(AC), a highly valued polypore mushroom native only to Taiwan, has been traditionally used as a medicine for the treatment of food and drug intoxication, diarrhea, abdominal pain, hypertension, skin itching, and cancer. In this study, both of solid-state-cultured AC (S-AC) and wood-cultured AC (W-AC) were evaluated the anti-inflammatory effects on hyperoxia-induced lung injury in NF-κB-luciferase transgenic mice. The homozygous transgenic mice (NF-κB-luciferase ) were randomly assigned to four groups for treatment ( = 6) including Normoxia/DMSO group, Hyperoxia/DMSO group, Hyperoxia/S-AC group, and Hyperoxia/W-AC group. After 72 h of hyperoxia, we examined the bioluminescence images, reactive oxygen species (ROS), the mRNA and protein expression levels of inflammation factors, and histopathological analyses of the lung tissues. Hyperoxia-induced lung injury significantly increased the generation of ROS, the mRNA levels of , , and , and the protein expression levels of IKKα/β, iNOS and IL-6. Pulmonary edema and alveolar infiltration of neutrophils was also observed in the hyperoxia-induced lung tissue. However, treatment with either S-AC or W-AC obviously decreased hyperoxia-induced generation of ROS and the expression of IL-6, TNF-α, IL-1β, IL-8, IKKα/β and iNOS compared to hyperoxia treatment alone. Lung histopathology also showed that treatment with either S-AC or W-AC significantly reduced neutrophil infiltration and lung edema compared to treatment with hyperoxia treated alone. To find out their major compounds, eburicoic acid and dehydroeburicoic acid were both isolated and identified from S-AC and W-AC by using HPLC, MS, and NMR spectrometry. These results demonstrated that methanolic extracts both of S-AC and W-AC have excellent anti-inflammatory activities and thus have great potential as a source for natural health products.
Accumulating evidences indicate that pulmonary exposure to carbon nanotubes (CNTs) is associated with increased risk of lung diseases, whereas the effect on the vascular system is less studied. We investigated vascular effects of 2 types of multiwalled CNTs (MWCNTs) in apolipoprotein E-/ mice, wild-type mice, and cultured cells. The ApoE(/) mice had accelerated plaque progression in aorta after 5 intracheal instillations of MWCNT (25.6 g/mouse weekly for 5 weeks). The exposure was associated with pulmonary inflammation, lipid peroxidation, and increased expression of inflammatory, oxidative stress, DNA repair, and vascular activation response genes. The level of oxidatively damaged DNA in lung tissue was unaltered, probably due to increased DNA repair capacities. Despite upregulation of inflammatory genes in the liver, effects on systemic cytokines and lipid peroxidation were minimal. The exposure to MWCNTs in cultured human endothelial cells increased the expression of cell adhesion molecules (ICAM1 and VCAM1). In cocultures, there was increased adhesion of monocytes to endothelial cells after exposure to MWCNT. The exposure to both types of MWCNT was also associated with increased lipid accumulation in monocytic-derived foam cells, which was dependent on concomitant oxidative stress because the antioxidant N-acetylcysteine inhibited the lipid accumulation. Collectively, our results indicate that exposure to MWCNT is associated with accelerated progression of atherosclerosis, which could be related to both increased adherence of monocytes onto the endothelium and oxidative stress-mediated transformation of monocytes to foam cells.
Our previous studies reported that methanol extract of Sanguisorbae radix from Sanguisorba officinalis L. (Rosaceae) prevented neuronal cell damage induced by Aβ (25-35) in vitro. The present study was carried out to investigate the effect of gallic acid isolated from Sanguisorbae radix on Aβ (25-35)-induced neurotoxicity using cultured rat cortical neurons. Gallic acid (0.1, 1 μM) showed a concentration-dependent inhibition on Aβ (25-35) (10 μM)-induced apoptotic neuronal death, as assessed by a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Pretreatment of gallic acid inhibited 10 μM Aβ (25-35)-induced elevation of cytosolic Ca2+ concentration ([Ca2+]c) and generation of reactive oxygen species (ROS), which were measured by fluorescent dyes. Gallic acid also inhibited glutamate release into medium induced by 10 μM Aβ (25-35), which was measured by HPLC. These results suggest that gallic acid prevents Aβ (25-35)-induced apoptotic neuronal death by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release and generation of ROS, and that these effects of gallic acid may be partly associated with the neuroprotective effect of Sanguisorbae radix.
Background and Purpose Angiotensin II (AngII) induces migration and growth of vascular smooth muscle cell (VSMC), which is responsible for vascular remodelling in some cardiovascular diseases. Ang II also activates a Cl− current, but the underlying mechanism is not clear. Experimental Approach The A10 cell line and primary cultures of VSMC from control, ClC‐3 channel null mice and WT mice made hypertensive with AngII infusions were used. Techniques employed included whole‐cell patch clamp, co‐immunoprecipitation, site‐specific mutagenesis and Western blotting, Key Results In VSMC, AngII induced Cl− currents was carried by the chloride ion channel ClC‐3. This current was absent in VSMC from ClC‐3 channel null mice. The AngII‐induced Cl− current involved interactions between ClC‐3 channels and Rho‐kinase 2 (ROCK2), shown by N‐ or C‐terminal truncation of ClC‐3 protein, ROCK2 siRNA and co‐immunoprecipitation assays. Phosphorylation of ClC‐3 channels at Thr532 by ROCK2 was critical for AngII‐induced Cl− current and VSMC migration. The ClC‐3 T532D mutant (mutation of Thr532 to aspartate), mimicking phosphorylated ClC‐3 protein, significantly potentiated AngII‐induced Cl− current and VSMC migration, while ClC‐3 T532A (mutation of Thr532 to alanine) had the opposite effects. AngII‐induced cell migration was markedly decreased in VSMC from ClC‐3 channel null mice that was insensitive to Y27632, an inhibitor of ROCK2. In addition, AngII‐induced cerebrovascular remodelling was decreased in ClC‐3 null mice, possibly by the ROCK2 pathway. Conclusions And Implications ClC‐3 protein phosphorylation at Thr532 by ROCK2 is required for AngII‐induced Cl− current and VSMC migration that are involved in AngII‐induced vascular remodelling in hypertension.
BACKGROUND AND PURPOSE Deficient transmission at the glutamate NMDA receptor is considered a key component of the pathophysiology of schizophrenia. However, the effects of antipsychotic drugs on the release of the endogenous NMDA receptor partial agonist, d‐serine, remain to be clarified. EXPERIMENTAL APPROACH We determined the interaction between antipsychotic drugs (clozapine and haloperidol) and transmission‐modulating toxins (tetanus toxin, fluorocitrate, tetrodotoxin) on the release of L‐glutamate and d‐serine in the medial prefrontal cortex (mPFC) of freely moving rats, using microdialysis, and primary cultures of astrocytes using extreme high‐pressure liquid chromatography. KEY RESULTS Release of L‐glutamate and d‐serine in the mPFC and in cultured astrocytes was inhibited by tetanus toxin (a synaptobrevin inhibitor) and fluorocitrate (a glial toxin), whereas tetrodotoxin (a voltage‐sensitive Na+ blocker) inhibited depolarization‐induced L‐glutamate release in the mPFC without affecting that of d‐serine. Clozapine (1 and 5 mg·kg−1), but not haloperidol (0.5 and 1 mg·kg−1), dose‐dependently increased L‐glutamate and d‐serine release from both astrocytes and mPFC. Clozapine‐induced release of L‐glutamate and d‐serine was also reduced by tetanus toxin and fluorocitrate. Tetrodotoxin reduced clozapine‐induced mPFC L‐glutamate release but not that of d‐serine. Clozapine‐induced L‐glutamate release preceded clozapine‐induced d‐serine release. MK‐801 (a NMDA receptor antagonist) inhibited the delayed clozapine‐induced L‐glutamate release without affecting that of d‐serine. CONCLUSIONS AND IMPLICATIONS Clozapine predominantly activated glial exocytosis of d‐serine, and this clozapine‐induced d‐serine release subsequently enhances neuronal L‐glutamate release via NMDA receptor activation. The enhanced d‐serine associated glial transmission seems a novel mechanism of action of clozapine but not haloperidol.
Hepatobiliary elimination can be a major clearance pathway dictating the pharmacokinetics of drugs. Here, we first compared the dose eliminated in bile in preclinical species (monkey, dog, and rat) with that in human and further evaluated single-species scaling (SSS) to predict human hepatobiliary clearance. Six compounds dosed in bile duct–cannulated (BDC) monkeys showed biliary excretion comparable to human; and the SSS of hepatobiliary clearance with plasma fraction unbound correction yielded reasonable predictions (within 3-fold). Although dog SSS also showed reasonable predictions, rat overpredicted hepatobiliary clearance for 13 of 24 compounds. Second, we evaluated the translatability of sandwich-cultured human hepatocytes (SCHHs) to predict human hepatobiliary clearance for 17 drugs. For drugs with no significant active uptake in SCHH studies (i.e., with or without rifamycin SV), measured intrinsic biliary clearance was directly scalable with good predictability (absolute average fold error [AAFE] = 1.6). Drugs showing significant active uptake in SCHH, however, showed improved predictability when scaled based on extended clearance term (AAFE = 2.0), which incorporated sinusoidal uptake along with a global scaling factor for active uptake and the canalicular efflux clearance. In conclusion, SCHH is a useful tool to predict human hepatobiliary clearance, whereas BDC monkey model may provide further confidence in the prospective predictions.
Umbilical cord blood (UCB) provides an alternative source for hematopoietic stem/progenitor cells (HSPCs) in the treatment of hematological malignancies. However, clinical usage is limited due to the low quantity of HSPCs in each unit of cord blood and defects in bone marrow homing. Hyperbaric oxygen (HBO) is among the more recently explored methods used to improve UCB homing and engraftment. HBO works by lowering the host erythropoietin before UCB infusion to facilitate UCB HSPC homing, because such UCB cells are not directly exposed to HBO. In this study, we examined how direct treatment of UCB-CD34 cells with HBO influences their differentiation, proliferation and transmigration. Using a locally designed HBO chamber, freshly enriched UCB-CD34 cells were exposed to 100% oxygen at 2.5 atmospheres absolute pressure for 2 h before evaluation of proliferative capacity, migration toward a stromal cell–derived factor 1 gradient and lineage differentiation. Our results showed that HBO treatment diminishes proliferation and transmigration of UCB-CD34 cells. Treatment was also shown to limit the ultimate differentiation of these cells toward an erythrocyte lineage. As a potential mechanism for these findings, we also investigated HBO effects on the relative concentration of cytoplasmic and nucleic reactive oxygen species (ROS) and on erythropoietin receptor (Epo-R) and CXCR4 expression. HBO-treated cells showed a relative increase in nucleic ROS but no detectable differences in the level of Epo-R nor CXCR4 expression were established compared with non-treated cells. In summary, HBO amplifies the formation of ROS in DNA of UCB-CD34 cells, potentially explaining their reduced proliferation, migration and erythrocytic differentiation.
The cell extracts of two cultured freshwater Nostoc spp., UIC 10279 and UIC 10366, both from the suburbs of Chicago, showed antiproliferative activity against MDA-MB-231 and MDA-MB-435 cancer cell lines. Bioassay-guided fractionation led to the isolation of five glycosylated cylindrocyclophanes, named ribocyclophanes A-E (1-5) and cylindrocyclophane D (6). The structure determination was carried out by HRESIMS and 1D and 2D NMR analyses and confirmed by single-crystal X-ray crystallography. The structures of ribocyclophanes A-E (1-5) contain a beta-D-ribopyranose glycone in the rare C-1(4) conformation. Among isolated compounds, ribocyclophane D (4) showed antiproliferative activity against MDA-MB-435 and MDA-MB-231 cancer cells with an IC50 value of less than 1 mu M.
Abstract Background aims For transplantation, hematopoietic stem cells (HSC) are obtained from bone marrow, cord blood and mobilized adult peripheral blood. HSCs are present in the blood of healthy adults and can be recovered in leuko-reduction system chambers, with a potential yield of 1 to 4 × 106 CD34+ cells per unit. Some groups have investigated this valuable source of stem cells; however, investigations are still needed to support their use. Methods CD34+ cells were purified from leuko-reduction system chambers and cultured with a defined custom medium without animal protein and supplemented with interleukin-3, interleukin-6, Fms-like tyrosine kinase 3, stem cell factor and thrombopoietin. Cells were cultured under 8% and 21% oxygen levels. With the use of multiparametric flow cytometry analysis, the phenotypes of emerging populations were compared between oxygen levels and resting CD34+ cells. Both conventional gating and clustering analysis were used to visualize the cellular outcome. Results A maximum expansion of 20-fold was obtained without major differences in viability, number of cells or cellular heterogeneity between atmospheric and physiologic oxygen conditions. Worthy of note, phenotype analysis revealed that megakaryocyte and erythrocyte progenitors were favored, albeit more moderately when submitted to 8% O2. Conclusions This study suggests that the bias of cultured blood CD34+ cells toward megakaryocyte and erythrocyte progenitor cells can be reduced by use of 8% pO2 . It also shows how clustering software, such as SPADE, can help visualize the complexity of stem cell differentiation.