Two-headed sphingolipids bear a certain similarity with sphingolipids in the structure and but differing from classical sphingolipids in α,ω-position of the basic groups. We analyzed the apoptotic effects of some two-headed sphingolipids including rhizochalin (Rhz) and its derivatives isolated from sponge Rhizochalina incrustata on human leukemia HL-60 cells. Direct addition of Rhz induced apoptosis of HL-60 cells. The aglycon of Rhz, which has no galactosyl residue, showed a stronger ability to induce apoptotic activity than Rhz. Rhz congeners with acetate derivatives only weakly induced apoptosis. The usual mitochondrial membrane permeability changes and the decrease of protein levels of procaspases-8, -9, and -3 correlated with the apoptotic activity of Rhzs. These results suggest that derivatives of two-headed sphingolipids potently induce apoptosis in mammalian cells when administered exogenously and this cell death was dependent on caspase activation pathways.
During the resolution phase of hepatic fibrosis, a crucial mechanism is the apoptosis of activated hepatic stellate cells (HSCs). It is necessary to find more anti-fibrosis drugs that would modulate HSCs to be more susceptible to apoptotic stimuli. Here we showed that A771726, the active metabolite of leflunomide, markedly enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in the human hepatic stellate cell line LX-2. A771726 could increase caspase activity in LX-2 cells in a dose-dependent manner. A771726 did not increase the expression of TRAIL receptors in LX-2 cells but could inhibit activation of the c-Jun NH2-terminal kinase (JNK) pathway through decreasing TRAIL-induced JNK and c-Jun phosphorylation. Moreover, A771726 could accelerate TRAIL-induced apoptosis via inhibiting nuclear factor-kappaB (NF-κB) activation in LX-2 cells. In conclusion, our results indicated leflunomide could enhance the sensitivity of LX-2 cells to TRAIL-induced apoptosis via inhibiting the survival pathways and provided a promising approach to anti-fibrotic therapy with leflunomide.
NDRG2, a new member of the N-Myc downstream-regulated gene (NDRG) family, is a focus for study at present. Up to now, its expression and function in carcinoma remain to be elucidated. In this study, using a colorectal cancer tissue array and a series of 213 colorectal cancer samples, the relationship between Ndrg2 and c-MYC expression and tumor differentiation level was investigated. Immunohistochemistry showed that Ndrg2 expression was reduced and that c-Myc was increased in colorectal carcinomas. In addition, Ndrg2 protein levels increased from poorly differentiated to well-differentiated carcinomas (p=0.005). Real-time polymerase chain reaction and Western blots demonstrated quantitatively that NDRG2 mRNA and protein levels were lower in colorectal carcinomas compared to the adjacent tissue and normal tissue from the same individual (p=3×10−8). Also, the NDRG2 expression level in adjacent carcinoma tissue was lower than that of normal tissue. However, the expression pattern of c-MYC was the inverse (p=5×10−8). Finally, we induced the differentiation of the colorectal carcinoma cell lines HT29, SW480 and SW620 and found that NDRG2 expression increased and that c-MYC expression declined with increasing differentiation. These novel data show a disparity in both the mRNA and protein expression levels of Ndrg2 and c-Myc between colorectal cancers and normal tissues. Taken together, NDRG2 may play a role during the differentiation of colorectal cancer cells, and the function of NDRG2 in the development of colorectal cancer should be further investigated.
The liquid crystal compounds have a common structure with the cell membrane, having both a hydrophilic and hydrophobic residue, thus suggesting an affinity to the cell membrane. However, little information regarding a biological effect by liquid crystal compounds has been reported. In order to view the biological potential of liquid crystal compounds, the present study evaluated the in vitro human hematopoietic promoting effects by 18 liquid crystal-related compounds. In particular, these compounds are evaluated regarding their potential for platelet production from mature megakaryocytes by the culturing of CD34+ cells derived from normal human peripheral blood. Often, in the case of severe thrombocytopenia there is no choice but to perform a transfusion of platelet concentrates. Three of the tested compounds promoted megakacyocyte generation in the culture stimulated with thrombopoietin alone. In addition, two compounds led to a significant increase in CD42a+ particles which seemed to be platelets. At the same time, interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), that were used as a positive control for in vitro megakaryocytopoiesis and thrombopoiesis, resulted in a dramatic increase in the total number of cells; however, their promoting activity on in vitro hematopoiesis was almost at a similar level with the compounds. These results suggest that some liquid crystal-related compounds have a promoting effect on human thrombopoiesis, and that these compounds act with a different mechanism from either IL-3 or GM-CSF since the compounds specifically stimulated thrombopoiesis. The liquid crystal compounds may therefore be useful to develop a new functional medicine or a medical application.
The renal proximal tubule (RPT) plays a crucial role in organic cation (OC) secretion and has a major impact on pharmacokinetics of OC drugs. Secretory transport is vectorial. Thus, it involves transporters located at both basolateral and apical membranes. Although sex hormones have been shown to regulate OC transport, there is little data on the effect of testosterone on OC secretion in a whole animal. Therefore, we determined the clearance of tetraethylammonium (TEA), a model OC substrate, in intact and castrated male mice. Castration significantly decreased renal TEA secretion by 30%, and testosterone supplementation returned TEA secretion to control levels in castrated mice. The mechanism of this effect was further examined in isolated mouse renal proximal tubules (mRPT). TEA uptake in isolated mRPT from castrated mice was reduced by 36%. This effect was reversed in tubules from castrated mice supplemented with testosterone. Kinetic analysis of [3H]-TEA uptake in isolated mRPT showed a decreased Vmax with no change in Km, implying that the decrease in transport rate was caused by lowering in the number of transporters in castrated mice rather than a change in transporter affinity. Quantitative real time polymerase chain reaction (real time PCR) revealed that organic cation transporter (OCT)2 is the major TEA transporter in male mice. Moreover, OCT2 mRNA level was significantly reduced after castration. Castrated mice also showed a modest increase in organic cation/carnitine transporter 1 (OCTN1) mRNA level, indicating that testosterone may also regulate apical OCTN1 expression. These data suggest that testosterone regulates transepithelial transport of OC through modulation of OCT2 expression in male mice.
We investigated the effect of rapamycin, a specific inhibitor of the mammalian serine/threonine kinase, mammalian target of rapamycin (mTOR), on the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Pretreatment of cells with rapamycin significantly inhibited LPS-induced nitrite production and the expression of iNOS protein in a dose-dependent manner. However, LPS-induced mRNA expression of iNOS and its concomitant activation of nuclear factor (NF)-κB remained unchanged by rapamycin. Intriguingly, LPS-induced nitrite production and iNOS protein expression were partially blocked at nanomolar concentrations of rapamycin, whereas phosphorylation of both p70 S6 kinase and 4E-BP1 was completely abolished. The suppression of LPS-induced iNOS expression by rapamycin was reversed by the protease inhibitor lactacystin. Furthermore, rapamycin treatment stimulated 20S proteasome activity, which was slightly elevated by LPS. Taken together, our findings strongly suggest that rapamycin down-regulates LPS-induced iNOS protein expression via proteasomal activation, as well as through inhibition of the mTOR signaling pathway.
AMP-activated protein kinase (AMPK) is a central player in glucose and lipid metabolism while its role in adipocyte differentiation remains obscure. A-769662, a novel activator of AMPK, has been implicated to reduce body weight gain and decrease liver and plasma triglyceride levels via increasing fatty acid oxidation and reducing fatty acid synthesis in vivo. In this study, we examined the effects of A-769662 on adipocyte differentiation using 3T3-L1 cells. We found that A-769662 significantly inhibited 3T3-L1 differentiation via down-regulation of adipogenesis-related transcription factors and adipogenic markers. The inhibitory effect mainly occurred at the early phase of differentiation through inhibition of mitotic clonal expansion (MCE), which was essential for adipogenesis. A-769662 also decreased cell viability of differentiating and mature 3T3-L1 adipocytes. Moreover, treatment of differentiating 3T3-L1 cells with A-769662 resulted in AMPK over-activation, which led to an increased phosphorylation and inactivation of acetyl-CoA carboxylase (ACC), an important enzyme for the synthesis and usage of fatty acids. Taken together, these results suggest that A-769662 inhibits 3T3-L1 adipogenic differentiation in several ways and therefore may be a promising compound for the treatment of obesity.
Pax6 genes are highly conserved and important for eye development. Vertebrates predominantly produce two alternatively spliced Pax6 isoforms, Pax6 and Pax6-5a. Pax6-5a differs from Pax6 by the presence of 14 additional amino acids encoded by exon 5a. These additional amino acids occur in the Pax6 paired domain and thus influence its DNA-binding properties. However, little is known about Pax6-5a's physiological functions. Here we establish murine embryonic stem (ES) cell lines in which expression of either the human Pax6 or Pax6-5a isoform is negatively controlled by tetracycline. We report that, in contrast to Pax6 expression, Pax6-5a expression strongly induces ES cells to differentiate into neurons. Moreover, using DNA microarray analysis, we have identified the transcription factor basic helix loop–helix domain containing, class b2 (bHLHb2) in Pax6-5a-expressing ES cells. Our Pax6 isoform-expressing ES cell lines may serve as useful models for identifying Pax6-regulated genes that are important for neurogenesis and/or eye development.
Interstitial extracellular matrix tenascin-X (iTNX) with about 450 kDa is prominently present in various tissues. Previously, we identified the serum form of TNX (sTNX) with 200 kDa in the mouse. In the present study, in order to investigate distinctive features and functions of sTNX, a plasmid encoding the recombinant mouse sTNX was constructed. As a control, we also constructed a plasmid encoding mouse 450-kDa iTNX and a plasmid encoding 250-kDa iTNX, which lacks the region of 200-kDa sTNX from 450-kDa iTNX. In cells stably expressing each recombinant TNX, a more than 7-fold larger amount of 200-kDa sTNX was released into conditioned medium than the amounts of 250-kDa iTNX and 450-kDa iTNX released into the medium. We previously reported that a splice isoform of iTNX (340-kDa iTNX) binds to vascular endothelial growth factor B (VEGF-B) as well as to VEGF-A. Therefore, the ability of VEGF-A and VEGF-B to bind to 200-kDa sTNX was examined by a co-immunoprecipitation assay in comparison with the binding abilities to 250-kDa iTNX and 450-kDa iTNX. It was found that sTNX strongly bound to VEGF-A and VEGF-B, compared with the binding abilities of other iTNX proteins. Based on the results of assays of incorporation of 5-ethynyl-2′-deoxyuridine (EdU), we found that purified recombinant 200-kDa sTNX both alone and in combination with VEGF-A or basic fibroblast growth factor (bFGF) can weakly promote DNA synthesis in proliferating vascular endothelial cells (UV♀2 cells). These results suggest that sTNX possesses weak activity for proliferation of endothelial cells.
This study was conducted to evaluate the protective mechanisms of Nelumbinis semen (NS) on lipopolysaccharide (LPS)-induced activation of BV-2 microglial cells. The anti-inflammatory effects of NS were determined by analyzing nitric oxide production and proinflammatory cytokines using enzyme-linked immunosorbent assay. The mechanism was evaluated in BV-2 cells with or without NS treated with LPS for various lengths of time using oligonucleotide microarray and real time reverse transcription-polymerase chain reaction. The oligonucleotide microarray analysis revealed that mitogen activated protein kinase (MAPK) signaling pathway-related genes such as Fgfr3, Fgf12, Rasal2, Nfkb2, Map2k5, Mapk1, Map3k7, and NFatc2 were down-regulated in LPS activated BV-2 cells by pretreatment with NS. In addition, significant decreases in Nos1ap gene expression were observed with NS pretreatment. Cluster linked pathway analysis using the Kyoto Encyclopedia of Genes and Genomes database revealed that the effects of NS were closely associated with the regulation of mitochondria functions. These results suggested that NS can affect the MAPK signaling pathway and mitochondrial functions in BV-2 cells activated with LPS.
Thymosin β4, a major G-actin-sequestering protein, is known to be involved in tumor metastasis. In the present study, we found that thymosin β4 expression promotes the formation of actin-based pseudopodia-like extensions, associated with cell migration, in human prostate cancer LNCaP cells. Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin and Cdc42/Rac1/RhoA inhibitor Clostridium difficile toxin B significantly reduced pseudopodia formation in thymosin β4-overexpressing LNCaP cells, suggesting that the pseudopodia formation by thymosin β4 is probably involved in PI3K and Rho family pathway. We recently reported that thymosin β4 expression is upregulated by androgen deprivation in prostate cancer cells. The increase in thymosin β4 may be one of the causes of prostate cancer progression after androgen ablation therapy.
Camptothecin (CPT) reversibly binds and stabilizes cleavable complexes formed between DNA and topoisomerase I (Top1), thereby activating many downstream signaling pathways. Although several pathways induced by CPT have been elucidated, there are additional proteins that represent targets of CPT pharmacological mechanisms and remain uncharacterized. Using two-dimensional electrophoresis analysis and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-MS/MS identification, we investigated the hepatocellular carcinoma cell line SMMC-7721 for changes of protein expression following CPT treatment. Proteomic results showed that CPT treatment caused decreased expression of galectin-1 in SMMC-7721 cells. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed mRNA expression changes in galectin-1. Protein expression levels of DNA methyltransferases (DNMTs) were downregulated in response to CPT. The DNMT inhibitor 5-aza-2′-deoxycytidine (DAC) sensitized SMMC-7721 cells to the cytotoxic effect of CPT. Our results indicate that inhibition of DNMT activity by CPT may play a role in CPT-induced cell proliferation inhibition and apoptosis.
Because of the lack of efficacious treatments for advanced melanoma, new approaches are necessary. Chalcones are contained in fruits and vegetables, and have been suggested to be cancer-preventive. In this study, effects of synthetic chalcone derivatives were investigated especially on the proliferation of human melanoma cells and peripheral blood mononuclear cells (PBMCs). Four out of the 12 synthetic chalcones: 4-trifluoromethyl-4′-methoxychalcone (CH-1), 4-trifluoromethyl-2′-methoxychalcone (CH-3), 3-trifluoromethyl-2′,4′-dimethoxychalcone (CH-4) and 3-trifluoromethyl-4′-methoxychalcone (CH-7) exhibited significant antiproliferative efficacies against the cultured cells of the human melanoma cell line A375. CH-1, CH-3, CH-4, and CH-7 induced cell cycle arrest at the S-G2/M phase within 24 h after the treatment. CH-3, CH-4, and CH-7 significantly activated caspase-3 at 12 h, subsequently induced apoptosis at 72 h. All chalcones inhibited concanavalin A-induced proliferation of PBMCs dose-dependently. Our results suggest that some methoxy- and/or fluoro-chalcones have antitumor efficacy by inducing apoptosis and the cell-cycle arrest.
In the current study we investigated the antibacterial activity of fragrance ingredients against Legionella pneumophila, a causative agent of severe pneumonia. Among the 41 different fragrance ingredients tested, we found that the natural fragrance ingredients oakmoss (OM) and birch tar oil (BT), which contain many components, exhibit potent antibacterial activity. The minimum inhibitory concentration (MIC, % (v/v)) of OM and BT were 0.0020 and 0.0024, respectively and were lower than that of cinnamic aldehyde (0.0078), which has been previously shown to possess high antimicrobial activity. In a time–kill assay of OM and BT at MIC and two times MIC, the colony forming units (CFU) of the microbe were reduced to between 10−3 to 10−4 of the original CFU after 1 h co-incubation. After this time, the CFU gradually decreased in number, but remained above detection levels even after a 48-h co-incubation, except for BT at two times MIC. In contrast, at a concentration of 0.1% OM and BT (approximately 50 times MIC), CFU were not detected after co-incubation for 1 h. Another 18 fragrance ingredients including ketone, aldehyde, lactone, acid, phenol derivative, aliphatic alcohol and quinoline also exhibited a lesser degree of antibacterial activity against L. pneumophila at a MIC of less than 0.10.
Many Chinese therapeutic herbs that are traditionally used in combination demonstrate significantly better pharmacological effects when used in the combination than when used alone. However, the pharmacological mechanism for this synergism is still not well understood. In the present study, the antioxidant activities of six herbs ((Paeonia lactiflora (PL), Atractylodes macrocephala (AMA), Angelica sinensis (AS), Astragalus membranaceus (AME), Glycyrrhiza uralensis (GU) and Rheum officinale (RO)), which were historically combined into eight traditional Chinese herb pairs (TCHPs) (AME-AS, AME-AMA, AME-RO, AME-GU, AME-PL, PL-AS, PL-AMA and PL-GU), were investigated in vitro by assessing the 1,1-diphenyl-2-picryl hydrazine (DPPH)-radical scavenging abilities of the herbs. The results of this study showed that all eight TCHPs had a significantly larger scavenging capacity than would be expected from the theoretical sum of those of the respective constituent herbs (p<0.05). Furthermore, the AME-GU, AME-PL and AME-AMA pairs not only showed a significant synergistic effect in the DPPH scavenging assay, but they also demonstrated similar results in hydroxyl radical and superoxide radical anion scavenging assays. Interestingly, the AME-AMA combination had a significantly higher superoxide anion (0.2 g/ml) and hydroxyl radical scavenging ability than the AME or AMA. The changes in the total phenolic and flavonoid contents were also investigated. Our study showed a significant correlation between the rate of enhancement in antioxidant capacity and the rate of increase in flavonoid content. Thus, the flavonoids are likely responsible for the synergistic effects present in TCHPs.
In the present study, we extracted Corydalis heterocarpa with various solvents in order to find the bioactive constituents that demonstrated anti-inflammatory effects. We isolated the active compound, Columbianetin. Anti-inflammatory effect of Columbianetin has been reported but the precise effects of Columbianetin in experimental models have remained unknown. In the present study, we investigate the effect of Columbianetin on the production of histamine, interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α and expression of cyclooxygenase-2 (COX-2) by using the human mast cell line (HMC-1). Various concentrations of Columbianetin were treated before the activation of HMC-1 cells with phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore, A23187. PMA plus A23187 significantly increased IL-1β, IL-6, IL-8, and TNF-α production compared with media control (p<0.05). We also show that the increased cytokines IL-1β, IL-6, IL-8, and TNF-α level was significantly inhibited by Columbianetin in a dose-dependent manner (p<0.05). Maximal inhibition rates of IL-1β, IL-6, IL-8, and TNF-α production by Columbianetin were about 102.6%, 101.1%, 95.8%, and 103.9%, respectively. Columbianetin inhibited expression of COX-2. In addition, the effect of Columbianetin was investigated on the histamine release from HMC-1 stimulated by substance P, which promotes histamine release. Columbianetin also inhibited the histamine release by substance P. In conclusion, these results indicate that Columbianetin may be helpful in regulating mast cell-mediated allergic inflammatory responses.
In the present study, we designed and synthesized a novel 1,5-diarylpyrazole derivative, 2-amino-N-(2-methyl-5-(1-(4-sulfamoylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)phenyl) acetamide hydrochloride (CC06), which was intended to act as a prodrug and would exert potent anti-inflammatory activity after being converted to its parent compound in vivo. In vitro cell-based biological assay, CC06 showed decreased inhibitory effects on cyclooxygenase (COX)-1 and COX-2 compared with its parent compounds, but it exhibited potent anti-inflammatory activity in vivo. The anti-inflammatory effect was evaluated in a carrageenan-induced rat paw edema model and CC06 (15, 30, 60 mg/kg, intragastrically) reduced rat paw edema in a dose-dependent manner. CC06 is also a selective inhibitor of COX-2 since it can reduce prostaglandin E2 (PGE2) production in the inflamed pouch dose-dependently without affecting PGE2 production in stomach in rat air pouch model. Furthermore, preliminary pharmacokinetics experiments were conducted using high performance liquid chromatography/mass spectrometry (HPLC/MS) to detect whether CC06 can convert to its parent compound or not. Our results supported the hypothesis that CC06 was actually converted to its parent compound. These suggested that CC06 served as an anti-inflammatory prodrug and actually converted to its parent compound to exert its anti-inflammatory effect. This finding will be of great benefit in carrying out structural modifications of prodrug-like selective COX-2 inhibitors.
Metallothionein (MT) is a low-molecular-weight cysteine-rich protein which has a high affinity for metals and plays important roles in the protection against metal toxicity. As little information is available concerning the mechanism of MT induction by lead (Pb) compounds, we investigated the induction of MT by Pb acetate both at mRNA and protein levels in mice. Administration of Pb increased the levels of MT-I mRNA in the liver and kidney in six strains of mice. However, MT protein was detected only in the liver, and little or no increases in MT protein were detected in the kidney of any strains of mice. Speciation of metals in the liver cytosol showed that the major metal bound to MT was zinc but not Pb. The increases in plasma concentrations of interleukin-6 suggest that the production of interleukin-6 by Pb administration is involved in the induction of MT in the liver. Treatment of renal cells with Pb in vitro also resulted in the increase in MT mRNA but little increase in MT protein. These data suggest that Pb exerts a dual effect on MT expression; enhancement of MT gene transcription both in the liver and kidney and suppression of MT mRNA translation in the kidney.
Genipin is an iridoid compound and an aglucon of geniposide isolated from Gardenia fructus. We have previously reported that genipin induces neurite outgrowth in PC12h and Neuro2a cells and protects against cytotoxicity induced by several conditions such as β-amyloid peptide, serum deprivation, and oxidative stress in rat primary hippocampal neurons and Neuro2a cells. In this paper, we examined the protective effect of genipin on A23187 (a calcium ionophore)-induced cytotoxicity in Neuro2a cells. A23187 induced cytotoxicity in concentration- and time-dependent manners as assayed by measurements of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetazolium bromide (MTT) reduction activity and lactate dehydrogenase (LDH) release. The cytotoxicity was significantly suppressed by genipin in a concentration-dependent manner. A23187 also significantly activated caspase3/7, which is known to be the critical mediator of apoptosis, after 1 h, and the cytotoxicity was clearly blocked by an inhibitor of caspase 3/7. Furthermore, A23187 induced the expression of immunoglobulin-binding protein/glucose-regulated protein of 78 kDa (BiP/GRP78) protein, which is an endoplasmic reticulum (ER) stress marker protein, and the expression was suppressed by genipin. These results suggest that genipin protects Neuro2a cells from A23187-induced cytotoxicity mediated by caspase 3/7 and ER stress. Therefore, genipin may be effective in preventing neurodegeneration observed in Alzheimer's disease and Parkinson's disease involving ER stress.
Heme oxygenase (HO)-1 is a well-known cytoprotectant against oxidative stress and exhibits an antiproliferative effect in vascular smooth muscle cells (VSMCs). The purpose of the present study was to test whether isoproterenol, one of the synthetic catecholamines having β-adrenergic activity, affected angiotensin II (Ang II)-induced cell proliferation and reactive oxygen species (ROS) production. Also, the presumptive underlying signaling pathways in VSMCs were studied. Aortic VSMCs from 11-week-old male Sprague-Dawley rats were used. Isoproterenol dose-dependently increased HO-1 expression through β2-adrenoceptor (AR) and protein kinase A (PKA) pathway, and isoproterenol concentration-dependently increased β2-AR mRNA expression. Isoproterenol attenuated Ang II-induced cell proliferation, as evidenced by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. This effect of isoproterenol was inhibited by pretreatment of the cells with β2-AR antagonist butoxamine, PKA inhibitor H-89 and HO inhibitor Tin Protoporphyrin IX (SnPP IX), respectively. Isoproterenol inhibited phosphorylation level of Ang II-induced extracellular signal-regulated kinase (ERK1/2). Isoproterenol significantly inhibited Ang II-induced ROS production through the ERK1/2 pathway. These findings suggest that isoproterenol, via induction of HO-1, inhibits Ang II-stimulated proliferation and ROS production in cultured VSMCs.
Gallotannins are plant secondary metabolites and are widely included to polyphenolic compounds. Gallotannins are water-soluble polyphenols with wide-ranging biological activities. Nitric oxide (NO) is well known as a mediator of inflammation. Macrophages express inducible nitric oxide synthase (iNOS) and produce NO after lipopoly saccharide (LPS) stimulation. In the present study, we examined the inhibitory effects of seven gallotannins isolated from Euphorbia species (Euphorbiaceae) on the LPS-induced NO production and underlying mechanisms of action. Among the seven gallotannins, 1,2,3,4,6-penta-O-galloyl-β-D-glucose (gallotannin 15) and 1,2,6-tri-O-galloyl-β-D-allose (gallotannin 23) significantly reduced LPS-induced NO production in macrophages. Gallotannin 15 and 23 (0.1—10 μg/ml) dose-dependently decreased gene expression and production of iNOS. In addition, gallotannin 15 and 23 (0.1—10 μg/ml) dose-dependently inhibited LPS-induced activation of nuclear factor (NF)-κB as indicated by inhibition of degradation of I-κBα, nuclear translocation of NF-κB, and NF-κB-dependent gene reporter assay. Our results suggest that gallotannins possess an inhibitory effect on the LPS-induced inflammatory reaction.
Bronchial asthma is characterized by chronic airway inflammation. Eosinophils are involved in airway inflammation and play crucial roles in asthma. There is accumulating evidence to suggest contributions of cysteinyl leukotrienes (cysLTs) and thromboxane (TX) A2 to the recruitment of eosinophils into lung in asthmatics. KP-496 is a novel dual antagonist for CysLT receptor type 1 and TXA2 receptors. The aim of this study was to evaluate the anti-inflammatory effects of KP-496 on Sephadex-induced airway inflammation. Sephadex suspension was intratracheally injected into rats. Amounts of regulated on activation, normal T cell expressed and secreted (RANTES) and eotaxin, and numbers of infiltrating cells in bronchoalveolar lavage fluid were measured 24 and 48 h after Sephadex injection, respectively. KP-496 (30, 100 μg/head) was intratracheally administered to rats 1 h before and 7 h after Sephadex injection. KP-496 and prednisolone (10 mg/kg, per os) exhibited significant inhibitory effects on infiltration of total cells and eosinophils into lung. Production of RANTES was significantly inhibited by KP-496 and prednisolone. Production of eotaxin was significantly inhibited by prednisolone. KP-496 also inhibited the production of eotaxin, though this effect was not significant. These results demonstrate that KP-496 exhibited the anti-inflammatory effects by inhibiting infiltration of inflammatory cells and productions of RANTES and eotaxin.
Ellagic acid is known to have anti-oxidant, anti-carcinogenic and anti-mutagenic effects. However, roles of ellagic acid in the immediate-type allergic reactions have not yet been investigated. In the present study, we have demonstrated that ellagic acid attenuates immunoglobulin (Ig)E-mediated allergic response in mast cells and in vivo. Oral administration of ellagic acid inhibited anti-dinitrophenyl (DNP) IgE-mediated passive cutaneous anaphylaxis. Ellagic acid dose-dependently reduced histamine release and the expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6 in rat peritoneal mast cells (RPMC) activated by anti-DNP IgE. Moreover, ellagic acid suppressed an increase in intracellular calcium ion concentration ([Ca2+]i) in RPMC. Furthermore, ellagic acid decreased the activation of nuclear factor-kappa B (NF-κB). Decreased NF-κB activity as well as reduced [Ca2+]i might be involved in the inhibitory effect of ellagic acid on the secretory response. Our findings provide possibility that ellagic acid may serve as an effective therapeutic agent for allergic diseases.
Fraxinellone is formed by the natural degradation of limonoids isolated from the root bark of Dictamnus dasycarpus. Fraxinellone has been reported to possess neuroprotective and vasorelaxing activities, but the effects and the mechanism of fraxinellone in inflammation have not been fully characterized. In the present study, the anti-inflammatory effect of fraxinellone was evaluated in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Fraxinellone was found to inhibit LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, and to reduce the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the mRNA and protein levels in a dose-dependent manner. Furthermore, fraxinellone significantly attenuated LPS-induced DNA binding activity and the transcription activity of nuclear factor-kappa B (NF-κB). Consistent with these findings, pretreatment with fraxinellone significantly suppressed the LPS-stimulated phosphorylation of inhibitory kappa B-α (IκB-α) and the subsequent translocation of p65 to the nucleus. Fraxinellone also suppressed the IκB kinase (IKK) activity and the phosphorylation of extracellular-signal-related kinase (ERK1/2), whereas the phosphorylations of Jun N-terminal kinase (JNK1/2) and p38 were unaffected. These results suggest that the anti-inflammatory properties of fraxinellone are related to the down-regulations of iNOS and COX-2 due to NF-κB inhibition through the negative regulations of IKK and ERK1/2 phosphorylations in RAW 264.7 cells.
A cDNA clone, designated Sd-racrop (969 bp), was isolated from seedlings of Scoparia dulcis. This gene contains an open reading frame encoding the protein of 197 amino acid residues with high homology to Rac/Rop small guanosine 5′-triphosphate-binding proteins from various plant sources. In Southern hybridization analysis, the restriction digests prepared from genomic DNA of S. dulcis showed a main signal together with a few weakly hybridized bands. The transcriptional level of Sd-racrop showed a transient decrease by exposure of the leaf tissues of S. dulcis to the ethylene-generating reagent 2-chloroethylphosphonic acid. However, an appreciable increase in gene expression was reproducibly observed upon treatment of the plant with methyl jasmonate. These results suggest that the Sd-racrop product plays roles in ethylene- and methyl jasmonate-induced responses of S. dulcis accompanying the change in the transcriptional level, however, the cellular events mediated by this protein toward these external stimuli would be regulated by various mechanisms.
In the present study, ginsenoside Rg1 (Rg1), a naturally occurring drug which is hardly absorbed in gastrointestinal (GI) tract due to its high hydrophilicity and low membrane permeability, was incorporated in different compositions of water-in-oil microemulsions (MEs). And parallel artificial membrane permeability assay (PAMPA) that have been mainly utilized for the evaluation of in vitro permeability of early drug candidates was introduced in present study, as well as rat in vivo pharmacokinetics and in vitro permeability measurements, to investigate the effect of w/o ME on Rg1 absorption. Correlation between various models as mentioned above was further performed to estimate the feasibility of PAMPA in the application of pharmaceutical preparation studies. After being administrated intraduodenally to rats, most of MEs can enhance the intestinal absorption of Rg1 to various extents with relative bioavailability (Fre) ranging from 268 to 1270% using drug solution as control. This enhanced absorption of Rg1 may be related to its increased membrane permeability induced by ME as exhibited in the PAMPA and rat in vitro permeability measurements. Meanwhile, rat in vivo pharmacokinetics–PAMPA correlation (r2=0.6082) is significant (p<0.05) for ME, representing a potential prospect for the application of PAMPA in the study of pharmaceutical preparation in some conditions.
Tong-Xie-Yao-Fang (TXYF) is a prescription in traditional chinese medicine (TCM), used for relieving abdominal pain associated with irritable bowel syndrome. The aim of the present study was to investigate the effects and mechanism of TXYF on experimental visceral hypersensitivity (VH) models. TXYF affected the abdominal withdrawal reflex produced by colonic distention in maternal separation-induced visceral hypersensitivity rats, in a dosage-dependent manner. TXYF significantly decreased serotonin (5-HT) levels in serum and corticotrophin releasing factor (CRF) concentrations in the brain. Moreover, it was found that VH alleviation by TXYF was dependent on the substance P (SP) expression in the colon mucosa. These results suggest that TXYF attenuates behavioral hyperalgesia by regulating substance associated with the brain–gut axis, including decreasing the expression of 5-HT and SP in the periphery and that of CRF in the center.
The effect on the bioavailability of the antimicrobial agents (ciprofloxacin and tetracycline), which are well known to form chelates with cationic metals such as calcium, was evaluated in 20 healthy male volunteers according to an open, random crossover fashion using a Kampo preparation, byakkokaninjinto (TJ-34) which contains various cationic metals including calcium. Each subject received a single oral dose of tetracycline (250 mg) alone or ciprofloxacin (200 mg) alone along with a single coadministration of one pack (3 g) of the Kampo preparation, at one-week intervals. Concentrations of the drugs in plasma and urine were analyzed by HPLC. Concomitant administration of the Kampo preparation significantly decreased the peak plasma concentration (Cmax) and area under the plasma concentration–time curves (AUC), but not time to reach Cmax (Tmax), of ciprofloxacin and tetracycline. However, the decrease in bioavailability of ciprofloxacin was slight (15%) compared with that of tetracycline (30%). The Kampo preparation significantly decreased the urinary recovery of tetracycline, but not ciprofloxacin, and it had no effect on the renal clearance of either ciprofloxacin or tetracycline. These results indicate that the Kampo preparation tested in this study reduces the extent of bioavailability of ciprofloxacin and tetracycline, but not renal excretion, by decreasing the gastrointestinal absorption due to the formation of insoluble chelates with calcium. We recommend that the dose timing of the Kampo preparation should be carefully controlled to avoid therapeutic failure especially for patients receiving the treatment with tetracycline.
A previous study showed that macelignan extracted from Myristica fragrans has anti-inflammatory properties using hippocampal neuronal and primary microglial cells. Subsequently, a study using animals with chronic lipopolysaccharide (LPS) infusion into the brain showed that oral treatments of macelignan reduced the hippocampal microglial activation and hippocampal-dependent spatial memory impairments induced by LPS. However, the molecular mechanisms responsible for the anti-inflammatory activity of macelignan have not been elucidated in the microglia. Therefore, the present study was conducted to determine if mitogen-activated protein kinase (MAPK) signaling and nuclear factor-kappa B (NF-κB) activities are related to the anti-inflammatory effects of macelignan on LPS-stimulated BV-2 microglial cells. The results show that macelignan suppresses both the phosphorylations of MAPKs and the degradation of inhibitory-kappa B (IκBα) and increases of nuclear NF-κB in LPS-stimulated BV-2 microglial cells. These results suggest that macelignan has an anti-inflammatory effect on the affected brain through regulation of the inflammation through the MAPK signal pathway.
To discover an active skin depigmenting agent, we isolated a novel inhibitor of melanin biosynthesis from the methanol extract of Erigeron breviscapus using a bioactivity-guided fractionation and identified it as (2Z,8Z)-matricaria acid methyl ester by means of spectroscopic analysis. The compound showed strong whitening activity in melan-a cell. Compared with arbutin (IC50=4.0 mM) as a positive control, the depigmentation IC50 value for (2Z,8Z)-matricaria acid methyl ester was 25.4 μM in B16F10 melanoma cell. Moreover, its inhibitory effect on tyrosinase, the key enzyme of melanogenesis, was examined by in vivo and in vitro tyrosinase assay and Western blot. The results indicate that (2Z,8Z)-matricaria acid methyl ester isolated from Erigeron breviscapus is a promising compound that could be useful for treating hyper-pigmentation as skin-whitening agents.
Ulifloxacin is a new quinolone antibiotic and it is effective against pneumonia. We previously showed that it is highly distributed into the epithelial lining fluid (ELF) in rats, which might be resulting from certain active transport. The transport system has not been, however, clarified yet. In this study, we attempted to characterize the distribution mechanism of ulifloxacin into the rat ELF. We also aimed to elucidate the feature of ulifloxacin uptake in rat lung and human lung adenocarcinoma cells (Calu-3). In infusion studies, ulifloxacin concentrations in the ELF and lung were higher than that in the plasma, and decreased by co-administration of sparfloxacin or azithromycin to the level of plasma concentration. Integration plot analysis showed that active uptake of ulifloxacin from the plasma to lung was also inhibited by sparfloxacin and azithromycin. In in vitro studies, time and temperature-dependent uptake into Calu-3 was observed, and this uptake was inhibited by sparfloxacin and azithromycin as observed in the rat lung. Additionally sparfloxacin inhibited the active uptake of ulifloxacin into Calu-3 more strongly than levofloxacin as observed in the rat lung. These results suggest that active uptake of ulifloxacin from the plasma to lung controls the distribution of ulifloxacin from the plasma to ELF, and that the uptake of ulifloxacin into Calu-3 has partly similar characteristics to its uptake into the rat lung. We believe our study will contribute to much better understanding of antibiotic efficacy against pathogens which cause pneumonia.
Fast-disintegrating tablets of furosemide (FS) were prepared by the novel direct compression method. FS, microcrystalline cellulose (MC), croscarmellose sodium (CC), xylitol (XL) and sucrose stearic acid esters (SSEs) with an hydrophilic–lipophilic balance (HLB) of 16, 15 and 11, named S1670, S1570 and S1170, were used. An FS/SSE/MC mixed powder was obtained by solvent evaporation of a suspension of MC in ethanol solution containing FS and SSE, and the resultant mixed powder was mixed with CC and XL, and directly compressed. The tablets with hardness of more than 40 N and disintegration time of less than 20 s were obtained at the addition of SSE at 0—0.5% (w/w). A tablet with S1670 at 0.1% (w/w), named TA2, dissolved faster than a commercial FS tablet, Lasix. TA2 tended to show higher plasma concentration than Lasix after intragastric administration to rats. It was demonstrated that the present direct compression using homogeneous FS/S1670/MC powder mixture could give an excellent fast-disintegrating tablet of FS.
The purpose of this study was to determine the high-density lipoprotein (HDL)-associated α-tocopherol (α-tocopherol-HDL) transport and clarify the contribution of scavenger receptor class B, type I (SR-BI) to the uptake in the human retinal pigment epithelial cell line (ARPE-19 cells). [3H]α-Tocopherol-HDL uptake into ARPE-19 cells seeded onto a transwell from both the apical (apical-to-cell) and basal compartment (basal-to-cell) exhibited a time-dependent increase for 90 min and there was a reduction at 4 °C. These results indicate the involvement of carrier-mediated process in α-tocopherol-HDL uptake in ARPE-19 cells. Immunoblot analysis revealed that SR-BI protein was expressed in ARPE-19 cells. However, the uptake of [3H]α-tocopherol from the apical or basal compartment was hardly inhibited by block lipid transport-1 (BLT-1), a specific inhibitor of the SR-BI-mediated lipid transfer. In conclusion, ARPE-19 cells have a carrier-mediated transport mechanism of [3H]α-tocopherol-HDL regardless of any SR-BI-mediated process.
Correction: In the manuscript described above, we have described the effect of plagiochin E alone and in combination with fluconazole on the ergosterol biosynthesis of Candida albicans. Now we found a fatal error occurred during the sterol content calculation according to Fig. 3A. Based on this wrong datum, the calculation of relative sterol composition of C. albicans CA10 has provided wrong results in Table 2. According to the correct data, for plagiochin E on the resistant strain CA10, ergosterol percentage was reduced, but, lanosterol and 14α-methylfecosterol were accumulated. So when FLC-resistant strain was treated by PLE, the accumulation of 14α-methylated sterols (lanosterol and 14α-methylfecosterol) and the down regulation of ERG11 may indicate the other mechanism for resistance reverse. We therefore deleted this publication of Biol. Pharm. Bull. Ling Mei SUN School of Pharmaceutical Sciences, Shandong University, China February 3, 2009
Authors Comments: We deeply regret that the experimental date appeared in the above-mentioned article were completely forged from those previously published in the Journal of Nanobiotechnology 5(3) doi:10.1186/1477-3155-5-3 (2007). Therefore, we wish that this article written by Dong-Myung KIM, Gee-Dong LEE, Seung-Hyun AUM, and Ho-Jun KIM should be totally deleted from Biol. Pharm. Bull. Ho-Jun KIM Department of Oriental Rehabilitation Medicine, Dongguk University, Korea February 16, 2009
Fig. 1C ↑ 2–3 10, 30, 100 and 300μM → 30 and 300μM left ↑ 3 exposure to 100-μM SC. → exposure to 300-μM SC. left ↑ 2 were -17.7±1.5 mV in the control and -19.8±1.6mV → were -17.2±0.6 mV in the control and -19.3±0.8mV right 1 6.8±0.2 and 6.5±0.2, respectively → 7.9±0.6 and 7.2±0.8, respectively right 2 100-μM SC → 300-μM SC right ↑ 7 -70.9±3.0mV → -66.8±3.3mV right ↑ 6 and -81.8±2.6mV in 100-μM SC (p<0.05, n=12) → and -90.1±3.5mV in 300-μMSC (p<0.001, n=12) right ↑ 5 -3.1±5.6 → -3.4±5.9 right ↑ 4 -13.8±5.5 in SC → -14.8±6.2 in SC Fig. 2 4 at an SC concentration of 100μM → at an SC concentration of 300μM ↑ 3 100-μM → 300-μM ↑ 1 *p<0.05, **p<0.01 vs. control. → *p<0.05, **p<0.01 ***p<0.001 vs. control. Fig. 3 ↑ 6 100-μM SC → 300-μM SC ↑5 (p>0.05) → (p<0.05) ↑ 3 100-μM SC → 300-μM SC left 2 100-μM SC → 300-μM SC left ↑ 6 100-μM SC → 300-μM SC right 9 100-μM SC → 300-μM SC right 13 100-μM SC → 300-μM SC