Apoptosis In Mouse Macrophages Research Articles

Molecular Characteristics and Potent Immunomodulatory Activity of Fasciola hepatica Cystatin.

Cystatin, a cysteine protease inhibitor found in many parasites, plays important roles in immune evasion. This study analyzed the molecular characteristics of a cystatin from Fasciola hepatica (FhCystatin) and expressed recombinant FhCystatin (rFhcystatin) to investigate the immune modulatory effects on lipopolysaccharide-induced proliferation, migration, cytokine secretion, nitric oxide (NO) production, and apoptosis in mouse macrophages. The FhCystatin gene encoded 116 amino acids and contained a conserved cystatin-like domain. rFhCystatin significantly inhibited the activity of cathepsin B. rFhCystatin bound to the surface of mouse RAW264.7 cells, significantly inhibited cell proliferation and promoted apoptosis. Moreover, rFhCystatin inhibited the expression of cellular nitric oxide, interleukin-6, and tumor necrosis factor-α, and promoted the expression of transforming growth factor-β and interleukin-10. These results showed that FhCystatin played an important role in regulating the activity of mouse macrophages. Our findings provide new insights into mechanisms underlying the immune evasion and contribute to the exploration of potential targets for the development of new drug to control F. hepatica infection.

Targeting Fas-associated protein with death domain gene with chitosan-lipid nanoparticles inhibits coronary heart disease progression in mice

Cell apoptosis or programmed cell death is a physiological phenomenon during the development of the body; however, under certain pathological conditions, insufficient, or excessive apoptosis can cause tissue damage and physiological dysfunction. This study investigated the inhibition of oxidized low-density lipoprotein (oxLDL)-induced macrophage apoptosis in mice with coronary heart disease by regulating the target Fasassociated protein with death domain (FADD) gene. We used Western blotting to measure the effect of oxLDL on mouse macrophages and observe the effects of adding simvastatin, 3-methyladenine (3-MA), self-blocking, and other drugs. The results indicated that the target FADD gene played an important role in inhibiting lactate dehydrogenase release by 13%, in the process of oxLDL-induced lipid aggregation in macrophages. The EC50 observed by dual acridine orange/ethidium bromide fluorescence-staining accounts for the proportion of FADD being reduced by 32%, indicating that the target FADD gene exhibited a significant inhibitory effect on oxLDL-induced macrophage apoptosis in coronary heart disease. This study used a novel chitosan-lipid nanoparticle as a gene carrier to significantly improve efficacy. Therefore, targeting the FADD gene using novel chitosan-lipid nanoparticles to deliver siRNA could be a potential clinical treatment in coronary heart disease.

Effects of miR-124-3p regulation of the p38MAPK signaling pathway via MEKK3 on apoptosis and proliferation of macrophages in mice with coronary atherosclerosis.

Atherosclerosis (AS) is the main cause of myocardial infarction and stroke. Macrophage apoptosis in the early stages can attenuate lesions, while in the late stage it is associated with AS plaque rupture. To explore the effects of miR-124-3p regulation of the p38MAPK signaling pathway via the MEKK3 gene on the apoptosis and proliferation of macrophages in mice with coronary AS. Fifty male apolipoprotein E (ApoE) -/- mice were equally assigned to a normal group and a coronary AS group. In the AS group, the mice were given a high-fat diet to establish a coronary AS model. The macrophages of the mice were isolated for culture and divided into 7 groups: normal, negative control (NC), control, miR-124-3p mimic, miR-124-3p inhibitor, si-MEKK3, and miR-124-3p inhibitor+si-MEKK3. Compared with the normal group, the AS group had lower expression levels of miR-124-3p and higher expression levels of MEKK3 and p-p38MAPK in the coronary artery tissue and peritoneal macrophages (all p < 0.050). We found that miR-124-3p could negatively regulate MEKK3 expression. Compared with the control group, the miR-124-3p mimic group and si-MEKK3 group had greater cell apoptosis rates and Bax levels, weaker cell proliferation and invasion abilities, slower cell cycle progression, and lower PCNA and Bcl-2 levels (all p < 0.050). This trend was also displayed in the miR-124-3p inhibitor+si-MEKK3 group when compared with the miR-124-3p inhibitor group, and in the si-MEKK3 group when compared with the miR-124-3p inhibitor+si-MEKK3 group (all p < 0.050). miR-124-3p overexpression can downregulate MEKK3 expression and inhibit the expression of the p38MAPK signaling pathway, thereby inhibiting macrophage proliferation and promoting macrophage apoptosis in mice with coronary AS.

Evaluation of antibacterial and cytotoxic activity of Artemisia nilagirica and Murraya koenigii leaf extracts against mycobacteria and macrophages

BackgroundArtemisia nilagirica (Asteraceae) and Murraya koenigii (Rutaceae) are widely distributed in eastern region of India. Leaves of Artemisia nilagirica plant are used to treat cold and cough by the local tribal population in east India. Murraya koenigii is an edible plant previously reported to have an antibacterial activity. Pathogenic strains of mycobacteria are resistant to most of the conventional antibiotics. Therefore, it is imperative to identify novel antimycobacterial molecules to treat mycobacterial infection.MethodsIn this study, ethanol, petroleum ether and water extracts of Artemisia nilagirica and Murraya koenigii were tested for antibacterial activity against Mycobacterium smegmatis and Mycobacterium bovis BCG in synergy with first line anti-tuberculosis (TB) drugs, and for cytotoxic activities on mouse macrophage RAW264.7 cells. Antibacterial activity was determined by colony forming unit (CFU) assay. Intracellular survival assay was performed by infecting RAW264.7 cells with M. smegmatis before and after treatment with plant extracts. Cytotoxity was checked by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. Genotoxicity was studied by DAPI staining and COMET assay using mouse macrophage RAW264.7 cell line. Cell apoptosis was checked by Annexin-V/FITC dual staining method. Reactive oxygen species and nitric oxide production was checked by DCFH staining and Griess reagent, respectively.ResultsEthanol extracts of A. nilagirica (IC50 300 μg/ml) and M. koenigii (IC50 400 μg/ml) were found to be more effective against Mycobacterium smegmatis as compared to petroleum ether and water extracts. M. koenigii extract showed maximum activity against M. bovis BCG in combination with a first line anti-TB drug rifampicin. M. koenigii leaf extract also exerted more cytototoxic (IC50 20 μg/ml), genotoxic and apoptosis in mouse macrophage RAW 264.7 cell line. Treatment of mouse macrophages with A. nilagirica extract increased intracellular killing of M. smegmatis by inducing production of reactive oxygen species and nitric oxide.ConclusionsEthanol extracts of A. nilagirica and M. koenigii were found to be more effective against mycobacteria. As compared to A. nilagirica, M. koenigii ethanol extract exhibited significant synergistic antibacterial activity against M. smegmatis and M. bovis BCG in combination with anti-tuberculosis drug rifampicin, and also showed increased cytotoxicity, DNA damage and apoptosis in mouse macrophages.

Resin monomer-induced differential activation of MAP kinases and apoptosis in mouse macrophages and human pulp cells

Triethylene glycol dimethacrylate (TEGDMA) is a resin monomer which is released from polymerized dental composite materials. It induced apoptosis in various target cells or inhibition of LPS-induced cytokine production in cells of the immune system after prolonged exposure. In these tissues, mitogen-activated protein kinases (MAPK) regulate signal transduction pathways that support cell survival and cytokine synthesis. The time-dependent regulation of MAPK as well as their linkage to the induction of apoptosis and cytokine release under the influence of resin monomers is unknown. It was the aim of the present study to investigate the kinetics of the up- or down-regulation of the MAPK p38, JNK, and ERK1/2, the induction of apoptosis and cytokine release in RAW264.7 mouse macrophages and human pulp-derived cells. ERK1/2, p38 and JNK were differentially activated by phosphorylation in the presence of lipopolysaccharide (0.1 μg/ml; LPS), a known inducer of MAPK activity, and TEGDMA (3 m m) as detected by Western blotting. In macrophages, ERK1/2 was activated about 6-fold by LPS, while no activation was observed in the presence of TEGDMA after 15 and 30 min. A slight activation of p38 was detected in cell cultures after short exposure to TEGDMA (30 min), but activated JNK was identified after LPS stimulation only. After a long 24 h exposure period, ERK1/2 and p38 were strongly activated by LPS, a combination of LPS/TEGDMA, and TEGDMA alone (15–20-fold). In human pulp-derived cells, ERK1/2 was phosphorylated after exposure to TEGDMA up to 2 h, and sustained activation of ERK1/2 as well as p38 (12–15-fold) was detected after prolonged exposure for 24 h. The LPS-induced, time-related increase in the secretion of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) as well as the anti-inflammatory IL-10 was instantaneously inhibited by TEGDMA in mouse macrophages. In parallel, the percentage of cells in macrophage cultures in the stage of apoptosis and necrosis increased with exposure period. Yet, in contrast to the inhibition of cytokine release, apoptosis and necrosis caused by LPS and TEGDMA was a late response in both mouse macrophages and human pulp-derived cells. From these data it appears as if MAPK activation, inhibition of cytokine release and the induction of apoptosis and necrosis by TEGDMA are tightly related. The direct causal correlation of these phenomena, however, requires further investigation.

Apoptosis induced by lipid-associated membrane proteins fromMycoplasma penetransis mediated by nuclear factor κB activation in mouse macrophage

Mycoplasma penetrans was shown to be involved in alteration of several eukaryotical cells functions and a causative agent in urogenital infectious diseases. Lipid-associated membrane proteins (LAMPs) may be responsible for the pathogenicity of some mycoplamas. In this study, we investigated whether M. penetrans LAMPs have pathogenic potential by inducing apoptosis in mouse macrophages. As analyzed by annexin-V - fluorescein isothiocyanate staining, significant early- and late-stage apoptosis was induced in M. penetrans LAMPs-challenged mouse macrophages. And agarose gel electrophoresis of the DNA of M. penetrans LAMPs-challenged cells revealed a ladder-like pattern of migration of DNA indicative of apoptosis. The possible molecular mechanisms responsible for the induction of apoptosis were also investigated by characterizing the activation of nuclear transcription factor kappaB (NFkappaB). NFkappaB was activated and translocated into the nucleus in mouse macrophages stimulated by M. penetrans LAMPs. The activation of NFkappaB and M. penetrans LAMPs-induced apoptosis in mouse macrophages was partially inhibited by the NFkappaB-specific inhibitor pyrrolidine dithiocarbamate. Thus, this study demonstrates that M. penetrans LAMPs may be an important etiological factor owing to their ability to induce apoptosis in mouse macrophages, which is probably mediated through the activation of NFkappaB.

TLR-dependent Bim phosphorylation in macrophages is mediated by ERK and is connected to proteasomal degradation of the protein

The pro-apoptotic Bcl-2 homology domain 3-only protein Bim has been shown to play an important role in immune cell homeostasis and various forms of apoptosis in the immune system. Bim is expressed in most immune cells, and regulation of Bim activity can occur on both transcriptional and post-translational levels. Toll-like receptor (TLR) stimulation has previously been shown to increase Bim expression and to cause Bim phosphorylation in the absence of apoptosis in mouse macrophages. Here we identify extracellular signal-regulated kinase as the major kinase responsible for TLR-dependent Bim phosphorylation. Three TLR-dependent serine phosphorylation sites, S55, S65 and S100, on mouse Bim were identified, two of them unique to the splice form Bim(EL) and one also present on Bim(L). A Bim mutant defective in these three phosphorylation sites showed slightly enhanced pro-apoptotic activity, which might indicate a protective effect of Bim phosphorylation in this system. Phosphorylation did not alter the association of Bim protein with the microtubule cytoskeleton. However, TLR-mediated phosphorylation led to accelerated degradation of Bim via the proteasome. Thus, TLR stimulation of macrophages can regulate Bim levels in opposing ways, namely by transcriptional induction and by phosphorylation-dependent degradation of the protein.

Inhibition of macrophage apoptosis in mice infected with intracellular protozoan parasites by 65 kilodalton heat shock protein

Inhibition of macrophage apoptosis in mice infected with intracellular protozoan parasites by 65 kilodalton heat shock protein

Inhibition of macrophage apoptosis in mice infected with intracellular protozoan parasites by 65 kilodalton heat shock protein

Inhibition of macrophage apoptosis in mice infected with intracellular protozoan parasites by 65 kilodalton heat shock protein