RESUMO
OBJECTIVES: Systemic sclerosis (SSc) is a rare rheumatic disease characterized by vascular damage, dysregulated immune response, and fibrosis. Interleukin-11 (IL-11) is upregulated in SSc. This study aimed to investigate the pathological and therapeutic role of the IL-11 trans-signaling pathway in SSc. METHODS: Plasma IL-11 level was evaluated in 32 patients with SSc and 15 healthy controls, while the expression levels of ADAM10, ADAM17, IL-11, IL-11 Rα, or IL-11 co-stained with CD3 or CD163 in the skin of SSc patients and healthy controls were analyzed. Fibroblasts were treated with IL-11 and ionomycin to evaluate the profibrotic effect of IL-11 trans-signaling pathway. TJ301 (sgp130Fc) and WP1066 (a JAK2/STAT3 inhibitor) intervention groups were set up to investigate the antifibrotic effect of targeting IL-11. RESULTS: Levels of plasma IL-11 were extremely low in most SSc patients and healthy controls. In contrast, levels of IL-11, IL-11 Rα, and ADAM10, but not ADAM17, were significantly elevated in the skin of SSc patients. Moreover, the numbers of IL-11+ CD3+ cells and IL-11+ CD163+ cells were increased in the skin of SSc patients. Besides, IL-11 and ADAM10 were also elevated in the skin and pulmonary of bleomycin-induced SSc mouse. Fibroblasts co-stimulated with IL-11 and ionomycin showed increased expression of COL3 and phosphorylation of STAT3, which could be inhibited by TJ301 or WP1066. TJ301 also ameliorated skin and lung fibrosis in BLM-induced SSc mouse. CONCLUSIONS: IL-11 induces fibrosis in SSc by regulating the trans-signaling pathway. Blockage of sgp130Fc or inhibition of the JAK2/STAT3 pathway could ameliorate the profibrotic effect of IL-11.
Assuntos
Interleucina-11 , Escleroderma Sistêmico , Humanos , Animais , Camundongos , Interleucina-11/efeitos adversos , Interleucina-11/metabolismo , Ionomicina/efeitos adversos , Ionomicina/metabolismo , Fibrose , Escleroderma Sistêmico/tratamento farmacológico , Pele/patologia , Transdução de Sinais , Fibroblastos/patologia , Janus Quinase 2/efeitos adversos , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
This study aimed to combine cellular and molecular analyses for better detail the effects of various stresses on a sentinel species of freshwater invertebrate. For this purpose, the hemocytes of the zebra mussel, Dreissena polymorpha, were exposed to different stresses at two different intensities, high or low: chemical (cadmium and ionomycin), physical (ultraviolet B), or biological ones (Cryptosporidium parvum and Toxoplasma gondii). After exposure, flow cytometry and droplet digital PCR analyses were performed on the same pools of hemocytes. Several responses related to necrosis, apoptosis, phagocytosis, production of nitric oxide and expression level of several genes related to the antioxidant, detoxification and immune systems were evaluated. Results showed that hemocyte integrity was compromised by both chemical and physical stress, and cellular markers of phagocytosis reacted to ionomycin and protozoa. While cadmium induced oxidative stress and necrosis, ionomycin tends to modulate the immune response of hemocytes. Although both biological stresses led to a similar immune response, C. parvum oocysts induced more effects than T. gondii, notably through the expression of effector caspases gene and an increase in hemocyte necrosis. This suggests different management of the two protozoa by the cell. This work provides new knowledge of biomarkers in the zebra mussel, at both cellular and molecular levels, and contributes to elucidate the mechanisms of action of different kinds of stress in this species.
Assuntos
Cádmio/efeitos adversos , Cryptosporidium parvum/fisiologia , Dreissena/imunologia , Hemócitos , Ionomicina/efeitos adversos , Toxoplasma/fisiologia , Raios Ultravioleta/efeitos adversos , Animais , Biomarcadores/análise , Citometria de Fluxo , Hemócitos/efeitos dos fármacos , Hemócitos/parasitologia , Hemócitos/efeitos da radiação , Reação em Cadeia da Polimerase , Estresse Fisiológico/imunologiaRESUMO
Artificial oocyte activation (AOA) has shown to improve fertility in severe male infertility following intracytoplasmic sperm insemination (ICSI). However, the effect of AOA on the health status of children has not been studied. This pilot historical cohort study aims to evaluate physical and mental health of 79 and 89 children from 275 and 406 couples undergoing ICSI-AOA using ionomycin and conventional ICSI, respectively. The outcomes assessed were clinical pregnancy, abortion, type of delivery, and health of children (major birth defect, mental and behavior status). No significant differences were observed between the ICSI-AOA and the ICSI groups for these parameters, and the rate of major birth defects were not significantly different between the 2 groups. In this study, AOA has not imposed a greater risk on physical and mental health of children born through AOA, but for such a solid conclusion, further trails with higher number of cases are required and conclusions drawn are limited to this study.
Assuntos
Ionóforos de Cálcio/uso terapêutico , Sinalização do Cálcio/efeitos dos fármacos , Nível de Saúde , Infertilidade Masculina/terapia , Ionomicina/uso terapêutico , Saúde Mental , Oócitos/efeitos dos fármacos , Injeções de Esperma Intracitoplásmicas , Fatores Etários , Ionóforos de Cálcio/efeitos adversos , Comportamento Infantil , Desenvolvimento Infantil , Pré-Escolar , Feminino , Humanos , Lactente , Infertilidade Masculina/diagnóstico , Infertilidade Masculina/fisiopatologia , Ionomicina/efeitos adversos , Masculino , Oócitos/metabolismo , Projetos Piloto , Gravidez , Complicações na Gravidez/etiologia , Fatores de Risco , Injeções de Esperma Intracitoplásmicas/efeitos adversos , Resultado do TratamentoRESUMO
Parthenotes have been proposed as a source of embryonic stem cells but they lack the centriole which is inherited through the sperm in all mammalian species, except for rodents. We investigated the centrosome of parthenotes and parthenogenetic embryonic stem cells using parthenogenetic and biparental pig pre-implantation embryos, human and pig parthenogenetic and biparental embryonic stem cells, sheep fibroblasts derived from post implantation parthenogenetic and biparental embryos developed in vivo. We also determined the level of aneuploidy in parthenogenetic cells. Oocytes of all species were activated using ionomycin and 6-dimethylaminopurine (6-DMAP). Over 60% of parthenogenetic blastomeres were affected by an excessive number of centrioles. Centrosome amplification, was observed by microscopical and ultrastructural analysis also in parthenogenetic cell lines of all three species. Over expression of PLK2 and down regulation of CCNF, respectively involved in the stimulation and inhibition of centrosome duplication, were present in all species. We also detected down regulation of spindle assembly checkpoint components such as BUB1, CENPE and MAD2. Centrosome amplification was accompanied by multipolar mitotic spindles and all cell lines were affected by a high rate of aneuploidy. These observations indicate a link between centrosome amplification and the high incidence of aneuploidy and suggest that parthenogenetic stem cells may be a useful model to investigate how aneuploidy can be compatible with cell proliferation and differentiation.
Assuntos
Aneuploidia , Blastômeros/metabolismo , Centrossomo/metabolismo , Instabilidade Cromossômica , Partenogênese , Fuso Acromático/metabolismo , Animais , Blastômeros/patologia , Ionóforos de Cálcio/efeitos adversos , Ionóforos de Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Centrossomo/patologia , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Ionomicina/efeitos adversos , Ionomicina/farmacologia , Proteínas Mad2 , Oócitos/metabolismo , Oócitos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Ovinos , Fuso Acromático/patologia , SuínosRESUMO
In previous studies, we have shown that mefloquine disrupts calcium homeostasis in neurons by depletion of endoplasmic reticulum (ER) stores, followed by an influx of external calcium across the plasma membrane. In this study, we explore two hypotheses concerning the mechanism(s) of action of mefloquine. First, we investigated the possibility that mefloquine activates non-N-methyl-d-aspartic acid receptors and the inositol phosphate 3 (IP3) signaling cascade leading to ER calcium release. Second, we compared the disruptive effects of mefloquine on calcium homeostasis to those of ionomycin in neuronal and nonneuronal cells. Ionomycin is known to discharge the ER calcium store (through an undefined mechanism), which induces capacitative calcium entry (CCE). In radioligand binding assays, mefloquine showed no affinity for the known binding sites of several glutamate receptor subtypes. The pattern of neuroprotection induced by a panel of glutamate receptor antagonists was dissimilar to that of mefloquine. Both mefloquine and ionomycin exhibited dose-related and qualitatively similar disruptions of calcium homeostasis in both neurons and macrophages. The influx of external calcium was blocked by the inhibitors of CCE in a dose-related fashion. Both mefloquine and ionomycin upregulated the IP3 pathway in a manner that we interpret to be secondary to CCE. Collectively, these data suggest that mefloquine does not activate glutamate receptors and that it disrupts calcium homeostasis in mammalian cells in a manner similar to that of ionomycin.
Assuntos
Antimaláricos/efeitos adversos , Cálcio/metabolismo , Ionomicina/efeitos adversos , Macrófagos/efeitos dos fármacos , Mefloquina/efeitos adversos , Neurônios/efeitos dos fármacos , Animais , Linhagem Celular , Células Cultivadas , Córtex Cerebral/citologia , Homeostase/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , RatosRESUMO
We have previously reported that a novel neuroprotective substance named serofendic acid was purified and isolated from ether extract of fetal calf serum. In the present study, we investigated the effect of serofendic acid on acute neurotoxicity induced by L-glutamate (Glu) using primary cultures of rat cortical neurons. Exposure of cortical cultures to Glu for 1 h caused a marked decrease in cell viability, as determined by trypan blue exclusion. This acute Glu neurotoxicity was prevented by N-methyl-D-aspartate (NMDA) receptor antagonists, extracellular Ca(2+) removal, nitric oxide (NO) synthase inhibitor and NO scavenger. Serofendic acid prevented acute Glu neurotoxicity in a concentration-dependent manner. Acute neurotoxicity was induced by ionomycin, a Ca(2+) ionophore, and S-nitroso-L-cysteine, an NO donor. Serofendic acid also prevented both ionomycin- and S-nitroso-L-cysteine-induced neurotoxicity. Moreover, the protective effect of serofendic acid on acute Glu neurotoxicity was not affected by cycloheximide, a protein synthesis inhibitor, and actinomycin D, an RNA synthesis inhibitor. These results indicate that serofendic acid protects cultured cortical neurons from acute Glu neurotoxicity by reducing the cytotoxic action of NO and de novo protein synthesis is not required for this neuroprotection.
Assuntos
Células Cultivadas , Córtex Cerebral/citologia , Cisteína/análogos & derivados , Diterpenos/uso terapêutico , Neurônios/patologia , Síndromes Neurotóxicas/prevenção & controle , Glutamato de Sódio/efeitos adversos , Valina/análogos & derivados , Animais , Cálcio/antagonistas & inibidores , Cálcio/farmacologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Cisteína/efeitos adversos , Cisteína/antagonistas & inibidores , Cisteína/metabolismo , Diterpenos/isolamento & purificação , Diterpenos/farmacologia , Maleato de Dizocilpina/farmacologia , Maleato de Dizocilpina/uso terapêutico , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Sangue Fetal/química , Feto/anatomia & histologia , Ionomicina/efeitos adversos , Ionomicina/antagonistas & inibidores , Neurônios/citologia , Neurônios/efeitos dos fármacos , Óxido Nítrico/efeitos adversos , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Quinoxalinas/farmacologia , Quinoxalinas/uso terapêutico , Ratos , S-Nitrosotióis/efeitos adversos , S-Nitrosotióis/antagonistas & inibidores , S-Nitrosotióis/metabolismo , Glutamato de Sódio/antagonistas & inibidores , Fatores de Tempo , Valina/farmacologia , Valina/uso terapêuticoRESUMO
We examined the mechanism of 17beta-estradiol (estrogen)-mediated inhibition of apoptosis in C6 (rat glioma) cells following exposure to hydrogen peroxide (H(2)O(2)). Cells were preincubated with 4 microM estrogen for 2 h and then exposed to 100 microM H(2)O(2) for 24 h. Exposure to H(2)O(2) caused significant increases in intracellular calcium (Ca(2+)), as determined by fura-2, which was attenuated by preincubation with estrogen. H(2)O(2) and ionomycin caused cell death in a dose-dependent manner, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Preincubation with estrogen restored viability in cells exposed to H(2)O(2) but not in cells exposed to ionomycin. Western blot analysis showed an increase in Bax/Bcl-2 ratio, calpain activity, and caspase-3 activity following treatment with H(2)O(2), and estrogen pretreatment decreased levels of all three. Cell morphology, as evaluated by Wright staining, indicated apoptosis in cells treated with H(2)O(2), and pretreatment with estrogen reduced apoptosis. Results from MTT and Wright staining were further supported by the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) assay. These results indicate a role for estrogen in preventing apoptosis in C6 glial cells exposed to H(2)O(2). Our results suggest that estrogen may have a protective role in minimizing glial cell apoptosis in neurological diseases such as demyelinating disease or central nervous system trauma.