RESUMO
These experiments aimed to understand the relationship between interleukin 10 (IL10), the IL10 receptor subunits, and progesterone (P4) at the time of parturition. We hypothesized that there is a biologic connection between IL10 and P4, supporting an immunomodulatory mechanism for the onset of labor. Using samples from control and P4-treated pregnant mice, we assessed the production of IL10 and its receptor subunits (IL10Rα and IL10Rß) in gestational tissues. After preliminary studies, P4-treated pregnant mice were compared with controls to assess for differences in IL10 and IL10 receptor subunit expression throughout gestation. To investigate the contribution of the P4 receptor at the onset of labor, we performed timed studies on pregnant mice after treatment with RU486. Samples collected included placentas, placentation sites, and maternal livers. IL10, IL10Rα, and IL10Rß levels were measured in homogenized tissue using ELISA assays; the cytokine results were normalized for homogenate protein concentration. Control mice delivered on gd 18-19, and P4 treatment prevented parturition to beyond gd 20, as expected. In treated mice, P4 not only prevented the anticipated nadir of IL10 at term, but maintained elevated levels of IL10 through gd 20 (p < 0.05). P4 also reversed the anticipated decrease of the IL10Rα, which was increased in P4-treated mice (p < 0.05). Treatment with RU486 did not modulate the expression of IL10 or IL10Rα, but showed a significant decrease in the level of IL10Rß (p < 0.05). Progesterone functions at least in part through the IL10 signaling pathway to prolong gestation.
Assuntos
Subunidade alfa de Receptor de Interleucina-10/metabolismo , Subunidade beta de Receptor de Interleucina-10/metabolismo , Interleucina-10/metabolismo , Parto/metabolismo , Progesterona/metabolismo , Animais , Feminino , Camundongos Endogâmicos C57BL , Parto/efeitos dos fármacos , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Progesterona/administração & dosagem , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
INTRODUCTION: Cell-free "fetal" DNA is released from the placenta. Because the fetal membranes also arise from the trophectoderm layer of the blastocyst, these studies sought to test the hypothesis that the membranes also release cell-free DNA (cfDNA). METHODS: Fetal membranes were harvested from pregnant CD-1 mice and cultured in 12-well plates containing media alone or with staurosporine and thapsigargin (apoptosis stimulators), Q-VD-OPh (caspase inhibitor), Trolox (vitamin E analog), and lipopolysaccharide and tumor necrosis factor α (TNFα; inflammatory mediators). The cfDNA in the media was extracted, quantified, and normalized for tissue weight. Media was used for a lactate dehydrogenase (LDH) assay. Membrane homogenates were used to assess activated caspase levels and the expression of DNA fragmentation factor B (DFFB) and BAX proteins. 5-Methylcytosine was assessed using a 5-mC DNA enzyme-linked immunosorbent assay. The cfDNA was used to stimulate interleukin 6 (IL6) release by J774A.1 mouse macrophage cells. RESULTS: Increased cfDNA release at 6 and 21 hours occurred in parallel with increasing LDH levels. The cfDNA concentrations were significantly suppressed by Q-VD-OPh and Trolox and increased by thapsigargin and TNFα. Increased caspase activity was suppressed by Q-VD-OPh and increased by TNFα, thapsigargin, and staurosporine. The expression of BAX and DFFB proteins significantly increased by 21 hours. 5-Methylcytosine levels were significantly lower in fetal membranes and placentas and below detectable in the cfDNA released by the explants. The cfDNA-stimulated IL6 release by macrophage cells was suppressed by chloroquine, a Toll-like receptor 9 (TLR9) inhibitor. CONCLUSIONS: These studies have confirmed cfDNA release by the mouse fetal membranes; cfDNA was markedly hypomethylated and a robust stimulator of TLR9.
Assuntos
Ácidos Nucleicos Livres/metabolismo , Membranas Extraembrionárias/metabolismo , Tapsigargina/farmacologia , 5-Metilcitosina/análise , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Inibidores de Caspase/farmacologia , Ácidos Nucleicos Livres/isolamento & purificação , Meios de Cultivo Condicionados/química , Membranas Extraembrionárias/química , Feminino , Camundongos , Placenta/química , Gravidez , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: The fetal fraction of cell-free DNA (cfDNA) in maternal plasma is decreased in obese women. The underlying mechanism is not well understood. The amount of cfDNA released from the placenta has not been directly examined in maternal obesity. OBJECTIVE: We sought to quantify release of cfDNA from the placenta and fetal membranes in maternal diet-induced obesity using explant cultures in an established mouse model. STUDY DESIGN: C57BL6/J females were fed either 60% high-fat diet or 10% fat-matched control diet for 14 weeks prepregnancy and throughout gestation. Placentas and fetal membranes were collected on e18 and randomly allocated to time 0-, 1-, or 6-hour culture times. The CfDNA was isolated from culture media, quantified, and normalized to tissue weight. RESULTS: Placentas from obese dams released significantly less cfDNA compared to those of lean dams at time 0 (45.8 ± 4.3 ng/mg vs 65.6 ± 7.9 ng/mg, P = .02). Absolute cfDNA levels increased with longer placental culture, with no significant differences between obese and lean dams at 1 and 6 hours. Membranes released significantly less cfDNA than did placentas at every time point. CONCLUSIONS: Maternal obesity is associated with decreased release of cfDNA from the placenta compared to lean controls immediately after tissue harvest. This may provide an alternative explanation for the lower fetal fraction of cfDNA noted in maternal obesity.
Assuntos
Ácidos Nucleicos Livres/metabolismo , Dieta Hiperlipídica , Desenvolvimento Fetal/fisiologia , Obesidade Materna/metabolismo , Placenta/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Camundongos , GravidezRESUMO
INTRODUCTION: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.
Assuntos
DNA/metabolismo , Inflamação/metabolismo , Parto , Placenta/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Feminino , Feto , Interleucina-6/metabolismo , Macrófagos/metabolismo , Camundongos , Gravidez , Células RAW 264.7 , Telômero/metabolismoRESUMO
Although suggested that "fetal" cell-free DNA (cfDNA) is derived from trophoblast cells, the exact origin is unclear. The studies in this report sought to demonstrate that placental tissue releases cfDNA in parallel with cell death, that the size range of cfDNA is similar to that found in maternal plasma, and that the cfDNA fragments are able to stimulate a proinflammatory cytokine response. Placentas were harvested from near term pregnant CD-1 mice and cultured in DMEM/Ham's F12/FBS media in 8% or 21% O2. After centrifugation to remove cells and cellular debris, the cfDNA was extracted from the media and quantified by DNA spectrophotometry. The cfDNA fragments were sized using a 1.5% TAE gel. Cell death was quantified by lactate dehydrogenase assay; and tissue homogenates were used to quantify caspase activity and BAX expression. Cultured RAW-264.7 macrophage cells were used to determine IL6 stimulation by cfDNA. The cfDNA levels released in 8% O2 (placental normoxia) were not significantly different from explants cultured in 21% O2 (placental hyperoxia). The cfDNA fragments ranged in size from < 100 -< 400 bp. The cfDNA release increased when cultured with LPS, whereas it decreased with trolox (vitamin E analog). Explant release of cfDNA increased in parallel with cell death. The cfDNA release and cell death of trophoblast appears to involve components of the apoptosis signaling pathway as suggested by LPS enhancement of placental caspase activity, suppression of cfDNA release by a pan-caspase inhibitor and the trend toward increased Bax protein expression. Studies with cultured macrophage cells confirmed the ability of cfDNA to stimulate an IL6 response. In summary, these studies have confirmed the ability of placental tissue to release significant amounts of cfDNA, a phenomenon that appears to be mediated, at least in part, by apoptosis; and that the cfDNA released by the placental explants is able to stimulate a significant proinflammatory response. Thus, these studies provide support for the hypothesis that cell-free fetal DNA released by placental tissue potentially plays a mechanistically important role during the events leading to the onset of parturition.
Assuntos
Lipopolissacarídeos/farmacologia , Placenta/metabolismo , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-6/metabolismo , Camundongos , Gravidez , Células RAW 264.7 , Proteína X Associada a bcl-2/metabolismoRESUMO
We investigated the effects of 25Mg-Porphyrin-Fullerene nanoparticles, (25MgPMC16) smart ferroporphyrin nanoparticles, on PC12 cells exposed to oxygen-glucose deprivation/reperfusion. In order to explore its effect on cells under oxygen-glucose deprivation conditions, the cultures were pretreated with 25MgPMC16 24 hours prior to oxygen-glucose deprivation/reperfusion. To initiate the oxygen-glucose deprivation/reperfusion, the cell culture medium was replaced with a glucose-free medium and the cells were transferred to a humidified incubation chamber in a mixture of 95% N2 and 5% CO2 at 37° C for 30, 60 and 120 min. Cell viability was assessed by MTT assay. Exposure of PC12 cells to 30, 60 and 120 min oxygen-glucose deprivation significantly decreased the cell viability. Pretreatment of the cultures with 25MgPMC16 significantly increased cell viability in a concentration-dependent manner. Pretreatment, the cultures with MK-801 (10 µM), a non-competitive NMDA antagonist, has attenuated the cell death after 30 min oxygen-glucose deprivation. We concluded that 25MgPMC16 could protect PC12 cells against oxygen-glucose deprivation/reperfusion-induced cell injury in a concentration-dependent manner. That could be due to the effect of 25MgPMC16 on ATP synthesis and the antioxidant effects of its components.