Reactive oxygen species (ROS) are produced in various cells and affect many biological processes. We previously reported that 2-methyl-1,4-naphtoquinone (menadione) inhibited Ca2+ influx from the extracellular medium and exocytosis evoked by antigen stimulation in the mast cell line, RBL-2H3. Mast cells release various inflammatory mediators such as leukotrienes (LTs) and cytokines in addition to the exocytotic secretion of histamine. In this study, we investigated the effects of menadione on LT release in RBL-2H3. Treatment of RBL cells with menadione inhibited LTC4 secretion induced by antigen stimulation. To elucidate the mechanism of this inhibition, we examined the effects of menadione on the activation process of 5-lipoxygenase that is responsible for the synthesis of LTs from arachidonic acid. Menadione did not affect the phosophorylation of mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) and p38, which regulates phosphorylation of 5-lipoxygenase. However, menadione inhibited the translocation of 5-lipoxygenase from the cytoplasm to the nuclear membrane. Together with the result that LT secretion was severely impaired in the absence of extracellular Ca22+, it is suggested that ROS produced by menadione inhibited LT secretion through impaired Ca22+ influx and 5-lipoxygenase translocation to the nuclear membrane.
The compound β-citryl-L-glutamate (β-CG) was initially isolated from developing brains, while it has also been found in high concentrations in testes and eyes. However, its functional roles are unclear. To evaluate its coordination with metal ions, we performed pH titration experiments. The stability constant, logβpqr for Mp(β-CG)qHr was calculated from pH titration data, which showed that β-CG forms relatively strong complexes with Fe(III), Cu(II), Fe(II) and Zn(II). β-CG was also found able to solubilize Fe more effectively from Fe(OH)2 than from Fe(OH)3. Therefore, we examined the effects of β-CG on Fe-dependent reactive oxygen species (ROS)-generating systems, as well as the potential ROS-scavenging activities of β-CG and metal ion-(β-CG) complexes. β-CG inhibited the Fe-dependent degradation of deoxyribose and Fe-dependent damage to DNA or plasmid DNA in a dose-dependent manner, whereas it had no effect on Cu-mediated DNA damage. In addition, thermodynamic data showed that β-CG in a physiological pH solution is an Fe(II) chelator rather than an Fe(III) chelator. Taken together, these findings suggest that β-CG is an endogenous low molecular weight Fe chelator.
A human aldose reductase-like protein, AKR1B10 in the aldo-keto reductase (AKR) superfamily, was recently identified as a tumor marker of several types of cancer. Tolrestat, an aldose reductase inhibitor (ARI), is known to be the most potent inhibitor of the enzyme. In this study, we compared the inhibitory effects of other ARIs including flavonoids on AKR1B10 and aldose reductase to evaluate their specificity. However, ARIs showed lower inhibitory potency for AKR1B10 than for aldose reductase. In the search for potent and selective inhibitors of AKR1B10 from other drugs used clinically, we found that non-steroidal antiinflammatory N-phenylanthranilic acids, diclofenac and glycyrrhetic acid competitively inhibited AKR1B10, showing Ki values of 0.35—2.9 μM and high selectivity to this enzyme (43—57 fold versus aldose reductase). Molecular docking studies of mefenamic acid and glycyrrhetic acid in the AKR1B10–nicotinamide adenine dinucleotide phosphate (NADP+) complex and site-directed mutagenesis of the putative binding residues suggest that the side chain of Val301 and a hydrogen-bonding network among residues Val301, Gln114 and Ser304 are important for determining the inhibitory potency and selectivity of the non-steroidal antiinflammatory drugs. Thus, the potent and selective inhibition may be related to the cancer chemopreventive roles of the drugs, and their structural features may facilitate the design of new anti-cancer agents targeting AKR1B10.
The coding region nucleotide sequences of rat, hamster, and bovine S-adenosylmethionine decarboxylase (AdoMetDC) cDNA exhibit over 90% homology with the human sequence. No N-terminal amino acid could be detected when either bovine or rat AdoMetDC was subjected to Edman degradation, suggesting that the β-subunit must be blocked since the pyruvate residue is located at the amino terminus of the α-subunit. In this study, we present the primary structure, including post-translational modification, of rat prostate AdoMetDC. Our strategy was to compare the molecular masses of peptides produced by five specific cleavage methods with peptides expected from the known cDNA-derived amino acid sequence of rat AdoMetDC using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). All AdoMetDC peptide fragments produced by the five cleavage methods could be assigned to theoretical peptides based on the rat cDNA sequence except for the peptides containing the N-terminus of the β- and α-subunits. The N-terminus of the α-subunit was assigned as pyruvoyl peptide. Liberation of acetylmethionine was demonstrated when the peptide containing the β-subunit N-terminal amino acid obtained by lysylendopeptidase digestion was reacted with acylamino acidreleasing enzyme. Furthermore, N-terminal acetylation of the β-subunit was confirmed by MALDI-post source decay analysis. In conclusion, the results of the present study on amino acid full sequence of rat prostate AdoMetDC determined by the combination of five specific cleavage methods demonstrate that the N-terminus of the β-subunit is acetylated, and the expected amino acid sequence based on the rat AdoMetDC cDNA sequence is correct.
CD133 is regarded as a marker of tumor initiating cells in many tumors, including colorectal cancer. O'Brien and Ricci et al. have proved that in primary colorectal tumors there are colorectal tumor stem cells (initiating cells) which are marked by CD133 antigen. Using a genetic knockin lacZ reporter mouse model, Shmelkov et al. challenged this increasingly influential viewpoint and drew two important conclusions that challenge former opinions. First, CD133 is widely distributed throughout the full range of tumor epithelial cells in the colon as opposed to being limited to a few cells. Second, CD133 negative cells of colon tumors are also tumorigenic, and are more inclined to metastasize. Based on these two opinions, we hypothesize that the expression of CD133 is different among tumor cells, and that quantitative but not qualitative analyses of CD133 abundance are necessary to determine the relationship between CD133 expression and tumor stem cell characteristics. To verify this hypothesis, colorectal cancer cell line SW620 was cultured and sorted into CD133Hi, CD133Mid and CD133Low subgroups using magnetic microbeads to compare their xenograft biological characteristics. The results showed that the CD133Hi subgroup of SW620 is more close to the tumor initiating cells in terms of biological characteristics than CD133Mid and CD133low subgroups, but the CD133low subgroup still maintains the ability of tumorigenicity. It supported that tumor initiating cells are more correlated to the abundance of CD133.
Recent research has shown that the Na+/K+-ATPase α1 subunit is a novel anti-cancer target, which plays pivotal roles in malignant cell ion transport, metabolism, migration and signal transduction. The purpose of the present study was to investigate the anti-cancer effects of ouabain and Na+/K+-ATPase α1 small interfering ribonucleic acid (siRNA) on HepG2 cell proliferation, apoptosis and cell cycle, and to explore the molecular mechanisms. The expression of Na+/K+-ATPase α1 subunit in human hepatocellular carcinoma (HCC), normal liver tissues and human HCC line (HepG2, SMMC-7721 and Bel-7402) has been investigated. Using the ouabain and Na+/K+-ATPase α1 subunit siRNA, which target the Na+/K+-ATPase, we have evaluated the effects of inhibiting Na+/K+-ATPase α1 in human HepG2 cells with respect to cell proliferation, morphology, cell cycle, impact on intracellular Ca2++, reactive oxygen species (ROS) concentration, and correlated gene expression level on messenger ribonucleic acid (mRNA) and protein. Our data showed that the expression Na+/K+-ATPase α1 subunit in HCC tissues is higher than that in normal liver tissues. Ouabain and Na+/K+-ATPase α1 siRNA could inhibit HepG2 cell proliferation. Ouabain could induce HepG2 cell apoptosis and generate S phase arrest, and siRNA could enhance the anti-cancer effect of ouabain that induced HepG2 cells apoptosis via an intracellular Ca2+ and ROS increase-mediated, and generated cell cycle S phase arresting by decreasing the CyclinA1/cyclin-dependent kinase 2 (CDK2)/proliferating cell nuclear antigen (PCNA) complex product and increasing the expression of cyclin-dependent kinase inhibitor 1A (P21CIP1). We believe that targeting of the Na+/K+-ATPase α1 subunit in human HCC cells could provide new sight into the treatment of HCC.
Radiation induces an important inflammatory response in the irradiated organs, characterized by leukocyte infiltration and vascular changes. Since adhesion molecules play an important role in facilitating the immune response at the inflammation sites, interfering with the expression of these molecules may be an important therapeutic target of radiation induced inflammation. Many adhesion molecules such as intercellular cell adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) have been identified in radiation. Ferulic acid (FA), an effective radioprotector during radiotherapy, is widely used in endothelium protection. The present study examined the effect of FA on the induction of adhesion molecules by gamma-radiation and the mechanisms of its effect in gamma-irradiated human umbilical vein endothelial cells (HUVECs). HUVECs were pretreated for 18 h with FA and then exposed to 10 Gy radiation. The result of cell adhesion assay showed FA inhibited radiation-induced U937 adhesion to HUVECs. FA prevented induction of ICAM-1 and VCAM-1 expression in a concentration-dependent manner after stimulation with radiation at the level of mRNA and protein. Inhibitors of the extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways were used to determine which pathway was involved in FA action; the result showed that the inhibitory effect of FA on adhesion molecule expression was mediated by the blockade of JNK. FA appears to be a potential therapeutic agent for treating various inflammatory disorders including radiation induced inflammation.
Reperfusion arrhythmias (RA) especially ventricular tachycardia (VT) and ventricular fibrillation (VF) remain the most important causes of sudden death following reperfusion. In isolated rat hearts grape seed proanthocyanidin extract (GSPE) had been proved to reduce the incidence of reperfusion-induced VF and VT. However the mechanism of this protection remained unclear. The aim of this study was to elucidate the potential mechanism of this protection of GSPE. The myocardial ischemia reperfusion (IR) model was induced by 30 min coronary occlusion and 120 min reperfusion in open chest anesthetized rats. The ultrastructure of ischemic cardiomyocytes was observed. An isobaric tag labeling for relative and absolute quantification (iTRAQ) proteomics was used to identify differentially expressed membrane proteins. Western blot was performed to verify the results of iTRAQ. The results demonstrated GSPE can significantly reduce the incidence of VT and VF induced by reperfusion in vivo. We identified 92 differentially expressed proteins. Western blot analysis confirmed GSPE increased the expression of Na+/K+-ATPase α1 subunit (p<0.01). We found the subunit distribution of Na+/K+-ATPase was changed after reperfusion. Na+/K+-ATPase α1 subunit was decreased in IR group (without GSPE-treated) compared to sham group while it was significantly increased in GSPE group. The decrease of free radical generation induced by GSPE may lead to the up-regulation of Na+/K+-ATPase α1 subunit. This change of subunit distribution may lead to the increase of activity of Na+/K+-ATPase which may result in the protection of GSPE against reperfusion arrhythmias. Our experiments provided new avenues for the treatment of reperfusion arrhythmias.
Probucol is a cholesterol-lowering drug with an anti-proliferative effect. Excessive growth of glomerular mesangial cells and overexpression of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) are the pathological features of diabetic nephropathy. In this study, human mesangial cells (HMCs) treated with high glucose showed the above-mentioned features through the activation of Janus kinase 2 (JAK2)/signal transducers and activators of transcription (STAT) pathway. Probucol can suppress cell proliferation, down-regulate mRNA and protein levels of TGF-β1 and CTGF in HMCs treated with high glucose. Phosphorylation of JAK2, STAT1 and STAT3 caused by high glucose was obviously prevented in HMCs pretreated with probucol, indicating that the protective effect of probucol on HMCs might be through the inhibition of JAK2/STAT pathway. Therefore, probucol could be a potential therapeutic agent for diabetic nephropathy, and this paper provides new insights into the molecular mechanisms underlying probucol's effects.
To elucidate molecular mechanisms of adipocyte differentiation, we previously isolated TC10-like/ TC10βLong (TCL/TC10βL), which was transiently expressed in the early phase of adipogenesis of 3T3-L1 cells and seemed to be a positive regulator of adipogenesis. By using TCL/TC10βL-overexpressing NIH-3T3 cells, we also isolated gelsolin as a gene whose expression was up-regulated by TCL/TC10βL. However, the roles of gelsolin in adipocyte differentiation are unclear. In this paper we characterized the function of gelsolin in adipogenesis in 3T3-L1 cells. The level of gelsolin changed during adipocyte differentiation. Knockdown of the expression of gelsolin using RNAi inhibited adipocyte differentiation, and impaired the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein (C/EBP) α. Interestingly, the knockdown also impaired mitotic clonal expansion (MCE), and increased cell size, though it reduced levels of C/EBPβ and C/EBPδ, markers for the early stage of adipogenesis, only slightly. Gelsolin plays a crucial role in the differentiation of 3T3-L1 cells into adipocytes.
Urban air pollution, especially in developing countries, is a crucial environmental problem. Urban aerosols may contain various kinds of substances and induce harmful effects such as allergic diseases. Therefore, it is critical to clarify the biological effects of urban aerosols on human health. In this study, we evaluated the induction of airway inflammation in vitro and in vivo due to exposure of urban aerosols. We investigated cytokine production and nuclear factor-κB (NF-κB) activation after stimulation of macrophage cells by exposure of urban aerosols. Urban aerosols were found to induce the production of interleukin (IL)-8, tumor necrosis factor-α and IL-1β on macrophage cells. In addition, we showed that NF-κB pathway regulated the urban aerosols-induced inflammatory cytokine response. Moreover, the intranasal administration of urban aerosols resulted in increases in the total cell number in bronchoalveolar lavage and infiltration of eosinophils in lung tissue. These results indicate that urban aerosols induce respiratory inflammation and onset of inflammatory disease due to an activation of the immune system.
Nuclear receptor liver receptor homolog-1 (LRH-1; NR5A2) plays a crucial role in the homeostasis of bile acids and cholesterol by controlling the expression of genes central to bile acid synthesis and efflux, reverse cholesterol transport, and high density lipoprotein-remodeling. However, the molecular mechanisms that modulate the transactivation activity of LRH-1 remain unclear. It is proposed that LRH-1's activity is regulated by post-modifications, the binding of small heterodimer partner (SHP), or the binding of coregulators. To search for cofactors that regulate the transactivation activity of LRH-1, we performed a pull-down assay using glutathione Stransferase (GST) fused to the N-terminal portion of LRH-1 and nuclear extracts from HeLa cells, and identified Ku proteins as interacting proteins with LRH-1. We also found that Ku proteins associate with LRH-1 through its DNA-binding domain and hinge region. Luciferase reporter assays revealed that Ku proteins repressed the SHP promoter activity mediated by LRH-1. Furthermore, Ku proteins suppressed the coactivating effect of peroxisome proliferator-activated receptor (PPAR) &gammma; coactivator-1α (PGC-1α), an LRH-1 coactivator, on the LRH-1-mediated SHP promoter activity. Previously, we showed that Ku proteins interacted with nuclear receptor farnesoid X receptor (FXR; NR1H4) and decreased the expression of its target gene. In this study, we demonstrated that Ku proteins also interacted with not only LRH-1 but various nuclear receptors, such as the estrogen receptor, PPAR, and Rev-erb. Ku proteins may function as corepressors for various nuclear receptors including LRH-1.
Previous studies have demonstrated that activation of thrombin receptor could promote endothelial progenitor cell (EPC) migration. As cAMP-response-element-binding-protein-binding protein (CBP) is involved in many cellular biological processes, we hypothesized that CBP mediates thrombin-induced EPC migration. In this study, we examined whether CBP silencing would affect EPC migration induced by thrombin using small interference RNA approach. EPC isolated from the bone marrow of femurs and tibias of Sprague-Dawley rats were cultured and identified, and then were treated by thrombin alone or combined with CBP-shRNA lentivirus. Transwell chamber assay was performed to measure EPC migration. Quantitative real-time polymerase chain reaction and Western blot were carried out to detect the expression of CBP and CXCR4. Thrombin induced CBP expression in a time- and dose-dependent manner. Small interference RNA for CBP downregulated thrombin-induced CBP expression. Thrombin-induced EPC migration was also attenuated by CBP downregulation. Western blot indicated that CXCR4 expression on EPC is upregulated by thrombin and this effect was blocked by CBP silencing. In conclusion, thrombin-induced EPC migration was inhibited by CBP silencing via downregulation of CXCR4 expression, indicating that CBP plays an important role in thrombin-induced EPC migration.
Nucleolin is an abundant non-ribosomal protein found in nucleolus and a major component of silver-stained nucleolar organizer region (AgNOR), a histopathological marker of cancer which is highly elevated in cancer cells. We recently reported that nucleolin on the cell surface of mouse gastric cancer cells acts as a receptor for tumor necrosis factor-α-inducing protein (Tipα), a new carcinogenic factor of Helicobacter pylori. In this study, we first examined the localization of nucleolin on cell surface of five gastric cancer cell lines by cell fractionation and flow cytometry: We found that large amounts of nucleolin were present on surface of MKN-45, KATOIII, MKN-74, and AGS cells, with smaller amounts on surface of MKN-1 cells. The membrane fraction of normal epithelial cells of mouse glandular stomach did not contain much nucleolin, suggesting that translocation of nucleolin to the cell surface occurs during carcinogenesis, making for easier binding with Tipα. AS1411, a nucleolin targeted DNA aptamer, inhibited growth of gastric cancer cell lines in this order of potency: MKN-45>KATOIII>AGS>MKN-74=MKN-1, associated with induction of S-phase cell cycle arrest. Fluorescein isothiocyanate (FITC)-AS1411 was more rapidly incorporated into MKN-45 and AGS than into MKN-1 cells, based on varying amounts of cell surface nucleolin. We think that AS1411 first binds to nucleolin on the cell surface and that the binding complex is then incorporated into the cells. All results indicate that nucleolin on the cell surface is a new and promising therapeutic target for treatment of gastric cancer.
The outer layer of the cell wall of pathogenic fungi, Candida species, consists of mannan, which plays an important role in infection. In this study, we synthesized several oligosaccharides using mannosyltransferases obtained from Candida parapsilosis and Candida albicans. Namely, we synthesized mannotetraoses [Manα1→ 2Manα1→3Manα1→2Man and Manα1→3(Manα1→6)Manα1→2Man] from mannotriose, Manα1→3Manα1→ 2Man, and mannohexaoses [Manα1→2Manα1→3Manα1→3Manα1→2Manα1→2Man and Manα1→3(Manα1→6)Manα1→3Manα1→2Manα1→2Man] from mannopentaose, Manα1→3Manα1→3Manα1→2Manα1→2Man. The linkage sequence of these oligosaccharides was identified by a sequential 1H-NMR assignment method combined with rotating frame nuclear Overhauser enhancement spectroscopy and relayed coherence transfer spectroscopy. The steric effect by the α-1,6-linked branching mannose residue to the H-1 proton chemical shift of the neighboring 3-O-substituted mannose residue was different from that of the 2-O-substituted mannose residue. These oligosaccharides having novel structures seem to be useful as the substrate or ligand for glycomics.
The effect of the proinflammatory cytokine interleukin (IL)-1β on the cellular proliferation of human osteoblastic cells (SaM-1) and osteosarcoma-derived cells (SaOS-2, HOS, and MG-63) was examined. IL-1β stimulated the proliferation of SaM-1 and MG-63 cells, but had no effect on that of SaOS-2 or HOS cells. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, the mRNA expression of IL-1 receptor type I (IL-1R1) was detected in SaM-1 and MG-63 cells consistently, but not in SaOS-2 or HOS cells in the proliferative stage. Neither the decoy inhibitory IL-1 receptor type II (IL-1R2) nor IL-1R antagonist mRNA was detected in any of the cell lines, suggesting that IL-1β stimulated proliferation via IL-1R1. The IL-1β -stimulated proliferation was inhibited by the MAPK kinase (MEK) inhibitor PD98059 but not by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 or the cyclooxygenase-2 specific inhibitor NS-398, suggesting that IL-1β stimulated proliferation via MEK, without affecting prostaglandin E2 synthesis. IL-1β stimulated cellular proliferation but inhibited the synthesis of osteocalcin containing γ-carboxylated glutamic acid (Gla-OSCAL). Both the increased proliferation and decreased Gla-OSCAL synthesis were suppressed by vitamin K2 (VK2), which is a cofactor for γ-carboxylase. Furthermore, the inhibitory effect of VK2 on IL-1β -stimulated proliferation was suppressed by warfarin. However, rifampicin the nuclear receptor steroid and xenobiotic receptor (SXR) ligand had no effect of IL-b , suggesting that IL-1β is involved in VK2 dependent γ-calboxylation but not SXR-activation. These results suggest that IL-1β stimulated cellular proliferation via MEK and inhibited Gla-OSCAL synthesis, which were both inhibited by VK2via γ-carboxylation.
For rats, glomerular filtration rate (GFR) and its relative GFR (ratio to normal GFR0) were estimated in order to classify their chronic kidney disease (CKD) into 5 stages like those in humans. The adenine-loaded rats, which were used to show the intrinsic antioxidant and creatinine (Cr) metabolite, NZ-419 (5-hydroxy-1- methylimidazolidine-2,4-dione), when taken orally, prevented the progression of chronic renal failure (CRF), were used as a model to reach the severest stage 5. In this report, we show that, by using both a tubular lesion and a glomerular lesion models (adenine-loaded and 5/6 nephrectomized rats, respectively), peroral NZ-419 might be a common tool to prevent the progression of CRF at CKD stages 3 and 4 under the condition that most rats in the control group still remained at stage 4 (0.15<GFR/GFR0<0.29) at the last. At the minimum effective dose (MED: 100 mg/kg/d) of NZ-419 in adenine-loaded rats, serum Cr and all oxidative stress markers were ameliorated. Two doses (80, 160 mg/kg/d), at around the MED, used for 5/6 nephrectomized rats with a similar CRF severity, gave significant inhibitory effects against the increases in blood urea nitrogen, decreases in renal blood flow and renal plasma flow, and nephrotic syndrome. Oxidative stress markers, the urinary methylguanidine and serum albumin level, were significantly ameliorated.
Shikonin, a major ingredient in the Chinese traditional herb Lithospermum erythrorhixon, exhibits multiple biological functions including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we delineated the molecular mechanisms of shikonin in the apoptosis of 143B osteosarcoma cells. Shikonin reduced the cell viability of 143B cells in a dose- and time-dependent manner. The IC50 at 24 h and 48 h for 143B cells was 4.55 and 2.01μM, respectively. A significantly elicited hypodiploid cell population was found in cells treated with 2, 4, and 8μM shikonin for 24 h. Moreover, treatment with shikonin induced reactive oxygen species (ROS) generation, increased extracellular signal-regulated kinase (ERK) phosphorylation, decreased B-cell lymphoma-2 (Bcl2) expression, and was accompanied by poly(ADP-ribose) polymerase (PARP) cleavage. Pretreatment with the antioxidant agent N-acetyl cysteine (NAC) not only reversed shikonin-induced ROS generation but also significantly attenuated the cytotoxic effects of shikonin in 143B cells. Furthermore, NAC attenuated shikonin-induced ERK phosphorylation. Taken together, our results reveal that shikonin increased ROS generation and ERK activation, and reduced Bcl2, which consequently caused the cells to undergo apoptosis. Therefore, shikonin may be a promising chemotherapeutic agent for osteosarcoma treatment.
To investigate the involvement of reactive oxidant species (ROS), presumably arising from cytochrome P-450 (CYP)-catalyzed metabolism of schisandrin B (Sch B), in triggering glutathione antioxidant response, Sch Binduced reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and CYP-catalyzed reaction and associated ROS production were examined in rat heart microsomes. Sch B analogs were also studied for comparison. Using rat heart microsomes as a source of CYP, Sch B and schisandrin C (Sch C), but not schisandrin A and dimethyl diphenyl bicarboxylate (an intermediate compound derived from the synthesis of Sch C), were found to serve as co-substrate for the CYP-catalyzed NADPH oxidation reaction, with concomitant production of ROS. The stimulation of CYP-catalyzed NADPH oxidation reaction and/or ROS production by Sch B or Sch C correlated with the increase in mitochondrial reduced glutathione level and protection against ischemia/reperfusion (I/R) injury in rat hearts. The involvement of ROS in Sch B-induced cardioprotection was further confirmed by the suppressive effect produced by N-acetylcysteine or α-tocopherol pretreatment. Taken together, these results suggest that Sch B-induced glutathione antioxidant response and cardioprotection may be mediated by ROS arising from CYP-catalyzed reaction.
Researchers see algae as a promising tool to discover both efficient and safe agents for pain therapy. We evaluated the antinociceptive and anti-inflammatory activities of lectin from the marine alga Pterocladiella capillacea lectin (PcL). PcL was purified and tested in classical models of nociception and inflammation. Male Swiss mice received PcL 30 min prior to receiving 0.8% acetic acid (10 μl/10 g, i.p.), 1% formalin (20 μl/intraplantar) or the hot plate test, and were compared to untreated animals or animals pretreated with indomethacin or morphine. PcL (0.9, 8.1 or 72.9 mg/kg, i.v.) significantly reduced the number of writhes (30%, 39%, and 52%, respectively). PcL (72.9 mg/kg, i.v.) also reduced (p<0.05) both the first and second phases of the formalin test by 58% and 87%, respectively. However, PcL (72.9 mg/kg) did not present significant antinociceptive effects in the hot plate test when compared to morphine, suggesting that its antinociceptive action occurs via peripheral rather than a central-acting mechanism. It was also observed that leukocyte migration was induced by carrageenan (500 μg/cavity) in male Wistar rats and that PcL (8.1 mg/kg, i.v.) significantly reduced neutrophil migration by 84%, as compared to untreated animals, suggesting inhibition of inflammatory mediators. The data indicated that PcL has peripheral actions with both anti-inflammatory and antinociceptive properties.
Elevated levels of β-amyloid (Aβ) in the brains being a hallmark of Alzheimer's disease (AD) have been believed to play a critical role in the cognitive dysfunction that occurs in AD. Recent evidence suggests that Aβ induces neuronal apoptosis in the brain and in primary neuronal cultures. In this study, we investigated the effects of β-asarone, the major ingredient of Acorus Tatarinowii Schott, on cognitive function and neuronal apoptosis in Aβ hippocampus injection rats and its mechanism of action. The results show that the Aβ (1—42) injection caused impairments in spatial reference memory in a Morris water maze task and apoptosis in hippocampus. Oral administration of β-asarone with three different dose (12.5, 25, or 50 mg/kg) for 28 d ameliorated Aβ (1—42)-induced cognitive impairment and reversed the increase of apoptosis in the hippocampus. Aβ-induced c-Jun N-terminal kinase (JNK) results in phosphorylation, subsequent down-regulation of Bcl-2 and Bcl-w expression, and caspase-3 activation. Beta-asarone attenuate Aβ (1—42)-induced neuronal apoptosis in hippocampus by reversal down-regulation of Bcl-2, Bcl-w, caspase-3 activation, and JNK phosphorylation. These results suggest that β-asarone may be a potential candidate for development as a therapeutic agent to manage cognitive impairment associated with conditions such as Alzheimer's disease.
The aim of this study was to investigate the vasorelaxant effect induced by cassiarin A, a novel antiplasmodial alkaloid from Cassia siamea, in rings cut from rat superior mesenteric arteries. In rings precontracted with phenylephrine, cassiarin A induced a concentration-dependent relaxation. This relaxation was attenuated: 1) after removal of the endothelium or after pretreatment of rings with 100 μM of NG-nitro-L-arginine (nitric oxide synthase inhibitor) or 10 μM of 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (guanylyl cyclase inhibitor), but not after pretreatment with 10 μM of indomethacin (cyclooxygenase inhibitor); and 2) after pretreatment of preparations with either a nonselective or selective inhibitor of large-conductance Ca2+-activated K+ (BKCa) channels [1 mM of tetraethylammonium or 100 nM of iberiotoxin, respectively]. The cassiarin A-induced relaxation was also attenuated by these BKCa inhibitors in endothelium-denuded preparations. The cassiarin Ainduced relaxation was not altered by treatment with the ATP-sensitive K+-channel inhibitor glibenclamide (10 μM) or with the voltage-dependent K+-channel inhibitor 4-aminopyridine (1 mM). In isolated mesenteric artery rings, cassiarin A tended to increase nitric oxide (NO) levels. These results suggest that in the rat mesenteric artery, cassiarin A-induced relaxation may be mediated by endothelial NO and may occur partly via BKCa-channel activation.
The present study was undertaken to investigate the effect of ethanol on the sleep-wake cycle in normal rats and sleep-disturbed rats. In normal rats, no significant difference was observed by ethanol in sleep latency, total awake time and total non-rapid eye movement (NREM) sleep time, except for total REM sleep time. On the other hand, in sleep-disturbed rats, ethanol at doses of 1 and 2 g/kg caused significant decreases in sleep latency and total wake time, and an increase in total NREM sleep time. In addition, ethanol showed a significant increase in delta activity in the sleep-disturbed model rat, different from triazolam. These results suggested that ethanol had not only a hypnotic but also a sleep-maintaining effect in sleep-disturbed rats at reasonable blood ethanol concentrations.
Benzylideneacetophenone analogues are known to have several significant biological activities, including antiinflammatory, antitumor, antibacterial, antiviral, and gastric-protective activities. However, the antiproliferative effects of benzylideneacetophenone analogues on vascular smooth muscle cells (VSMCs) are unknown. The aim of this study was to elucidate the antiproliferative effects and molecular mechanism of BST406, a newly synthesized benzylideneacetophenone derivative, on platelet-derived growth factor (PDGF)-BB-stimulated rat aortic VSMCs. BST406 inhibited [3H]-thymidine incorporation into DNA in VSMCs following treatment with PDGFBB 25 ng/ml. PDGF-BB-stimulated DNA synthesis was significantly reduced. Moreover, pretreatment with BST406 (0—10μM) suppressed the proliferation of PDGF-BB-stimulated cells in a concentration-dependent manner. We also investigated the mechanism of the antiproliferative effects of BST406 in PDGF-BB-stimulated VSMCs. In Western blot analysis, PDGF-BB-stimulated (25 ng/ml) phospholipase-C (PLC)γ1 and Akt phosphorylation was inhibited by BST406 (0—10μM). However, BST406 did not inhibit the PDGF-receptor β-chain (PDGF-Rβ) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation induced by PDGF-BB. To confirm that the inhibitory effects of BST406 are mediated through the inhibition of PLCγ1 or Akt, the effects of inhibitors on cell viability were examined. U73122 completely inhibited PDGF-BB-induced proliferation of VSMCs. However, LY294002 10μM had no significant effects on PDGF-BB-induced proliferation. These findings suggest that the inhibitory effects of BST406 on the proliferation of PDGF-BB-stimulated VSMCs are mediated by suppression of the PLCγ1 signaling pathways. Our observations may explain, in part, the mechanistic basis for the prevention of cardiovascular disease (such as atherosclerosis and restenosis after coronary angioplasty) by BST406.
The relationship of chemical structures of 6-formylpterin (6FP) and its derivatives with scavenging activity of singlet oxygen (1O2) was examined. First, effects of pterin derivatives on 1O2 released from activated human neutrophils were examined. The neutrophils, stimulated with opsonized zymosan, released 1O2 that was detected by chemiluminescence using a 1O2 specific probe, trans-1-(2′-methoxyvinyl)pyrene. 6FP and its derivatives suppressed the 1O2 release. 6FP and other commercially available pterin derivatives, such as biopterin and neopterin, which have different substitutions at the 6-position, suppressed the 1O2 release with similar extent. On the other hand, newly synthesized pterin derivatives, which have different substitutions at the 2- and/or 3-position, such as 2-amino-6-formyl-3-methylpteridin-4-one, suppressed the 1O2 release in a dose-dependent manner and more potently than 6FP. Then, the 1O2 scavenging activity of pterin derivatives was examined photochemically by direct analysis of near-infrared luminescence at 1270 nm, the most sensitive method for the detection of 1O2. When rose Bengal, a photosensitizer, in D2O solution, was irradiated by 514 nm laser beam, the emission spectrum of 1O2 was observed. 6FP suppressed this emission spectrum of 1O2, and the newly synthesized pterin derivatives with different substituent at the 2- and/or 3-position suppressed the spectrum more potently than 6FP. The order of potency was similar to that obtained from biological assays. These findings indicate that the substitutions at the 2- and/or 3-position play an important role in 1O2 scavenging activity of pterin derivatives.
In this study, the antipruritic effect of the methanol extract of Ganoderma lucidum (MEGL) was studied in mice. Oral administration of MEGL (10—1000 mg/kg) produced a dose-dependent inhibition of scratching, an itch-related response, induced by intradermal 5-hydroxytryptamine (5-HT) (100 nmol/site), α-methyl-5-HT (100 nmol/site), and proteinase-activated receptor-2 (PAR2)-activating peptide SLIGRL-NH2 (50 nmol/site). However, MEGL (100—1000 mg/kg) did not inhibit the scratching induced by histamine (100 nmol/site), substance P (100 nmol/site), and compound 48/80 (10 μg/site). These results raise the possibility that MEGL is effective against pruritus mediated by proteinases and 5-HT and that primary afferents expressing PAR2 and 5-HT2A receptors are the sites of its action.
The physiological and pathological role of oxidized polyunsaturated fatty acids (PUFAs) has been extensively studied, whereas those of hydroxy monounsaturated fatty acids (MUFAs) are not well understood. This study demonstrated that 11-hydroxy-(9Z)-octadecenoic acid ((9Z)-11-HOE), which was isolated from adlay seeds (Coix lacryma-jobi L. var. ma-yuen STAF.), can activate peroxisome proliferator-activated receptor (PPAR)α, δ and γ in luciferase reporter assays more efficiently than (9Z)-octadecenoic acid (oleic acid), and to the same degree as linoleic acid. (9Z)-11-HOE increased the mRNA levels of UCP2 and CD36 in C2C12 myotubes and THP- 1 cells, respectively, and these effects were blocked by the PPARδ- and γ-specific antagonists GSK0660 and T0070907, respectively. Evaluation of the structure.activity relationship between hydroxy MUFAs and PPAR activation revealed that (9E)-11-HOE, the geometrical isomer of (9Z)-11-HOE, activated PPARs more potently than (9Z)-11-HOE, and that PPAR activation by hydroxyl MUFAs was not markedly influenced by the position of the hydroxy group or the double bond, although PPARδ seemed to possess ligand specificity different to that of PPARα or γ . Additionally, the finding that 11-hydroxy octadecanoic acid, the hydrogenated product of (9E)-11- HOE, was also capable of activating PPARs to a similar extent as (9E)-11-HOE indicates that the double bond in hydroxy MUFAs is not essential for PPAR activation. In conclusion, (9Z)-11-HOE derived from alday seeds and hydroxy MUFAs with a chain length of 16 or 18 acted as PPAR agonists. Hydroxylation of MUFAs may change these compounds from silent PPAR ligands to active PPAR agonists.
We investigated the protective effect of several heterocyclic pyrimidine compounds against ultraviolet B (UVB)-induced damage in human keratinocyte HaCaT cells, as well as the inhibitory effect on melanogenesis in B16 melanoma cells. One of the compounds examined, 2-piperadino-6-methyl-5-oxo-5,6-dihydro(7H)pyrrolo[3,4d]pyrimidine maleate (MS-818), showed low cytotoxicity even at 500 μM. At 50—500 μM, MS-818 dose-dependently suppressed the UVB (100 mJ/cm2)-induced elevation of tumor necrosis factor alpha (TNF-α), one of the trigger cytokines for cell death, in HaCaT cells. In addition, MS-818 (100 μM) markedly inhibited melanogenesis in B16 melanoma cells via downregulation of tyrosinase expression mediated by microphthalmia-associated transcription factor (MITF) and extracellular signal-regulated kinase (ERK). In conclusion, MS-818 protected epidermal cells from UVB-induced damage and also suppressed melanogenesis in melanoma cells. It appears to be a good candidate for a new UVB-protective and whitening agent for application in cosmetics.
Synthetic metalloporphyrins, in the presence of monooxygen donors, are known to mimetize various reactions of cytochrome P450 enzymes systems in the oxidation of drugs and natural products. The oxidation of piperine and piplartine by iodosylbenzene using iron(III) and manganese(III) porphyrins yielded mono- and dihydroxylated products, respectively. Piplartine showed to be a more reactive substrate towards the catalysts tested. The structures of the oxidation products were proposed based on electrospray ionization tandem mass spectrometry.
We evaluated the inhibitory effects of components from the root of Glycyrrhiza uralensis (G. uralensis) on aldose reductase (AR) and sorbitol formation in rat lenses with high levels of glucose as part of our ongoing search of natural sources for therapeutic and preventive agents for diabetic complications. In order to identify the bioactive components of G. uralensis, 5 prenylated flavonoids (semilicoisoflavone B, 7-O-methylluteone, dehydroglyasperin C, dehydroglyasperin D, and isoangustone A), three flavonoids (liquiritigenin, isoliquiritigenin, and licochalcone A), and two triterpenoids (glycyrrhizin and glycyrrhetinic acid) were isolated; their chemical structures were then elucidated on the basis of spectroscopic evidence and comparison with published data. The anti-diabetic complication activities of 10 G. uralensis-derived components were investigated via inhibitory assays using rat lens AR (rAR) and human recombinant AR (rhAR). From the 10 isolated compounds, semilicoisoflavone B showed the most potent inhibition, with the IC50 values of rAR and rhAR at 1.8 and 10.6μM, respectively. In the kinetic analyses using Lineweaver.Burk plots of 1/velocity and 1/concentration of substrate, semilicoisoflavone B showed noncompetitive inhibition against rhAR. The results clearly indicated that the presence of a γ,γ-dimethylchromene ring is partly responsible for the AR inhibitory activity of isoprenoid-type flavonoids. Further, semilicoisoflavone B inhibited sorbitol formation of rat lens incubated with a high concentration of glucose, indicating that this compound may be effective for preventing osmotic stress in hyperglycemia.
All-trans retinoic acid (atRA) is an active metabolite of vitamin A with antioxidant effects. There have been few reports on the effects of atRA on liver ischemia/reperfusion (I/R) injury. Here we have used a rat liver ischemia/ reperfusion model to analyze the protective effect of atRA. Rats were administered with different does (5—15 mg/kg/d) of atRA intraperitoneally (i.p.) for 14 d before I/R. Partial (70%) hepatic ischemia was induced by clamping the hepatic artery, portal vein, and bile duct to the left and median lobes of the liver using a vascular clamp for 60 min, followed by 24 h of reperfusion. The serum aminotransferase (ALT and AST) and hepatic pathology were used to evaluate I/R injury. The results demonstrate that atRA pretreatment attenuates liver I/R injury by inhibiting the release of malondialdehyde (MDA) and by enhancing the activity of superoxide dismutase (SOD). To gain insight into the mechanism of the SOD up-regulation by atRA, the activity of p38 mitogenactivated protein kinase (p38MAKP) and Akt was measured. The results showed that the phosphorylation of p38MAPK and Akt paralleled the expression of manganese superoxide dismutase (MnSOD). That these activities are related was demonstrated by the addition of a p38 inhibitor which markedly decreased MnSOD levels. Taken together, our data reveal that atRA can protect liver from I/R injury by increaseing MnSOD, which is associated with an increased activity of p38MAPK and Akt.
Effect of sebum and ointment rubbing was evaluated on the permeation of a model steroidal drug, triamcinolone acetonide (TA), from white petrolatum ointment through excised hairless rat skin. Two kinds of white petrolatum ointment containing TA at a concentration of 1.0% were made using a “pre-applied ointment,” which was made by applying and recovering white petrolatum on and from the rat skin, respectively, and the “original ointment.” Solubility and skin permeation of TA in and from both the ointments were measured and compared. TA solubility in the pre-applied ointment was about twice to that of the original ointment. Full-thickness skin and stripped skin permeabilities of TA from pre-applied ointment were about 2.3- and 1.5-times higher than those from the original ointment. In addition, the calculated partition coefficient of TA from pre-applied ointment to full-thickness skin was 2.7-times that of the original ointment. Next, the ointment-rubbing effect was determined. Skin permeation of TA from 1.0% TA original ointment with a 30 s-rubbing was 9-times higher than that after drug-free ointment-rubbing and application of 1.0% TA original ointment. The increase in solubility and skin permeation of TA due to pre-applied ointment is probably due to dissolution of TA by skin lipids extracted from the skin surface. Ointment rubbing increases the transient decrease in skin impedance and enhanced delivery of ointment base to the skin. These results are useful for development of ointment formulations and skin penetration mechanisms from an ointment base.
Intestinal ischemia-reperfusion (I/R) causes gut dysfunction characterized by decreased basement membrane integrity and decreased barrier function. Indeed, it has been reported that the absorption of several drugs is altered after intestinal I/R. Intestinal I/R also promotes multi-organ failure (MOF). The liver and kidney can be affected by MOF after intestinal I/R. However, little is known about the alteration of pharmacokinetics after intravenous administration in intestinal I/R injury. In the present study, we investigated pharmacokinetics of digoxin after oral administration and intravenous administration in intestinal I/R injury. Plasma digoxin concentration in I/R rats after oral administration was not significantly altered at any time compared with that in sham-operated rats. Plasma digoxin concentration in rats reperfused for 1 h after intravenous administration was significantly higher than that in sham-operated rats. Plasma digoxin concentrations in rats reperfused for 6 and 24 h were the same as those in sham-operated rats. The area under the concentration.time curve after intravenous administraion (AUCi.v.) and total clearance (CLtot) in rats reperfused for 1 h was 1.89- and 0.57-fold higher than that in sham-operated rats. However, elimination rate (ke) and half-life (t1/2) in rats reperfused for 1 h were not altered. Distribution volume (Vd) in rats reperfused for 1 h was decreased than that in sham-operated rats, but there was not statistical difference. These results suggest that intestinal I/R affected the Vd of digoxin, and plasma concentration of digoxin was increased. The present study suggests that understanding pharmacokinetics of drug after intravenous administration in intestinal I/R injury is important to provide valuable information for safe drug therapy for intestinal I/R patients.
The utility of using a multifunctional envelope-type nano device (MEND) for delivering a gene to the liver was examined. Lipotrust, a commercially available transfection reagent whose lipid composition is DC6- 14 :DOPE: cholesterol=4 : 3 : 3, was used as a reference. When Lipotrust was administrated intravenously, luciferase activity of the lung was 25 times higher than that of the liver. The luciferase activity of the lung was greatly reduced when a MEND was administered, even though the lipid composition of the lipid envelope was the same in both devices. Furthermore, the luciferase activity of the liver was 5 times higher than that for lipotrust, suggesting that the encapsulation of plasmid DNA (pDNA) in liposomes is more advantageous for delivering pDNA to the liver than complex formation. The isolation of parenchymal cells (PCs) and non-parenchymal cells (NPCs) showed that the MEND system is capable of expressing the luciferase protein more preferentially in NPCs than the lipoplex system. In addition, when the surface was modified with a pH-sensitive fusogenic peptide (GALA) used as a device for endosomal escape, overall liver luciferase activity was greatly enhanced. This suggests that endosomal escape is a limiting step for the MEND system. In the case of the GALA-modified MEND, the luciferase activity of PCs and NPCs was 18 times and 11 times higher than MEND system, while the transfection efficiency of NPCs was significantly higher compared to that of PCs. Collectively, these data show that a GALA-modified MEND prepared with DC6-14 :DOPE: cholesterol represents a promising device for NPCtargeting gene delivery in vivo.