Murine Macrophage J774A Research Articles

Tanreqing Injection Inhibits Activation of NLRP3 Inflammasome in Macrophages Infected with Influenza A Virus by Promoting Mitophagy.

To investigate the inhibitory effect of Tanreqing Injection (TRQ) on the activation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome in macrophages infected with influenza A virus and the underlying mechanism based on mitophagy pathway. The inflammatory model of murine macrophage J774A.1 induced by influenza A virus [strain A/Puerto Rico/8/1934 (H1N1), PR8] was constructed and treated by TRQ, while the mitochondria-targeted antioxidant Mito-TEMPO and autophagy specific inhibitor 3-methyladenine (3-MA) were used as controls to intensively study the anti-inflammatory mechanism of TRQ based on mitophagy-mitochondrial reactive oxygen species (mtROS)-NLRP3 inflammasome pathway. The levels of NLRP3, Caspase-1 p20, microtubule-associated protein 1 light chain 3 II (LC3II) and P62 proteins were measured by Western blot. The release of interleukin-1β (IL-1β) was tested by enzyme linked immunosorbent assay, the mtROS level was detected by flow cytometry, and the immunofluorescence and co-localization of LC3 and mitochondria were observed under confocal laser scanning microscopy. Similar to the effect of Mito-TEMPO and contrary to the results of 3-MA treatment, TRQ could significantly reduce the expressions of NLRP3, Caspase-1 p20, and autophagy adaptor P62, promote the expression of autophagy marker LC3II, enhance the mitochondrial fluorescence intensity, and inhibit the release of mtROS and IL-1β (all P<0.01). Moreover, LC3 was co-localized with mitochondria, confirming the type of mitophagy. TRQ could reduce the level of mtROS by promoting mitophagy in macrophages infected with influenza A virus, thus inhibiting the activation of NLRP3 inflammasome and the release of IL-1β, and attenuating the inflammatory response.

Amoebicidal Effect of COVID Box Molecules against Acanthamoeba: A Study of Cell Death.

Acanthamoeba spp. can cause a sight threatening disease. At present, the current treatments used to treat Acanthamoeba spp. Infections, such as biguanide-based antimicrobials, remain inefficacious, with the appearance of resistant forms and high cytotoxicity to host cells. In this study, an initial screening was conducted against Acanthamoeba castellanii Neff and murine macrophages J774A.1 using alamarBlue™. Among the 160 compounds included in the cited box, 90% exhibited an inhibition of the parasite above 80%, while only 18.75% of the compounds inhibited the parasite with a lethality towards murine macrophage lower than 20%. Based on the amoebicidal activity, the cytotoxicity assay, and availability, Terconazole was chosen for the elucidation of the action mode in two clinical strains, Acanthamoeba culbertsoni and Acanthamoeba castellanii L10. A fluorescence image-based system and proteomic techniques were used to investigate the effect of the present azole on the cytoskeleton network and various programmed cell death features, including chromatin condensation and mitochondria dysfunction. Taking all the results together, we can suggest that Terconazole can induce programmed cell death (PCD) via the inhibition of sterol biosynthesis inhibition.

Inhibition of macrophage infectivity potentiator in Burkholderia pseudomallei suppresses pro-inflammatory responses in murine macrophages.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a disease endemic in many tropical countries globally. Clinical presentation is highly variable, ranging from asymptomatic to fatal septicemia, and thus the outcome of infection can depend on the host immune responses. The aims of this study were to firstly, characterize the macrophage immune response to B. pseudomallei and secondly, to determine whether the immune response was modified in the presence of novel inhibitors targeting the virulence factor, the macrophage infectivity potentiator (Mip) protein. We hypothesized that inhibition of Mip in B. pseudomallei would disarm the bacteria and result in a host beneficial immune response. Murine macrophage J774A.1 cells were infected with B. pseudomallei K96243 in the presence of small-molecule inhibitors targeting the Mip protein. RNA-sequencing was performed on infected cells four hours post-infection. Secreted cytokines and lactose dehydrogenase were measured in cell culture supernatants 24 hours post-infection. Viable, intracellular B. pseudomallei in macrophages were also enumerated 24 hours post-infection. Global transcriptional profiling of macrophages infected with B. pseudomallei by RNA-seq demonstrated upregulation of immune-associated genes, in particular a significant enrichment of genes in the TNF signaling pathway. Treatment of B. pseudomallei-infected macrophages with the Mip inhibitor, AN_CH_37 resulted in a 5.3-fold reduction of il1b when compared to cells treated with DMSO, which the inhibitors were solubilized in. A statistically significant reduction in IL-1β levels in culture supernatants was seen 24 hours post-infection with AN_CH_37, as well as other pro-inflammatory cytokines, namely IL-6 and TNF-α. Treatment with AN_CH_37 also reduced the survival of B. pseudomallei in macrophages after 24 hours which was accompanied by a significant reduction in B. pseudomallei-induced cytotoxicity as determined by lactate dehydrogenase release. These data highlight the potential to utilize Mip inhibitors in reducing potentially harmful pro-inflammatory responses resulting from B. pseudomallei infection in macrophages. This could be of significance since overstimulation of pro-inflammatory responses can result in immunopathology, tissue damage and septic shock.

Salvadoran Celastraceae Species as a Source of Antikinetoplastid Quinonemethide Triterpenoids

Chagas disease and leishmaniasis are among the most widespread neglected tropical diseases, and their current therapies have limited efficacy and several toxic side effects. The present study reports the chemical and antikinetoplastid profiles of extracts from five Salvadoran Celastraceae species against the Trypanosoma cruzi epimastigotes stage and Leishmania amazonensis and Leishmania donovani promastigote forms. The phytochemical profile evinced the presence of flavonoids, tannins, sterols, and triterpenes as the main components in all plant species, whereas quinonemethide triterpenoids (QMTs) were restricted to the root bark of the studied species. Antikinetoplastid evaluation highlights the root bark extracts from Zinowewia integerrima, Maytenus segoviarum, and Quetzalia ilicina as the most promising ones, exhibiting higher potency against T. cruzi (IC50 0.71–1.58 µg/mL) and L. amazonensis (IC50 0.38–2.05 µg/mL) than the reference drugs, benznidazole (IC50 1.81 µg/mL) and miltefosine (IC50 2.64 µg/mL), respectively. This potent activity was connected with an excellent selectivity index on the murine macrophage J774A.1 cell line. These findings reinforce the potential of QMTs as antikinetoplastid agents for the development of innovative phytopharmaceuticals and the plant species under study as a source of these promising lead compounds.

N-acetyl-l-tryptophan (NAT) ameliorates radiation-induced cell death in murine macrophages J774A.1 via regulating redox homeostasis and mitochondrial dysfunction.

Ionizing radiation interacts with the immune system and induces molecular damage in the cellular milieu by generating reactive oxygen species (ROS) leading to cell death. The present study was performed to investigate the protective efficacy of N-acetyl-L-tryptophan (NAT) against gamma-radiation-induced cell death in murine macrophage J774A.1 cells. The radioprotective efficacy of NAT was evaluated in terms of cell survivability, effect on antioxidant enzyme activity, and free radicals inhibition. Radioprotective efficacy of NAT pretreatment to irradiated cells was assessed via cell cycle progression, mitochondrial membrane potential (MMP) perturbation, and apoptosis regulation using flow cytometry. Results of the study demonstrated significant radioprotective efficacy (>80%) of NAT in irradiated cells as estimated by sulforhodamine B(SRB), MTT, and clonogenic assay. Significant (p < 0.001) reduction in ROS, xanthine oxidase, and mitochondrial superoxide levels along with increment in catalase, glutathione-s-transferase, glutathione, and ATPase activities in NAT pretreated plus irradiated cells was observed as compared to the gamma-irradiated cells. Further, significant (p < 0.001) stabilization of MMPand reduction in apoptosis was also observed in NAT pretreated plus irradiated cells as compared to irradiated cells that not pretreated with NAT. The current study demonstrates that NAT pretreatment to irradiated cells protects against gamma radiation-induced cell death by reducing oxidative stress, stabilizing MMP, and inhibiting apoptosis. These observations conclusively highlight the potential of developing NAT as a prospective radioprotective agent upon further validation using in-depth preclinical assessment in cellular and animal models.

A Fluorescence-Based In Vitro Method to Assess Cholesterol Efflux.

Cholesterol efflux (ChE) capacity is associated with the incidence of cardiovascular events and has been proposed as an emerging cardiovascular risk factor. ChE has been traditionally assessed by in vitro radioactive methods but these are not appropriate when assessing a large number of samples. Therefore, alternative, reproducible nonradioactive methods have been developed. This chapter describes a robust nonradioactive method using a fluorescent tracer to assess ChE in vitro.The measurement of ChE in vitro requires three main components: a cholesterol-loaded donor cell, a cholesterol tracer, and a cholesterol acceptor. This method involves labeling of murine macrophage J774A.1 cells using the fluorescent sterol dipyrromethene boron difluoride (BODIPY)-cholesterol. The cholesterol acceptors from humans or animals include lipid-free apolipoprotein (ApoA)-1, high-density lipoprotein (HDL), HDL2 and HDL3 subfractions, serum, plasma or ApoB-depleted serum or plasma. While lipid-free ApoA-1 mediates ChE via only ATP-binding cassette (ABC)A1 transporter, the remaining acceptors mediate ChE via ABCA1 , ABCG1 and scavenger receptor class B type 1 (SRB1) transporters. The reproducibility of this BODIPY-ChE assay is excellent as the intra-assay coefficients of variation (CVs) were <10% (30 replicates on the same day) and the interassay CVs were <14% (10 experiments performed on different days, with 3 replicates each). The fluorescent method therefore represents a reproducible, safe and useful tool to evaluate ChE as an emerging cardiovascular risk factor.

In vivo anti-inflammatory action evaluation of protease inhibitors from Cecropia hololeuca and their protein profile by electrophoretic and spectroscopic techniques

This work describes the electrophoretic and spectroscopic assessment of bioactive proteins from Cecropia hololeuca through SDS-PAGE, CE-UV, FT-IR and NMR, and their anti-inflammatory potential. In vitro assays were performed on murine macrophages J774A.1 and RAW 264.7 cells, and the in vivo anti-inflammatory potential on acute carrageenan edema in mice. The protein extract significantly inhibited the levels of NO, TNF-α, IL-1β and IL-6, and the expression of NF-κB in J774A.1 and RAW 264.7 cells, without causing toxicity to cells. In in vivo and ex vivo assay, the protein extract also reduced the formation of paw edema in animals, and the expression of IL-12 in macerate in the soft tissue of the paw. In the analysis of genotoxicity, the protein extract decreased the frequency of micronucleated erythrocytes when compared to the group treated with Dexamethasone. The analysis by SDS-PAGE, CE-UV, FT-IR and NMR corroborated the presence of proteins in the analyzed samples.

Cecropia hololeuca: A new source of compounds with potential anti-inflammatory action

Our objective is to investigate the phytochemical components, antioxidant capacity and in vitro and in vivo anti-inflammatory action from Cecropia hololeuca bark aqueous extract (AECh). The chemical characterization of AECh was performed through CE-UV, FTIR and NMR Spectroscopy. In vitro assays were performed with the AECh on murine macrophages J774A.1 cells in order to analyse cell viability, NO, TNF-α and IL-1β productions and the in vivo anti-inflammatory potential in acute carrageenan paw oedema in mice. The AECh showed a decrease in the production of NO, TNF-α and IL-1β, without altering the cell viability and reduction of the paw thickness in the 2nd, 3rd and 4th hour. The extract presented 72% free radical scavenging, 0.60% flavonoid content and showed the presence of gallic acid, caffeic acid and catechin as major constituents. The C. hololeuca bark extract showed important antioxidant and anti-inflammatory activity, emphasizing the industrial and pharmacological potential of this plant.

MicroRNA profile datasets of murine macrophages infected with different strains of Leptospira spp

MicroRNAs play an important role in the regulation of immune responses. The influence of epigenetic mechanisms, particularly RNA-mediated post-transcriptional regulation of host immune responses has been proven vital following infections by different pathogens, and bacteria can modulated host miRNAs. Global miRNA expression analysis from macrophages infected in vitro with different strains of Leptospira spp was performed using miRNA 4.1 microarray strips. Leptospirosis is a bacterial zoonosis of global importance, responsible for significant morbidity and mortality worldwide. Despite considerable advances, much is yet to be discovered about disease pathogenicity, particularly in regards to host-pathogen interactions. We present here a high-quality dataset examining the microtranscriptome of murine macrophages J774A.1 following 8h of infection with virulent, attenuated and saprophyte strains of Leptospira. Metadata files were submitted to the Gene Expression Omnibus (GEO) repository.

In Vitro and In Vivo Activity of Peptidomimetic Compounds That Target the Periodontal Pathogen Porphyromonas gingivalis.

ABSTRACTThe interaction of the periodontal pathogen Porphyromonas gingivalis with oral streptococci is important for initial colonization of the oral cavity by P. gingivalis and is mediated by a discrete motif of the streptococcal antigen I/II protein. A synthetic peptide encompassing this motif functions as a potent inhibitor of P. gingivalis adherence, but the use of peptides as topically applied therapeutic agents in the oral cavity has limitations arising from the relatively high cost of peptide synthesis and their susceptibility to degradation by proteases expressed by oral organisms. In this study, we demonstrate the in vitro and in vivo activity of five small-molecule mimetic compounds of the streptococcal peptide. Using a three-species biofilm model, all five compounds were shown to effectively inhibit the incorporation of P. gingivalis into in vitro biofilms and exhibited 50% inhibitory concentrations (IC50s) of 10 to 20 μM. Four of the five compounds also significantly reduced maxillary alveolar bone resorption induced by P. gingivalis infection in a mouse model of periodontitis. All of the compounds were nontoxic toward a human telomerase immortalized gingival keratinocyte cell line. Three compounds exhibited slight toxicity against the murine macrophage J774A.1 cell line at the highest concentration tested. Compound PCP-III-201 was nontoxic to both cell lines and the most potent inhibitor of P. gingivalis virulence and thus may represent a novel potential therapeutic agent that targets P. gingivalis by preventing its colonization of the oral cavity.

N-Acetyl-tryptophan glucoside (NATG) protects J774A.1 murine macrophages against gamma radiation-induced cell death by modulating oxidative stress

Immune system is amongst the most radiosensitive system to radiation-induced cellular and molecular damage. Present study was focused on the evaluation of radioprotective efficacy of a novel secondary metabolite, N-acetyl tryptophan glucoside (NATG), isolated from a radioresistant bacterium Bacillus sp. INM-1 using murine macrophage J774A.1 cells experimental model. Radioprotective efficacy of NATG against radiation-induced DNA damage and apoptosis was estimated using phosphatidyl-serine-externalization Annexin V-PI and Comet assay analysis. Radiation-induced cell death is the outcome of oxidative stress caused by free radicals. Therefore, perturbations in antioxidant enzymes i.e., superoxide dismutase (SOD), catalase, glutathione-s-transferase (GST) and GSH activities in irradiated and NATG pre-treated irradiated J774A.1 cells were studied. Results of the present study demonstrated that NATG pre-treated (0.25µg/ml) irradiated (20Gy) cells showed significant (p < 0.05) reduction in apoptotic cells index at 4-48h as compared to radiation alone cells. Comet assay exhibited significant protection to radiation-induced DNA damage in J774A.1 cells. Significantly shortened DNA tail length, increased % Head DNA contents and lower olive tail moment was observed in NATG pre-treated irradiated cells as compared to radiation alone cells. Further, significant increase in catalase (~ 3.9 fold), SOD (67.52%), GST (~ 1.9 fold), and GSH (~ 2.5 fold) levels was observed in irradiated cells pre-treated with NATG as compared to radiation-alone cells. In conclusion, current study suggested that NATG pre-treatment to irradiated cells enhanced antioxidant enzymes in cellular milieu that may contribute to reduce oxidative stress and decrease DNA damage which resulted to significant reduction in the cell death of irradiated macrophages.

Synthesis, characterization, and antileishmanial activity of neutral N-heterocyclic carbenes gold(I) complexes

A series of five new mononuclear neutral gold(I) complexes containing N-heterocyclic carbenes (NHCs) was synthesized and fully characterized by spectroscopic methods. The X-ray structures of four complexes are presented. These gold(I) complexes together with four other neutral gold(I)-NHC complexes previously described were evaluated in vitro against Leishmania infantum promastigotes and axenic amastigotes. Moreover, their cytotoxicity was assessed on the murine macrophages J774A.1. Except one complex (10), eight gold(I)-NHC-Cl complexes show potent activity against the pathological relevant form of L. infantum amastigote with IC50 in the low micromolar and submicromolar range and five of them exhibit a SI close to 10. The lead-complex 11 displays a very high and selective activity (IC50 = 190 nM, SI = 40.29) and constitutes the best promising gold(I)-based drug of this series.

Differential Regulation of IL-1β and IL-6 Release in Murine Macrophages.

Asbestos and silica (exogenous danger) and adenosine triphosphate (ATP, endogenous danger-signaling molecule) synergistically increase IL-1β release from endotoxin-primed macrophage, which is mediated by NOD-like receptor protein 3 (NLRP3) inflammasome. However, the conversion of pro-IL-1β to its active form seems to depend on the macrophage cell types. In the present study, bone marrow-derived macrophages (BMM) and three murine macrophage cell lines, J774.1, J774A.1, and RAW264.7 were exposed to ATP or fibrous titanium dioxide (FTiO2) in the presence or absence of lipopolysaccharide (LPS), and the concentrations of IL-1β and IL-6 in both cell lysates and in the culture media were measured by immunoblotting to differentiate active form of IL-1β from pro-IL-1β. IL-1β release was synergistically increased when the cells were exposed to both LPS and ATP or FTiO2, while IL-6 was readily released by LPS alone. IL-1β released into the culture medium was pro-IL-1β in J774.1 and RAW264.7, and most of the pro-IL-1β remained inside the cells. In contrast, the active form of IL-1β was released together with pro-IL-1β from J774A.1 and BMM after the co-stimulation. J774A.1 and BMM express apoptosis-associated speck-like protein contains a carboxyl-terminal CARD (ASC) while J774.1 and RAW264.7 do not or only faintly express ASC, and accordingly, caspase-1, which converts pro-IL-1β to its active form, is activated only in J774A.1 and BMM. Collectively, the canonical inflammasome pathway is not activated in J774.1 and RAW264.7, and the apparent synergistical increase of IL-1β in the culture medium mostly reflects the leakage of pro-IL-1β from these cells.

N-acetyl tryptophan glucopyranoside (NATG) as a countermeasure against gamma radiation-induced immunosuppression in murine macrophage J774A.1 cells

Radiation exposure to immune system induces imbalance in cytokines expression involved in Th1/Th2 homeostasis perturbations. In the present study, N-acetyl tryptophan glucoside (NATG), a bacterial secondary metabolite, was evaluated for its possible radioprotective potential to immune system using J774A.1 murine macrophages. In this study, expression of IFN-γ, TNF-α, IL-10, IL-2, IL-12, IL-13 and IL-17A cytokines was analyzed in irradiated and NATG pretreated cells using ELISA assay. Results of the study indicated that irradiated macrophages (NK-1R+ cells) pretreated with NATG showed higher (p < .05) survival at all observed time-intervals (2 h-48 h) as compared to irradiated (20Gy) cells that were not pretreated with NATG. However, NATG pretreatment to irradiated HEK293T cells (that did not express NK-1Receptor) did not provide significant survival, suggesting NK-1R involvement in NATG-mediated radioprotection. Cytokine expression analysis demonstrated that NATG pre-treated plus irradiated J774A.1 murine macrophages exhibited increased IFN-γ levels (∼90%) with significant decrease in TNF-α at 24h as compared to irradiated cells. Further, significant decrease (∼20%) in IL-10 and IL-2 (∼26%) levels was observed in irradiated macrophages pretreated with NATG as compared to only irradiated cells. A sharp improvement in IL-17A (∼92%) and IL-12 (∼116%) expression was observed in irradiated macrophages pretreated with NATG as compared to only irradiated cells. Hence, NATG pre-treatment to irradiated macrophages induced IFN-γ, IL-17A and IL-12 expression, but suppresses TNF-α, IL-10 and IL-2 expressions. Conclusively, NATG pretreatment overcomes radiation-induced Th2 immune response by improving Th1 responsive cytoprotective cytokines IFN-γ, IL-17A and IL-12 in irradiated macrophages possibly by NK-1R antagonistic mechanism, and thus contributes to radioprotection.

Resistance of mitochondrial DNA-depleted cells against oxidized low-density lipoprotein-induced macrophage pyroptosis.

Oxidized low-density lipoprotein (Ox-LDL)-induced macrophage pyroptosis is critical in atherosclerosis inflammation and plaque instability. It has been reported that mitochondrial (mt)DNA-depleted (rho0) cells demonstrate resistance to apoptosis. However, little is known about the susceptibility of rho0 cells to Ox-LDL-induced macrophage pyroptosis. Pyroptosis, a caspase-1-dependent programmed cell death, which compromises membrane integrity, cleaves pro-interleukin (IL)‑1β and pro‑IL‑18 into IL‑1β and IL‑18, respectively and releases damage‑associated molecular pattern molecules, is triggered by a variety of stimuli, including Ox‑LDL. In the present study, the expression levels of cleaved caspase‑1 and IL‑1β in Ox‑LDL‑treated J774A.1 rho0 cells were observed to be significantly decreased when compared with Ox‑LDL‑treated J774A.1 normal cells. Furthermore, J774A.1 rho0 cells exhibited a significant reduction in the ratios of dead cells and lactate dehydrogenase release following Ox‑LDL stimulation compared with the J774A.1 normal cells. In addition, the loss of mtDNA did not influence Ox‑LDL‑induced cholesterol accumulation in J774A.1 rho0 cells, which was observed by Oil RedO staining and CHOD‑PAP assay. Finally, J774A.1 rho0 cells exhibited reduced reactive oxygen species (ROS) production and were capable of maintaining the mitochondrial membrane potential following Ox‑LDL treatment. Thus, the results indicate that the loss of mtDNA potentially rendered murine macrophage J774A.1 resistant to Ox‑LDL‑induced pyroptosis by mitigating NACHT, LRR and PYD domains-containing protein3 inflammasome activation through reducing ROS production. In addition, mtDNA depletion did not interrupt Ox-LDL-induced intracellular lipid accumulation and continued to maintain the mitochondrial membrane potential.

Oral Administration of Polymyxin B Modulates the Activity of Lipooligosaccharide E. coli B against Lung Metastases in Murine Tumor Models.

IntroductionPolymyxin B (PmB) belongs to the group of cyclic peptide antibiotics, which neutralize the activity of LPS by binding to lipid A. The aim of this study was to analyze the effect of PmB on the biological activity of lipooligosaccharide (LOS E. coli B,rough form of LPS) in vitro and in experimental metastasis models.ResultsCultures of murine macrophage J774A.1 cells and murine bone marrow-derived dendritic cells (BM-DC) stimulated in vitro with LOS and supplemented with PmB demonstrated a decrease in inflammatory cytokine production (IL-6, IL-10, TNF-α) and down-regulation of CD40, CD80, CD86 and MHC class II molecule expression. Additionally, PmB suspended in drinking water was given to the C57BL/6 mice seven or five days prior to the intravenous injection of B16 or LLC cells and intraperitoneal application of LOS. This strategy of PmB administration was continued throughout the duration of the experiments (29 or 21 days). In B16 model, statistically significant decrease in the number of metastases in mice treated with PmB and LOS (p<0.01) was found on the 14th day of the experiments, whereas the most intensive changes in surface-antigen expression and ex vivo production of IL-6, IL-1β and TNF-α by peritoneal cells were observed 7 days earlier. By contrast, antigen expression and ex vivo production of IL-6, IL-10, IFN-γ by splenocytes remained relatively high and stable. Statistically significant decrease in LLC metastases number was observed after the application of LOS (p<0.01) and in the group of mice preconditioned by PmB and subsequently treated with LOS (LOS + PmB, p<0.01).ConclusionsIn conclusion, prolonged in vivo application of PmB was not able to neutralize the LOS-induced immune cell activity but its presence in the organism of treated mice was important in modulation of the LOS-mediated response against the development of metastases.

Trimethylamine-N-Oxide Treatment Induces Changes in the ATP-Binding Cassette Transporter A1 and Scavenger Receptor A1 in Murine Macrophage J774A.1 cells.

Recently, trimethylamine N-oxide was introduced as a risk factor for atherosclerosis in terms of helping foam cell formation and worsening atherosclerosis complications. The present study was performed to investigate whether/how trimethylamine N-oxide is involved in regulation of ATP-binding cassette transporter A1 and scavenger receptor A1 in macrophages at both mRNA and protein levels. Murine macrophage J774A.1 cells were treated with micromolar concentrations of trimethylamine N-oxide and 4-phenylbutyric acid, a chemical chaperon, for different time intervals. Tunicamycin was also used as a control for induction of endoplasmic reticulum stress. Similar to tunicamycin, trimethylamine N-oxide increased scavenger receptor A1 in all treatment periods, whereas ATP-binding cassette transporter A1 was only reduced 24h post-treatment with trimethylamine N-oxide at both mRNA and protein levels. In contrast, 4-phenylbutyric acid failed to induce such changes in either scavenger receptor A1 or ATP-binding cassette transporter A1. The results of this study, in agreement with previous studies, confirm the mechanistic role of trimethylamine N-oxide in the upregulation of scavenger receptor A1, which potentially can promote its proatherogenic role. The results also showed downregulation of ATP-binding cassette transporter A1 in trimethylamine N-oxide treated macrophages which may indicate another possible proatherosclerotic mechanism for foam cell formation.

Synthesis, characterization, and antileishmanial activities of gold(I) complexes involving quinoline functionalized N-heterocyclic carbenes

A series of new mononuclear cationic or neutral gold(I) complexes containing quinoline functionalized N-heterocyclic carbene(s) (NHC(s)) were synthesized and fully characterized by spectroscopic methods. The X-ray structures of two key compounds are presented. Proligands and their corresponding gold(I) complexes together with previously described silver(I) and gold(I) bis(NHC-quinoline) and gold(I) bis(NHC-methylbipyridine) complexes were evaluated in vitro towards Leishmania infantum. In parallel, the in vitro cytotoxicity of these molecules was assessed on the murine macrophages J774A.1. All gold(I) compounds show potent antileishmanial activity against L. infantum promastigotes and three of them are also efficient against L. infantum intracellular amastigotes. Structure-activity and toxicity relationships enables to evidence a lead-compound (6) displaying both a high activity and a good selectivity index.

In vitro anti-inflammatory, mutagenic and antimutagenic activities of ethanolic extract of Clerodendrum paniculatum root.

Clerodendrum paniculatum L. (Family Verbenaceae) has been used as an antipyretic and anti-inflammatory drug in traditional Thai medicine. This present study investigated the in vitro anti-inflammatory, mutagenic and antimutagenic activities of the ethanolic extract of C. paniculatum (CPE) dried root collected from Sa Kaeo Province of Thailand. Murine macrophage J774A.1 cells were stimulated by lipopolysaccharide (LPS) to evaluate nitric oxide (NO), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) production in the anti-inflammatory test while the mutagenic and antimutagenic potential was performed by the Ames test. The outcome of this study displayed that the CPE root significantly inhibited LPS-induced NO, TNF-α, and PGE2 production in macrophage cell line. In addition, the CPE root was not mutagenic toward Salmonella typhimurium strain TA98 and TA100 with and without nitrite treatment. Moreover, it inhibited the mutagenicity of nitrite treated 1-aminopyrene on both strains. The findings suggested the anti-inflammatory and antimutagenic potentials of CPE root.

English

Plasmid pRST98 is a hybrid antibiotic resistance-virulence plasmid isolated fromSalmonella enterica serovar Typhi (S. Typhi). Previously, we transferred pRST98 into attenuated Salmonella enterica serovar Typhimurium (S. typhimurium) strain RIA to create a transconjugant pRST98/RIA and indicated that pRST98 could enhance apoptosis of infected murine macrophages J774A.1. However, S. typhimurium-mice interaction cannot reflect all characteristics of S. typhi-human interaction. The present study Human-derived macrophage-like cell line THP-1 was infected with wild-type (ST8), pRST98-deletion (ST8-ΔpRST98) and complemented (ST8-c-pRST98) S. typhi strains to gain more precise and further insight into the role of pRST98 in interaction between S. typhi and human macrophage. Macrophage apoptosis, caspase-3 activity and nitric oxide (NO) production were assayed. The results demonstrated that macrophages infected with ST8 and ST8-c-pRST98 displayed more extensive apoptosis than those infected with ST8-ΔpRST98. Further studies showed that pRST98 could increase caspase-3 activity and suppress NO production. Pretreated macrophages with NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME) enhanced S. Typhi-induced macrophage apoptosis. The research data indicate that the plasmid pRST98 can enhance caspase-3 mediated macrophage apoptosis by suppressing NO production. Key words: Salmonella enterica serovar Typhi, plasmid pRST98, macrophage, apoptosis, nitric oxide, caspase-3 activity.