-300 μM) or the NO donor SIN-1 (1-10-1OOμM). The level of TNF-α in the serum of LPS-challenged rats (6mg/kg/i.p.) was increased in animals pre-treated s.c. with L-NMMA (10 and 50mg/kg) and reduced in those given L- arginine (100 and 300mg/kg). These results show a negative feedback mechanism exhibited by NO on TNF-α synthesis suggesting an important regulatory link between NO and TNF-α in pathological processes.     

The Na+/H+‐exchanger (NHE1) generates pH nanodomains at focal adhesions

Many tumor cells are characterized by an increased net acid production. They extrude the excess protons mainly through the Na⁺/H⁺‐exchanger NHE1. An increased NHE1 activity elevates the metastatic potential of tumor cells. Cell migration, a key step in the metastatic cascade, requires the formation and release of integrin‐mediated cell–matrix contacts (focal adhesions). As NHE1 has been localized to focal adhesion sites, the present study tests the hypothesis that NHE1 generates measurable pH nanodomains right at focal adhesions. In order to ratiometrically measure pH close to the plasma membrane, we established a novel application of the total internal reflection fluorescence microscopy (TIRFM). Human melanoma cells were transfected with DsRed2‐paxillin to identify focal adhesion sites. The pH‐sensitive dyes BCECF and WGA‐fluorescein were used to measure the submembranous cytosolic and the pericellular pH, respectively. Distinct pH nanodomains were found at focal adhesions, particularly at those located at the cell front, where NHE1 was concentrated. These sites featured a remarkably alkaline cytosolic and an acidic pericellular pH and thus a much steeper proton gradient across the plasma membrane compared to the rest of the cell. The generation of pH nanodomains could be assigned to NHE1‐mediated H⁺ export because such pH domains could not be detected in NHE1‐deficient cells. Given that both integrin avidity and mechanisms contributing to adhesion turnover are pH‐sensitive, we propose that pH nanodomains at focal adhesions, locally created and maintained by NHE1 activity especially at the cell front, modulate adhesion dynamics in migrating cells. J. Cell. Physiol. 228: 1351–1358, 2013. © 2012 Wiley Periodicals, Inc.     

Mammalian target of rapamycin inhibition in macrophages of asymptomatic HIV+ persons reverses the decrease in TLR-4-mediated TNF-α release through prolongation of MAPK pathway activation

TLR-4-mediated signaling is significantly impaired in macrophages from HIV(+) persons, predominantly owing to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. In this study we assessed in these macrophages if the blunted increase in TLR-4-mediated TNF-α release induced by lipid A (LA) is associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR-4-mediated TNF-α release, but suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in LA-induced TNF-α and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88/IL-1R-associated kinase interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (short hairpin RNA) and mTOR (RNA interference) inhibition resulted in TLR-4-mediated 70-kDa ribosomal protein S6 kinase activation and enhanced TNF-α release, whereas IL-10 release was inhibited in both silenced and nonsilenced HIV(+) macrophages. Furthermore, mTOR inhibition augmented LA-induced TNF-α release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAPK, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR-4-mediated TNF-α release in HIV(+) macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MAPK phosphatase 1 stabilization, which shortens and blunts MAPK activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV(+) persons.     

Inhibition of LPS-induced TNF-α production by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages

The purpose of this study was to examine whether rCGRP has effects on TNF-α produced by mouse resident peritoneal macrophages. Macrophages were obtained from the peritoneal exudate of male Balb/c mouse. The cells were plated on culture dishes at a density of 2.5 × 10⁵ cells per well and allowed to adhere for 2 hr. Pretreatment with rCGRP (10 nM − 1 μM) for 24 hr, the macrophages were cultured with LPS 1 μg/ml for another 24 h. The medium was harvested for measuring TNF-α by ELISA kits. The results showed that rCGRP had no direct effects on TNF-α production, but it inhibited LPS-induced TNF-α production in a concentration-dependent manner. When rCGRP was at a concentration of 1 μM, the LPS-induced TNF-α production was inhibited by 39%. The effect of rCGRP was reversed by hCGRP8-37 (10 μM), an antagonist of CGRP₁ receptor. The LPS-induced TNF-α production from macrophages was also inhibited by forskolin 3 μM, an activator of adenylate cyclase. Furthermore, pretreatment with H-89 1 μM or Rp-cAMPS 100 μM, the inhibitors of cAMP-dependent protein kinase, the effect of rCGRP was abolished. These data suggest that the LPS-induced TNF-α production is inhibited by rCGRP via activation of cAMP responses in mouse resident peritoneal macrophages.     

Bronchial epithelial cell-derived prostaglandin E2 dampens the reactivity of dendritic cells

Airway epithelial cells regulate immune reactivity of local dendritic cells (DCs), thus contributing to microenvironment homeostasis. In this study, we set out to identify factors that mediate this regulatory interaction. We show that tracheal epithelial cells secrete soluble factors that downregulate TNF-α and IL-12p40 secretion by bone marrow-derived DCs but upregulate IL-10 and arginase-1. Size exclusion chromatography identified small secreted molecules having high modulatory activity on DCs. We observed that airway tracheal epithelial cells constitutively release the lipid mediator PGE(2). Blocking the synthesis of PGs within airway epithelial cells relieved DCs from inhibition. Cyclooxygenase-2 was found to be expressed in primary tracheal epithelial cell cultures in vitro and in vivo as shown by microdissection of epithelial cells followed by real-time PCR. Paralleling these findings we observed that DCs treated with an antagonist for E-prostanoid 4 receptor as well as DCs lacking E-prostanoid 4 receptor showed reduced inhibition by airway epithelial cells with respect to secretion of proinflammatory cytokines measured by ELISA. Furthermore, PGE(2) mimicked the effects of epithelial cells on DCs. The results indicate that airway epithelial cell-derived PGE(2) contributes to the modulation of DCs under homeostatic conditions.     

黄芪甲苷对马兜铃酸诱导的巨噬细胞M1型极化的作用及机制研究

目的:探讨黄芪甲苷对马兜铃酸诱导的RAW264.7细胞向M1型极化的影响,并初步探索其可能的作用机制.方法:分别采用马兜铃酸和脂多糖(LPS)刺激RAW264.7细胞24h,伴或不伴黄芪甲苷进行药物干预处理.采用细胞计数检测试剂盒-8(CCK8)检测细胞活性变化,流式细胞仪检测巨噬细胞分型,酶联免疫吸附试验(ELISA...     

Regulation of dendritic cell function by insulin/IGF-1/PI3K/Akt signaling through klotho expression

Insulin or insulin-like growth factor 1 (IGF-1) promotes the activation of phosphoinositide 3 kinase (PI3K)/Akt signaling in immune cells including dendritic cells (DCs), the most potent professional antigen-presenting cells for naive T cells. Klotho, an anti-aging protein, participates in the regulation of the PI3K/Akt signaling, thus the Ca²⁺-dependent migration is reduced in klotho-deficient DCs. The present study explored the effects of insulin/IGF-1 on DC function through klotho expression. To this end, the mouse bone marrow cells were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs). Cells were treated with insulin or IGF-1 and followed by stimulating with lipopolysaccharides (LPS). Tumor necrosis factor (TNF)-α formation was examined by enzyme-linked immunosorbent assay (ELISA). Phagocytosis was analyzed by FITC-dextran uptake assay. The expression of klotho was determined by quantitative PCR, immunoprecipitation and western blotting. As a result, treatment of the cells with insulin/IGF-1 resulted in reducing the klotho expression as well as LPS-stimulated TNF-α release and increasing the FITC-dextran uptake but unaltering reactive oxygen species (ROS) production in BMDCs. The effects were abolished by using pharmacological inhibition of PI3K/Akt with LY294002 and paralleled by transfecting DCs with klotho siRNA. In conclusion, the regulation of klotho sensitive DC function by IGF-1 or insulin is mediated through PI3K/Akt signaling pathway in BMDCs.     

Mitochondrial density contributes to the immune response of macrophages to lipopolysaccharide via the MAPK pathway

We investigated the role of mitochondrial reactive oxygen species (ROS) in the response of macrophages to lipopolysaccharide (LPS) using RAW 264.7 cells and their ρ(o) cells lacking mitochondria. Mitochondrial density, respiratory activity and related proteins in ρ(o) cells were significantly lower than those in RAW cells. LPS rapidly stimulated mitochondrial ROS prior to cytokine secretion, such as TNF-α and IL-6, from RAW 264.7 cells by activating the MAPK pathway, while the response was attenuated in ρ(o) cells. Exposure of ρ(o) cells to H(2)O(2) partially restored the secretion of cytokines induced by LPS. These results suggest that mitochondrial density and/or the respiratory state contribute to intracellular oxidative stress, which is responsible for the stimulation of LPS-induced MAPK signaling to enhance cytokine release from macrophages.     

The sodium/hydrogen exchanger: a possible mediator of immunity

Immune cells such as macrophages and neutrophils provide the first line of defence of the immune system using phagocytosis, cytokine and chemokine synthesis and release, as well as Reactive Oxygen Species (ROS) generation. Many of these functions are positively coupled with cytoplasmic pH (pHi) and/or phagosomal pH (pHp) modification; an increase in pHi represents an important signal for cytokine and chemokine release, whereas a decrease in pHp can induce an efficient antigen presentation. However, the relationship between pHi and ROS generation is not well understood. In immune cells two main transport systems have been shown to regulate pHi: the Na+/H+ Exchanger (NHE) and the plasmalemmal V-type H+ ATPase. NHE is a family of proteins which exchange Na+ for H+ according to their concentration gradients in an electroneutral manner. The exchanger also plays a key role in several other cellular functions including proliferation, differentiation, apoptosis, migration, and cytoskeletal organization. Since not much is known on the relationship between NHE and immunity, this review outlines the contribution of NHE to different aspects of innate and adaptive immune responses such as phagosomal acidification, NADPH oxidase activation and ROS generation, cytokine and chemokine release as well as T cell apoptosis. The possibility that several pro-inflammatory diseases may be modulated by NHE activity is evaluated.     

Design, synthesis and biological assessment of novel N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines and 3-substituted 2,6-dioxopiperidines for TNF-α inhibitory activity

Eight novel 2-(2,6-dioxopiperidin-3-yl)phthalimidine EM-12 dithiocarbamates 9 and 10, N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines 11-14 and 3-substituted 2,6-dioxopiperidines 16 and 18 were synthesized as tumor necrosis factor-α (TNF-α) synthesis inhibitors. Synthesis involved utilization of a novel condensation approach, a one-pot reaction involving addition, iminium rearrangement and elimination, to generate the phthalimidine ring required for the creation of compounds 9-14. Agents were, thereafter, quantitatively assessed for their ability to suppress the synthesis on TNF-α in a lipopolysaccharide (LPS)-challenged mouse macrophage-like cellular screen, utilizing cultured RAW 264.7 cells. Whereas compounds 9, 14 and 16 exhibited potent TNF-α lowering activity, reducing TNF-α by up to 48% at 30 μM, compounds 12, 17 and 18 presented moderate TNF-α inhibitory action. The TNF-α lowering properties of these analogs proved more potent than that of revlimid (3) and thalidomide (1). In particular, N-dithiophthalimidomethyl-3-(phthalimidin-2-yl)-2,6-dioxopiperidine 14 not only possessed the greatest potency of the analogs to reduce TNF-α synthesis, but achieved this with minor cellular toxicity at 30 μM. The pharmacological focus of the presented compounds is towards the development of well-tolerated agents to ameliorate the neuroinflammation, that is, commonly associated with neurodegenerative disorders, epitomized by Alzheimer's disease and Parkinson's disease.     

Mitochondrial permeability transition in rat hepatocytes after anoxia/reoxygenation: role of Ca2+-dependent mitochondrial formation of reactive oxygen species

Onset of the mitochondrial permeability transition (MPT) is the penultimate event leading to lethal cellular ischemia-reperfusion injury, but the mechanisms precipitating the MPT after reperfusion remain unclear. Here, we investigated the role of mitochondrial free Ca(2+) and reactive oxygen species (ROS) in pH- and MPT-dependent reperfusion injury to hepatocytes. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 h and then reoxygenated at pH 7.4 to simulate ischemia-reperfusion. Some cells were loaded with the Ca(2+) chelators, BAPTA/AM and 2-[(2-bis-[carboxymethyl]aono-5-methoxyphenyl)-methyl-6-methoxy-8-bis[carboxymethyl]aminoquinoline, either by a cold loading protocol for intramitochondrial loading or by warm incubation for cytosolic loading. Cell death was assessed by propidium iodide fluorometry and immunoblotting. Mitochondrial Ca(2+), inner membrane permeability, membrane potential, and ROS formation were monitored with Rhod-2, calcein, tetramethylrhodamine methylester, and dihydrodichlorofluorescein, respectively. Necrotic cell death increased after reoxygenation. Necrosis was blocked by 1 μM cyclosporin A, an MPT inhibitor, and by reoxygenation at pH 6.2. Confocal imaging of Rhod-2, calcein, and dichlorofluorescein revealed that an increase of mitochondrial Ca(2+) and ROS preceded onset of the MPT after reoxygenation. Intramitochondrial Ca(2+) chelation, but not cytosolic Ca(2+) chelation, prevented ROS formation and subsequent necrotic and apoptotic cell death. Reoxygenation with the antioxidants, desferal or diphenylphenylenediamine, also suppressed MPT-mediated cell death. However, inhibition of cytosolic ROS by apocynin or diphenyleneiodonium chloride failed to prevent reoxygenation-induced cell death. In conclusion, Ca(2+)-dependent mitochondrial ROS formation is the molecular signal culminating in onset of the MPT after reoxygenation of anoxic hepatocytes, leading to cell death.     

小鼠骨髓源性树突状细胞体外诱导培养方法的比较研究

目的探讨最佳体外诱导培养小鼠成熟树突状细胞（dendritic cells,DC）的方法。方法分离、纯化6周龄C57BL／6小鼠骨髓单核细胞,以含10％胎牛血清、20ng／ml重组小鼠粒细胞-巨噬细胞集落刺激因子（GM—CSF）和10ng／ml重组小鼠白细胞介素-4（IL-4）的RPMI-1640培养基培养7d,然后将细胞分成对照未刺激组、肿瘤坏死因子-α（TNF-α）刺激组和TNF-α＋脂多糖（lipopolysaccharides,LPS）刺激组。继续培养48h后,观察各组细胞形态,检测IL-12、IL-6浓度及细胞表面标志CD11c、CD80、CD86和MHC II。结果培养9d后,两刺激组培养的细胞经相差显微镜观察有DC生长。TNF—α刺激组细胞培养上清液中IL-6、IL-12含量显著高于对照组（P〈0．01）,但显著低于TNF—α＋LPS刺激组（P〈0．05）。3组均高表达CD11c,各组间无显著差异;而CD80、CD86和MHC II表达阳性率TNF-α刺激组显著高于对照组（P〈0．01）,TNF-α＋LPS刺激组显著高于单纯TNF—α刺激组（P〈0．05）。结论联合使用TNF-α与LPS刺激可使DC成熟度提高,分泌IL-6、IL-12增加。     

Taurine attenuates lipopolysaccharide-induced disfunction in mouse mammary epithelial cells

The intent of this study was to evaluate the active defense reaction of mouse mammary epithelial cells and the cytoprotective and anti-inflammatory properties of taurine to lipopolysaccharide (LPS)-induced disfunction in mouse mammary epithelial cells. (1) Primary cultured mouse mammary epithelial cells were stimulated with LPS for 24 h (final concentration=0, 5, 10, 20 μg/mL). Western blotting demonstrated a significant decrease in the secretion of β-casein in the 20 μg/mL LPS treatment group (P<0.05), while nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), lactoferrin (LF) and N-acetyl-β-D-glucosaminidase (NAGase) were all significantly increased following LPS treatment (P<0.01). Furthermore, cell survival was significantly inhibited after treatment with 20 μg/mL LPS; however, neither 5 μg/mL nor 10 μg/mL LPS had any effect on cell survival. Therefore, a level of 10 μg/mL LPS was selected to test the protective effect of taurine on mouse mammary epithelial cells. (2) Primary cultured mouse mammary epithelial cells were treated with 0, 5, 15 or 45 mmol/L taurine for 3 h, followed by 10 μg/mL LPS for 24 h. Taurine significantly attenuated the LPS-induced increase in NAGase activity, NO concentrations and the level of TNF-α, IL-1β, IL-6 and LF. Taurine at 45 mmol/L markedly increased β-casein secretion in response to LPS-induced disfunction. This study demonstrated that the addition of taurine to a culture medium significantly inhibited the LPS-induced release of inflammatory factors and increased β-casein secretion from mammary epithelial cells, thereby providing a possible explanation for the protective effect proposed for taurine in the prevention of LPS-induced disfunction in mammary epithelial cells.