Mouse Peritoneal Macrophage Cells Research Articles

Cathepsin D interacts with adenosine A2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

Adenosine A2A receptor (A2AR)–dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A2AR targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A2AR and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A2AR-interacting partners have in macrophages. Here, we aimed to identify A2AR-interacting partners in macrophages that may effect receptor trafficking and activity. To this end, we performed a yeast two-hybrid screen using the C-terminal tail of A2AR as the “bait” and a macrophage expression library as the “prey.” We found that the lysosomal protease cathepsin D (CtsD) was a robust hit. The A2AR–CtsD interaction was validated in vitro and in cellular models, including RAW 264.7 and mouse peritoneal macrophage (IPMΦ) cells. We also demonstrated that the A2AR is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A2AR in macrophages. Conversely, we demonstrate that A2AR activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A2AR-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes.

Abstract P111: Effects Of Magnetic Field On Abca1-mediated Cellular Lipid Release And Adult Brain Cell Generation In Mice

Aim: This project aims to explore a novel magnetic field (MF) health care based on the cholesterol metabolism. Methods: Mouse peritoneal macrophages foam cells were incubated in CO 2 -independent culture medium containing apoA-I and were exposed in a MF (0.4 T) using the electromagnet of EPR spectrometer. C57BL/6N mice (8 week-old) were peritoneally injected F-ara-EdU (133μg/g mouse body weight) then exposed to MF under the identical condition. Mouse plasma, cerebrospinal fluid, liver and brain were harvested on the 28 th day. Lipoprotein profiles were examined enzymatic detection followed by a gel-permeable-HPLC (Skylight Biotech Inc.). Amyloid β40 and β42 levels were determined by WAKO ELISA kit. Fixed mouse brain sections (40um) were prepared by Leica CM1900 cryostat. Alexa fluor® 488 conjugates were link to F-ara-EdU by a click chemistry method and fluor positive nucleus images were captured by a confocal super resolution SpinSR10 (Olympus, Inc.). Results: ABCA1-apoA-I mediated cellular cholesterol release was not affected or reduced by the exposure in the MF of 0.4 T. The Pcsk9 expression showed significant reduction in the hepatocytes. Reduction of Abca1 expression and increase of Scarb1 were obtained non significantly. The newly generated hippocampal dentate gyrus cells in the control mice group and the MF exposed group were 1.6±1.17, and 0.47±0.62 cells per dentate gyrus section, respectively (P=0.003) despite bulk of new cells in olfactory bulbs in the both groups. Discussions: No substantial increase in cellular cholesterol export was observed by MF irradiation of mouse peritoneal macrophage cells. In addition, the lipoprotein profile showed a decrease in HDL cholesterol even after 28 days. As a result of gene expression in the liver, a decrease in ABCA1 expression and an increase in SR-BI expression inferred reduction of HDL. The number of new cells detected in the granular zone and the subgranular zone in the dentate gyrus of the hippocampus was significantly reduced in the mice subjected to the MF treatment. In the future, it is necessary to investigate in detail whether these newborn cells are neuron or glial cells.

Elsholtzia ciliata (Thunb.) Hyl. Extracts from Different Plant Parts: Phenolic Composition, Antioxidant, and Anti-Inflammatory Activities.

Polyphenols play an important role on the health-promoting properties of humans. Plants belonging to Lamiaceae family are known as rich source of phenolic compounds. The current work aimed to evaluate the phenolic compounds, antioxidant, and anti-inflammatory activity of Elsholtzia ciliata (Thunb.) Hyl. ethanolic extracts from leaf, stem, flower, and whole herb. Twelve compounds were identified in ethanolic extracts using high-performance liquid chromatography (HPLC). The HPLC analysis revealed that chlorogenic acid, rosmarinic acid, and rutin were predominant compounds in ethanolicic extracts. Using HPLC-ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) post-column assay, avicularin, chlorogenic, and rosmarinic acids were identified as the predominant radical scavengers in all ethanolic extracts. All tested preparations significantly reduced the level of secretion of proinflammatory cytokines TNF-α, IL-6, and prostaglandin E2 induced by lipopolysaccharide treatment in mouse peritoneal macrophage cell culture. Stem and flower extracts were most efficient in reducing cytokine release, but leaf extract demonstrated stronger effect on prostaglandin E2 secretion. This is the first study exploring antioxidant efficiency by HPLC–ABTS post-column method and investigating anti-inflammatory activity of ethanolic extracts from E. ciliata different plant parts.

The oxidation state of cysteine thiols on the ectodomain of TLR2 and TLR4 influences intracellular signaling.

Signal transduction by the Toll-like receptors (TLRs) is a key component of innate immunity against many pathogens and also underlies a large burden of human diseases. Therefore, the mechanisms and regulation of signaling from the TLRs are of considerable interest. Here we seek to determine the molecular mechanism by which TLR2 and TLR4, members of the Toll-like receptor family, are activated by bacterial LPS, hyperoxia, and zymosan respectively. Our central hypothesis is that the oxidation state of cysteine thiols on the ectodomain of TLR2 and TLR4 are critical for pathogen-initiated intracellular signaling as well in hyperoxia. Cysteine thiols of TLR4 and its co-receptor MD2 have been shown to aid binding between the two molecules and also bacterial LPS binding to the receptor complex. We extend these findings by demonstrating the oxidation of free thiols on the ectodomain of hTLR4, after exposure to LPS or hyperoxia suggesting that the cysteines on the ectodomain of TLR4 could form intra- or intermolecular disulfide bonds. We also demonstrated blockade of intracellular signaling from TLR4 and TLR2 by thiol-modifying compounds which suggest a novel therapeutic intervention for sepsis, hyperoxia-induced cell injury and yeast infection. In these experiments CHO-3E10, HEK293 cells expressing hTLR2 or hTLR4 and mouse peritoneal macrophages cells were pretreated with cell impermeable maleimides to alkylate thiols on the extracellular domain of TLRs, cells were then exposed to LPS, hyperoxia or zymosan. In all of these models, we detected decreased intracellular signaling from TLR2 or TLR4. Furthermore, incubation with phenyl arsine oxide - which forms stable complexes with vicinal cysteine residues - prevented LPS induced HEK293/hTLR4 intracellular signaling which was reversed by DMPS. Sequence analysis of different TLRs revealed Leucine-Rich Repeat C-terminal (LRRCT) domain that contains 4 conserved cysteines. Further work is required to pinpoint the role of each cysteine in receptor dimerization, pathogen binding, hyperoxia modulation, and intracellular signaling.

Anti-Inflammatory Effects of Canavalia gladiata in Macrophage Cells and DSS-Induced Colitis Mouse Model.

Canavalia gladiata, known as sword bean, has been used as a Chinese traditional medicine for anti-inflammatory effects. However, the action mechanisms of sword bean have not yet been clearly defined. In the present study, the whole parts of a ripened sword bean (RSB) and the green sword bean (GSB) containing bean pod were extracted with ethanol by reflux extraction. The two crude extracts (RSBE and GSBE) from RSB and GSB were validated by a liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis of gallic acid as a reference chemical. The anti-inflammatory effects of two sword bean extracts were extensively investigated using LPS-stimulated macrophage cells. First, RSBE and GSBE significantly inhibited the production of pro-inflammatory mediators, such as tumor necrosis factor- (TNF-), interleukin-6 (IL-6), prostaglandinE2 (PGE2), and nitric oxide (NO) in LPS-induced RAW264.7 cells. RSBE and GSBE showed no cytotoxicity to RAW264.7 cells and mouse peritoneal macrophage cells. In addition, the overexpression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) induced by LPS in RAW264.7 cells was significantly decreased by RSBE and GSBE. Western blotting and immunostaining analysis showed that RSBE and GSBE inhibited the nuclear translocation of NF-B subunits, which correlated with the inhibitory effects on inhibitor kappa B (IB) degradation. In dextran sulfated sodium (DSS)-induced colitis mice model, RSBE restored body weight, colon length, and the levels of pro-inflammatory cytokines, such as TNF-, IL-6, interleukin-1 (IL-1), and interferon- (IFN-). In addition, RSBE significantly suppressed the expression of COX-2, iNOS, and NF-B.

Nitric Oxide Is Involved in Activation of Toll-Like Receptor 4 Signaling through Tyrosine Nitration of Src Homology Protein Tyrosine Phosphatase 2 in Murine Dextran Sulfate-Induced Colitis.

Ulcerative colitis is characterized by colonic mucosal bleeding and ulceration, often with repeated active and remission stages. One factor in ulcerative colitis development is increased susceptibility to commensal bacteria and lipopolysaccharide (LPS). LPS activates macrophages to release nitric oxide (NO) through Toll-like receptor 4 (TLR4) signaling. However, whether NO is beneficial or detrimental to colitis remains controversial. In this study, we investigated whether NO enhances the development of colitis in mice treated with dextran sulfate sodium (DSS) and inflammation in cells treated with low-dose LPS. An NO donor, NOC18, induced colitis and increased CD14 protein and nitrotyrosine levels in colonic macrophages from mice treated with DSS for 7 d (molecular weight: 5000). In the mouse peritoneal macrophage cell line RAW264.7 stimulated with 3 ng/mL LPS, NO activated the CD14-TLR4-nuclear factor kappa B (NF-κB) axis. Low-dose LPS stimulation did not change the levels of signal transducer and activator of transcription (STAT) 3 phosphorylation, CD14, inducible NO synthase, interleukin (IL)-6, or NF-κB. In addition, low-dose LPS increased phosphorylation of src homology protein tyrosine phosphatase 2 (SHP2), a negative regulator of STAT3 phosphorylation. However, NO decreased SHP2 phosphorylation and significantly activated the downstream signaling molecules. NO increased SHP2 nitration in LPS-stimulated RAW264.7 cells and DSS-treated mice. These results indicate that SHP2 nitration in macrophages might be involved in activation of the CD14-TLR4-NF-κB axis through STAT3 signaling in mice with DSS-induced colitis.

60 - Aegeline, targets LOX1, the receptor for oxidized LDL to mitigate hypercholesterolemia: a new perspective in its anti-atherosclerotic action

Overview Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is one of the major receptors for oxidized low-density lipoprotein (Ox-LDL) in the aorta of aged rats. Ox-LDL initiates LOX-1 activation in the endothelium of lipid accumulating sites of both animal and human subjects of hypercholesterolemia. This study has explored the binding affinity of Aegeline to LOX1 for the first time. Targeting LOX-1 may provide a novel diagnostic strategy for hypercholesterolemia and vascular diseases. Objective This study was planned to address whether an Aegeline (AG) could bind to LOX-1 with a higher affinity and modulate the uptake of ox-LDL in hypercholesterolemia. Methods Thirty-six Wistar rats were divided into six groups. The pathology group rats were fed with High Cholesterol Diet (HCD) for 45 days and treatment group rats were fed with HCD and AG/AV for last 30 days. In vivo and in vitro experiments were carried out to assay the markers of atherosclerosis like Ox-LDL and LOX1 levels. Histopathological examination of aorta tissue sections was performed. Oil red O staining was carried out in the mouse peritoneal macrophage cell line IC21. Docking studies were performed to find out the binding affinity of AG and AV to LOX1. Results AG administration effectively decreased the lipid levels induced by HCD as evidenced by lipid profile. Further, the lowered levels of Ox-LDL and LOX1 in AG administered rats deem it to be a potent anti-hypercholesterolemic agent. Conclusion This study thus validates AG to be an effective stratagem in bringing down the lipid stress induced by HCD and by down-regulating LOX1 mediated uptake of Ox-LDL.

In vitro antiplasmodial, antitrypanosomal and antileishmanial activities of selected medicinal plants from Ugandan flora: Refocusing into multi-component potentials

Ethnopharmacological relevanceSeven medicinal plants from Ugandan flora, namely Entada abyssinica, Khaya anthotheca, Vernonia amygdalina, Baccharoides adoensis, Schkuhria pinnata, Entandropragma utile and Momordica foetida, were selected in this study. They are used to treat conditions and infections ranging from inflammations, pains and fevers to viruses, bacteria, protozoans and parasites. Two of the plants, V. amygdalina and M. foetida, are also used as human food or relish, while others are important in ethnoveterinary practices and in zoopharmacognosy in the wild. The aim of this study was to evaluate the in vitro antiplasmodial, antitrypanosomal and antileishmanial activities, along with cytotoxicity of the multi-component extracts of these plants. Materials and methodsDifferent parts of the plants were prepared and serially extracted with hexane, petroleum ether, dichloromethane, ethyl acetate, methanol and double distilled water. Solvent free extracts were assayed for in vitro inhibition against four reference parasite strains, Plasmodium falciparum (K1), Trypanosoma brucei rhodesiense (STIB 900), Trypanosoma cruzi (Talahuen C2C4) and Leishmania donovani (MHOM-ET-67/L82) using standard methods. Toxicity was assessed against L6 skeletal fibroblast and mouse peritoneal macrophage (J774) cells and selectivity indices (SIs) calculated for the most active extracts. ResultsThe strongest activities, demonstrating median inhibitory concentration (IC50) values ≤ 2 μg/ml, were observed for the dichloromethane and petroleum ether extracts of K. anthotheca, B. adoensis and S. pinnata. Overall, IC50 values ranged from < 1 μg/ml to > 90 μg/ml. Out of 22 extracts demonstrating IC50s < 20 μg/ml, seven were against T. b. rhodesiense (IC50: 1.6–16.2 μg/ml), six against T. cruzi (IC50: 2.1–18.57 μg/ml), none against L. donovani (IC50: falling > 3.3 and >10 μg/ml), and nine against P. falciparum (IC50: 0.96 μg/ml to 4.69 μg/ml). Selectivity indices (SI) calculated for the most active extracts ranged from <1.00 to 94.24. However, the B. adoensis leaf dichloromethane extract (a) was equipotent (IC50 = 3.3 μg/ml) against L. donovani and L6 cells respectively, indicating non-specific selection. Trypanosome and Plasmodium parasites were comparatively more sensitive to the test extracts. ConclusionsThe benefits achieved from the seven tested plant species as traditional ethnomedicinal and ethnoveterinary therapies or in zoopharmacognosy against infections and conditions of animals in the wild are strongly supported by results of this study. The synergy of plant extracts, so achieved by concerted actions of the ligands, produces adequate perturbation of targets in the four parasite genera, resulting in the strong potencies exhibited by low IC50 values. The total inhibitory effect, achieved as a sum of perturbations contributed by each participating compound in the extract, minimises toxic effects of the compounds as seen in the high SI's obtained with some extracts. Those extracts demonstrating SI ≥ 4 form promising candidates for further cell-based and system pharmacology studies.

Molecular targeting of breast and colon cancer cells by PAR1 mediated apoptosis through a novel pro-apoptotic peptide.

A novel activating peptide was designed and synthesized from V. cholerae hemagglutinine protease (HAP) mediated cleavage site of mouse PAR1. The peptide "PFISED" interacts with PAR1 in a new site which is different from its thrombin mediated conventional activation site and induced a series of new downstream signaling pathways. The peptide showed apoptosis in human and mouse breast (MCF-7 and EAC) and colon (HT29 and CT26) cancer cells where as in the same peptide concentration in normal human breast epithelial cells (MCF-10A), normal human fibroblast cells (MRC-5), normal mouse peritoneal macrophage cells and normal mouse breast and colon tissues did not show any effect. Treatment with this peptide enhanced the survival kinetics of EAC induced mice. The peptide mediated apoptosis was inhibited in presence of PAR1 inhibitor and was significantly reduced in si-PAR1 treated cells that indicate the activating peptide "PFISED" induced PAR1 mediated apoptosis of colon and breast cancer cells. This peptide induced over expression and activation of PAR1 and its downstream MAP kinase and NFκB signaling pathways. These signaling pathways enhanced the cellular ROS level to kill malignant cells. We report a novel pro-apoptotic peptide which can selectively kill malignant cells via its specific target receptor PAR1 which is over expressed in the malignant cells and can be used as a molecular target therapy for cancer treatment.

On the effects of NLRP3 inflammasome on mice pancreatic β-cell damage induced by advanced glycation end products

Objective To explore the role of the pyrin domain-containing 3 (NLRP3) inflammasome in advanced glycation end products (AGEs)-induced mice pancreatic β-cell damage. Methods AGEs were administered intraperitoneally for 6 weeks in NLRP3 knockout mice or C57BL/6J mice. Intraperitoneal glucose tolerance test and insulin releasing test were performed. Pancreatic sections were stained with haematoxylin and eosin, or with F4/80 and NLRP3 antibodies. Insulin and pancreatic tissue monocyte chemotactic protein 1 (MCP-1) as well as interleukin-1β (IL-1β) levels were measured with ELISA kits. Expression of MCP-1 protein was determined by western blot. MIN6 cells and mouse peritoneal macrophages cells were treated with AGEs and different interventions (antioxidant NAC, adenovirus NLRP3 shRNA or NLRP3 knockout). Reactive oxygen species production, NLRP3 mRNA expression, IL-1β secretion, caspase 1 activity, apoptosis and glucose stimulated insulin release were determined. Results Injection of AGEs induced an abnormal response to glucose, enhanced the insulitis score, and increased the levels of pancreatic tissue MCP-1 and IL-1β, as well as raised the expression of NLRP3 and F4/80 in pancreatic islet. Remarkably, co-localization of NLRP3 and macrophage marker F4/80 was observed in islet. The damages were improved in NLRP3 knockout mice. After incubation with AGEs, reactive oxygen species production and cell apoptosis was enhanced, NLRP3 inflammasome activated, with glucose-stimulated insulin release impaired in MIN6 cells. NAC treatment alliviated the above damages, but NLRP3 gene silencing had no effect on ROS level, apoptosis, and insulin secretion. Finally NAC treatment and NLRP3 gene knockout inhibited activation of NLRP3 inflammasome induced by AGEs in mouse peritoneal macrophages cells. Conclusion NLRP3 knockout ameliorates the islet β-cell damage induced by AGEs. These effects were associated with AGEs-induced islets macrophage infiltrating by up-regulation of MCP-1 expression, and AGEs-induced activation of NLRP3 inflammasome in macrophage through ROS pathway, which results in the release of active IL-1β and leads to the lesions of β-cell. (Chin J Endocrinol Metab, 2018, 34: 690-695) Key words: Advanced glycation end products; NLRP3 inflammasome; β-cell damage

Abstract 626: Bromodomain inhibition in ovarian cancer and the tumor microenvironment to improve PARP inhibitor response

Abstract Background: Ovarian cancer is the most lethal gynecologic malignancy. While women with BRCA-deficient tumors show sensitivity to PARP inhibitors (PARPi), new treatment options are needed for PARPi-resistant tumors. An emerging strategy to improve PARPi response is combination therapy with epigenetic drugs. A newly recognized epigenetic drug target in ovarian cancer is the bromodomain and extraterminal (BET) protein family. BET proteins such as BRD4 promote oncogenic transcription of progrowth and survival genes, including the established link between inflammation and cancer, nuclear factor-kappaB (NF-κB). A complementary strategy to targeting cancer cells with cytotoxic drugs is to activate normal immune processes in the tumor microenvironment (TME). In syngeneic mouse ovarian cancer models, we have shown that M2-like protumor macrophages are a prominent component of the TME, and that NF-κB inhibition reduces the M2 population. Thus, BET inhibitors (BETi) have the potential to induce transcriptional reprogramming in both tumors and macrophages for therapeutic benefit. Objective: To determine the cellular and molecular effects of combining BETi and PARPi in mouse ovarian cancer and peritoneal macrophage cell lines. Methods: Cultured wild-type and CRISPR-modified (TP53 and TP53/BRCA2 knockout) ID8 mouse ovarian cancer cells, and PMJ2-PC mouse peritoneal macrophages, were treated with vehicle, the PARPi olaparib, the first-in-class BETi JQ1 or the JQ1/olaparib combination for 24-72h. Sulforhodamine B (SRB) assays assessed cell growth. Immunofluorescence assays assessed adherent cell number, DNA damage (pH2AX) and cell cycle indices. Protein levels of pH2AX and the apoptosis marker cleaved PARP were assessed by Western blot. NF-κB activity was measured by luciferase assays of a transiently transfected reporter plasmid. Results: Combined JQ1 and olaparib treatment synergistically reduced cell growth in SRB assays in wild-type and TP53 knockout ID8 cells. TP53/BRCA2 knockout cells showed greater responses to PARPi alone and no synergism was observed. Consistent with these results, the JQ1/olaparib combination cooperatively reduced the number of adherent cells and cells in S phase, and increased the G0/G1 population, DNA damage and apoptosis. In contrast, the drug combination had minimal effects on DNA damage or apoptosis in PMJ2-PC macrophages, while NF-κB activity was reduced in both cancer cells and macrophages. Conclusions: BETi sensitize mouse ovarian cancer cells to the cytotoxic effects of PARPi. Combined drug treatment also has potential to inhibit NF-κB in both cancer cells and macrophages. Our novel immunomodulatory strategy will be tested in ID8 syngeneic ovarian cancer models. We believe BETi combination treatment could expand the use of PARPi in ovarian cancer patients, with the potential to benefit a substantial number of women with this devastating disease. Citation Format: Andrew J. Wilson, Alyssa Hoover, Whitney Harris, Esther Liu, Dineo Khabele, Fiona Yull. Bromodomain inhibition in ovarian cancer and the tumor microenvironment to improve PARP inhibitor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 626.

Fetal and Placental DNA Stimulation of TLR9: A Mechanism Possibly Contributing to the Pro-inflammatory Events During Parturition.

While there is evidence for a relationship between cell-free fetal DNA (cffDNA) and parturition, questions remain regarding whether cffDNA could trigger a pro-inflammatory response on the pathway to parturition. We hypothesized that placental and/or fetal DNA stimulates toll-like receptor 9 (TLR9) leading to secretion of pro-inflammatory cytokines by macrophage cells. Four in vitro DNA stimulation studies were performed using RAW 264.7 mouse peritoneal macrophage cells incubated in media containing the following DNA particles: an oligodeoxynucleotide (ODN2395), intact genomic DNA (from mouse placentas, fetuses and adult liver), mouse DNA complexed with DOTAP (a cationic liposome forming compound), and telomere-depleted mouse DNA. Interleukin 6 (IL6) secretion was measured in the media by enzyme-linked immunosorbent assay; and the cell pellet was homogenized for protein content (picograms IL6/mg protein). Robust IL6 secretion was observed in response to ODN2395 (a CpG-rich TLR9 agonist), mouse DNA-DOTAP complexes, and telomere-depleted mouse DNA in concentrations of 5 to 15 μg/mL. In contrast, ODN A151 (containing telomere sequence motifs), intact genomic mouse DNA, and restriction enzyme-digested DNA had no effect on IL6 secretion. The IL6 response was significantly inhibited by chloroquine (10 μg/mL), thereby confirming the important role for TLR9 in the response by macrophage cells. DNA derived from mouse placentas and fetuses, and depleted of telomeric sequences, stimulates a robust pro-inflammatory response by macrophage cells, thereby supporting the hypothesis that cffDNA is able to stimulate an innate immune response that could trigger the onset of parturition. These findings are of clinical importance, as we search for effective treatment/prevention of preterm parturition.

Flavonoid Enriched Fraction of Campylandra aurantiaca Attenuates Carbon Tetrachloride Induced Oxidative DNA Damage in Mouse Peritoneal Macrophages in Animal Model.

Recent studies have sought to draw attention of biological activity of Campylandra aurantiaca. The aim of the present study was to evaluate the effect of flavonoid enriched fraction of Campylandra aurantiaca (FEFCA) on in vitro and in vivo antioxidant and DNA protective effect in mouse peritoneal macrophages cells. FEFCA was characterized by HPLC analysis. The in vitro antioxidant activities of FEFCA was measured by different in vitro assays like 1, 1-diphenyl-2-picrylhydrazil radical (DPPH), superoxide anions, nitric oxide and hydroxyl radicals scavenging methods. Isolated mouse peritoneal macrophages were oxidized by carbon tetra chloride (CCl4) in animal model; subsequently the protective effect of FEFCA was determined in terms of estimation of antioxidant enzyme and the damage to DNA of the cells. FEFCA exhibited both in vitro antioxidant activities in a concentration dependent manner. FEFCA significantly (*p<0.05) attenuated the oxidative DNA damage of mouse peritoneal macrophage cells induced by CCl4 in an in vivo assay. Therefore FEFCA showed good free radical scavenging activity as well as reduced oxidative DNA damage in mouse peritoneal macrophages in animal model.

Development and evaluation of a cedrol-loaded nanostructured lipid carrier system for in vitro and in vivo susceptibilities of wild and drug resistant Leishmania donovani amastigotes

Leishmaniasis is an epidemic in various countries, and the parasite Leishmania donovani is developing resistance against available drugs. In the present study the antileishmanial action of cedrol was evaluated in vitro and in vivo. Activity potentiation was achieved via nanostructured lipid carrier (NLC) complexation of cedrol. Cedrol-loaded NLC was prepared through the hot-melting emulsification-ultrasonication method. The cedrol- NLC prepared did not require the use of any organic solvents. The characterization of NLC-C1 and NLC-C2 revealed that particle size was 46.62nm and 54.73nm for 3.85%, and 7.48% drug loading, respectively and negative charge of −19.2mV and −23.7mV. The cedrol-loaded NLC were found to be spherical with a smooth surface. Drug-carrier interactions were clearly visualized in FT-IR studies. Incorporation of cedrol in NLC was ascertained in DSC and XRD analysis. Antileishmanial activities of free cedrol and cedrol-NLC were performed against L. donovani wild-type, sodium stibogluconate, paromomycin and field isolated resistant strains in axenic amastigotes and amastigotes in macrophage model. Coumarin-6 loaded NLC nanoparticles were assessed for macrophage internalization in confocal microscopic studies. Cedrol showed significant antileishmanial activity in wild-type (IC50=1.5μM), sodium stibogluconate resistant (IC50=2μM), paromomycin resistant (IC50=1.8μM) and field isolated resistant (IC50=1.35μM) strains in macrophage together with cytotoxicity (CC50=74μM) in mouse peritoneal macrophage cells. Incorporation of cedrol in NLC-C2 resulted in 2.1-fold and 2-fold increase in selectivity indexes (CC50/IC50) for wild-type and drug resistant strains, respectively. In addition, in vivo studies revealed that bioactivity of NLC-C2 were 2.3 to 3.8-fold increased in wild-type and 3 to 4.9-fold increased in drug resistant strains when compared with free cedrol; administered orally in mouse leishmaniasis model. Overall, NLC-C2 showed superior antileishmanial activity to free cedrol and miltefosine in oral dose. These findings support the use of NLCs for oral delivery of poorly water-soluble antileishmanial drugs in treatment of leishmaniasis. Chemical compoundsCedrol (PubChem CID: 65575); Compritol® 888 ATO (PubChem CID: 62726); Triolein (PubChem CID: 5497163); Pluronic F68 (PubChem CID: 24751); Soya lecithin (PubChem CID: 57369748); Sodium deoxycholate (PubChem CID: 23668196); Miltefosine (PubChem CID: 3599); Paromomycin (PubChem CID: 165580); Amphotericin B (PubChem CID: 5280965); Sodium stibogluconate (PubChem CID: 16683012).

In Vitro susceptibilities of wild and drug resistant Leishmania donovani amastigotes to piperolactam A loaded hydroxypropyl-β-cyclodextrin nanoparticles

Leishmaniasis is an epidemic in various countries, and the parasite Leishmania donovani is developing resistance against available drugs. In the present study the antileishmanial action of piperolactam A (PL), isolated after bioactivity guided fractionation from root extracts of Piper betle was accentuated in detail. Activity potentiation was achieved via cyclodextrin complexation. Crude hydro-ethanolic extract (PB) and three fractions obtained from PB and fabricated PL-hydroxypropyl-β-cyclodextrin (HPBCD) nanoparticles were evaluated for antileishmanial activity. Tests were performed against L. donovani wild-type, sodium stibogluconate, paromomycin and field isolated (GE1) resistant strains in axenic amastigote and amastigote in macrophage models. PL-HPBCD complex was characterized and FITC loaded HPBCD nanoparticles were assessed for macrophage internalization in confocal microscopic studies. Isolated and purified PL from most potent, alkaloid rich ethyl acetate fraction of PB showed high level of antileishmanial activities in wild-type (IC50=36μM), sodium stibogluconate resistant (IC50=103μM), paromomycin resistant (IC50=91μM) and field isolated resistant (IC50=72μM) strains together with cytotoxicity (CC50=900μM) in mouse peritoneal macrophage cells. Inclusion of PL in HPBCD nanoparticles resulted in 10-fold and 4–10-fold increase in selectivity indexes (CC50/IC50) for wild-type and drug resistant strains, respectively. Drug-carrier interactions were clearly visualized in FT-IR studies. Complete incorporation of PL in HPBCD cavity was ascertained in DSC and XRD analyses. 180nm size stable nanospheres showed macrophage internalization within 1h of incubation. Piperolactam A (PL), a representative of the inchoate skeleton of aristolactam chassis might be the source of safe and affordable antileishmanial agents for the cure of deadly Leishmania infections.

HepG2 cells acquire stem cell-like characteristics after immune cell stimulation.

The presence of cancer stem cells (CSCs) is currently regarded as one of the main culprits of tumor formation and therapy failure. It is known that chronic inflammation is associated with CSCs, but it is not clear yet how inflammation affects the development of CSCs. In the present study we aimed to examine the relationship between cancer cell stimulation mediated by immune cells and the acquisition of a CSC-like phenotype. Cancer cells derived from single hepatocarcinoma HepG2 cells were treated with mouse splenic B cells (MSBCs) and mouse peritoneal macrophage cells (MPMCs), respectively. The stem cell-like characteristics of the resulting HepG2 cells (MSBC-HepG2 and MPMC-HepG2) were evaluated using different assays, including biomarker assays, in vitro tumoroid and colony forming assays, in vivo tumor forming assays and signal transduction pathway activation assays. Various stemness characteristics of HepG2 cells, including self-renewal, proliferation, chemoresistance and tumorigenicity were evaluated. The expression levels of stemness-related genes and its encoded proteins in the MSBC-HepG2 and MPMC-HepG2 cells were assessed using RT-PCR and FACS analyses. We found that MSBC-HepG2 and MPMC-HepG2 cells possess hepatic CSC properties, including persistent self-renewal, extensive proliferation, drug resistance, high tumorigenic capacity and over-expression of CSC-related genes and proteins (i.e., EpCAM, ALDH, CD133 and CD44), compared to the parental cells. We also found that 1x10(3) MSBC-HepG2 and MPMC-HepG2 cells were able to form tumors in NOD/SCID mice and that the Notch and SHH signaling pathways were highly activated in MSBC-HepG2 cells. We conclude that the immune system may have a double-edge effect on cancer development. On one hand, immune cells such as B lymphocytes and macrophages may recognize, attack and eliminate cancer cells, whereas on the other hand, they may promote a subset of cancer cells to acquire stem cell-like characteristics.

Effects of Various Carbon Nanotube Suspensions on A549, THP-1, and Peritoneal Macrophage Cells

The effects of iron content, fiber length, and stability of carbon nanotube (CNT) suspension on cells were examined. Five kinds of single-wall carbon nanotube (SWCNT) suspensions were prepared: with catalytic iron, without iron, long SWCNTs (stable), short SWCNTs (stable), and short SWCNT (unstable). These suspensions were applied to A549, THP-1, and mouse peritoneal macrophage cells. After a 24-h exposure, the mitochondrial activity, cell membrane damage, intracellular oxidative stress, and expression of cytokine genes were determined. Among these properties of SWCNTs, stability of CNT suspension had the most influence on the cells, whereas the effects of iron content and fiber length were small. The unstable SWCNT suspension caused a substantial increase in intracellular ROS levels. Additionally, the cellular effects of stable multi-wall carbon nanotubes (MWCNTs) were examined. The MWCNT suspension did not show any cellular effects. Overall, influences of CNT suspension on mitochondrial activity and cell membrane damage were small. These results suggest that the physical properties of CNT suspension are important factors for their cellular effects. Thus, CNT suspensions prepared with the same material but having different physical properties would differ in the cellular effects they exert, including cytotoxicity. Therefore, physical characterization of CNT suspensions is essential to the evaluation of CNT toxicity. In particular, stability of CNT suspension notably influenced the intracellular ROS level.

Rivaroxaban, a novel oral anticoagulant, attenuates atherosclerotic plaque progression and destabilization in ApoE-deficient mice

ObjectiveActivated factor X (FXa) plays a key role in the coagulation cascade, whereas accumulating evidence suggests that it also contributes to the pathophysiology of chronic inflammation on the vasculature. In this study, we assessed the hypothesis that rivaroxaban (Riv), a direct FXa inhibitor, inhibits atherogenesis by reducing macrophage activation. Methods and resultsExpression levels of PAR-1 and PAR-2, receptors for FXa, increased in the aorta of apolipoprotein E-deficient (ApoE-/-) mice compared with wild-type mice (P < 0.01, P < 0.05, respectively). Administration of Riv (5 mg/kg/day) for 20 weeks to 8-week-old ApoE-/- mice reduced atherosclerotic lesion progression in the aortic arch as determined by en-face Sudan IV staining compared with the non-treated group (P < 0.05) without alteration of plasma lipid levels and blood pressure. Histological analyses demonstrated that Riv significantly decreased lipid deposition, collagen loss, macrophage accumulation and matrix metallopeptidase-9 (MMP-9) expression in atherosclerotic plaques in the aortic root. Quantitative RT-PCR analyses using abdominal aorta revealed that Riv significantly reduced mRNA expression of inflammatory molecules, such as MMP-9, tumor necrosis factor-α (TNF-α). In vitro experiments using mouse peritoneal macrophages or murine macrophage cell line RAW264.7 demonstrated that FXa increased mRNA expression of inflammatory molecules (e.g., interleukin (IL)-1β and TNF-α), which was blocked in the presence of Riv. ConclusionsRiv attenuates atherosclerotic plaque progression and destabilization in ApoE-/- mice, at least in part by inhibiting pro-inflammatory activation of macrophages. These results indicate that Riv may be particularly beneficial for the management of atherosclerotic diseases, in addition to its antithrombotic activity.

Abstract 13438: Rivaroxaban, a Direct Factor Xa Inhibitor, Attenuates Neointima Formation after Mechanical Vascular Injury

Bachground: Activated factor X (FXa) is a key member in the coagulation cascade responsible for thrombin generation, although accumulating evidence suggests it also has various biological functions in many cell types, contributing to the pathogenesis of neointima formation after vascular injury. In this study, we assessed the hypothesis that rivaroxaban, a direct FXa inhibitor, attenuates neointima formation after mechanical vascular injury by inhibiting both pro-inflammatory activation of macrophages and proliferative activation of vascular smooth muscle cells (VSMCs). Methods and Results: Rivaroxaban (5 mg/kg/day) was administered for 4 weeks to C57BL/6 mice after wire-mediated vascular injury. Plasma lipid levels and blood pressure were similar between rivaroxaban group and the control group. Rivaroxaban significantly reduced neointima area (30452 ± 2531 μm2 vs. 19582 ± 2531 μm2; P &lt; 0.05) and intima/media ratio (4.37 ± 0.50 vs. 2.60 ± 0.50; P &lt; 0.05) in the injured arteries at four weeks after injury, as determined by elastica van Gieson staining, compared with the control group. In vitro experiments using mouse peritoneal macrophages or murine macrophage cell line RAW264.7 demonstrated that FXa increased mRNA expression of inflammatory molecules (e.g., IL-1β and TNF-α) and vasoproliferative molecules (e.g., PDGF and BMP-2), which was blocked in the presence of rivaroxaban. In vitro experiments using rat VSMCs demonstrated that FXa stimulation promoted both mitogenesis and migration of this cell type, as determined by MTS assay and scratch-wound assay, respectively. Rivaroxaban attenuated these FXa-induced mitogenesis and migration. Conclusions: Rivaroxaban attenuates neointima formation in a mouse model of vascular injury. Our analyses suggest that FXa contributes to both pro-inflammatory activation of macrophages and proliferative activation of VSMCs, participating in the progression of neointima formation after vascular injury. FXa may serve as a potential therapeutic target for neointima formation after vascular injury.

ERK pathway is activated in bare-FeNPs-induced autophagy

Iron oxide nanoparticles (FeNPs) are known to be one of the most biocompatible and safe nanoparticles. However, their long-term persistence remains a problem, and macrophages play as an important mediator in continuous stimulation of the immune system due to biopersistence of nanoparticles. In the present study, we identified the mechanisms underlying the uptake and toxicity of bare-FeNPs using RAW264.7 cells, a mouse peritoneal macrophage cell line. The bare-FeNPs penetrated the cell membrane through electrostatic interactions together with the general phagocytic pathway. At 24 h after exposure, they distributed freely in the cytosol or within autophagosome-like vacuoles. Bare-FeNPs induced decrease in the cell viability along with the cell cycle arrest in G1 phase. In addition, they increased the generation of ROS and the secretion of NO and TNF alpha as well as the expression of SOD-1 and SOD-2 proteins, which are an antioxidant. While the mitochondrial calcium level, the intensity of labeled mitochondria, and ATP production decreased, the levels of autophagy-related proteins such as p62, beclin 1, ATG5, and LC3B increased in a dose-dependent manner together with the levels of ATF 3, p-EGFR, and p-ERK proteins. However, the level of p-JNK protein clearly decreased. TEM images also showed that damaged organelle exist within autophagosome-like vacuoles with bare-FeNPs. On the basis of these results, we suggest that bare-FeNPs induce autophagy by initiating oxidative stress in RAW264.7 cells. Furthermore, ERK, but not JNK, pathway is activated in bare-FeNPs-induced autophagy.