RESUMO
Influenza A virus (IAV) H1N1 infection is a constant threat to human health and it remains so due to the lack of an effective treatment. Since melatonin is a potent antioxidant and anti-inflammatory molecule with anti-viral action, in the present study we used melatonin to protect against H1N1 infection under in vitro and in vivo conditions. The death rate of the H1N1-infected mice was negatively associated with the nose and lung tissue local melatonin levels but not with serum melatonin concentrations. The H1N1-infected AANAT-/- melatonin-deficient mice had a significantly higher death rate than that of the WT mice and melatonin administration significantly reduced the death rate. All evidence confirmed the protective effects of melatonin against H1N1 infection. Further study identified that the mast cells were the primary targets of melatonin action, i.e., melatonin suppresses the mast cell activation caused by H1N1 infection. The molecular mechanisms involved melatonin down-regulation of gene expression for the HIF-1 pathway and inhibition of proinflammatory cytokine release from mast cells; this resulted in a reduction in the migration and activation of the macrophages and neutrophils in the lung tissue. This pathway was mediated by melatonin receptor 2 (MT2) since the MT2 specific antagonist 4P-PDOT significantly blocked the effects of melatonin on mast cell activation. Via targeting mast cells, melatonin suppressed apoptosis of alveolar epithelial cells and the lung injury caused by H1N1 infection. The findings provide a novel mechanism to protect against the H1N1-induced pulmonary injury, which may better facilitate the progress of new strategies to fight H1N1 infection or other IAV viral infections.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Lesão Pulmonar , Melatonina , Infecções por Orthomyxoviridae , Humanos , Animais , Camundongos , Lesão Pulmonar/tratamento farmacológico , Lesão Pulmonar/metabolismo , Mastócitos/metabolismo , Melatonina/farmacologia , Melatonina/metabolismo , Síndrome da Liberação de Citocina/metabolismo , PulmãoRESUMO
BACKGROUND: Dorper and Tan sheep are renowned for their rapid growth and exceptional meat quality, respectively. Previous research has provided evidence of the impact of gut microbiota on breed characteristics. The precise correlation between the gastrointestinal tract and peripheral organs in each breed is still unclear. Investigating the metabolic network of the intestinal organ has the potential to improve animal growth performance and enhance economic benefits through the regulation of intestinal metabolites. RESULTS: In this study, we identified the growth advantage of Dorper sheep and the high fat content of Tan sheep. A transcriptome study of the brain, liver, skeletal muscle, and intestinal tissues of both breeds revealed 3,750 differentially expressed genes (DEGs). The genes PPARGC1A, LPL, and PHGDH were found to be highly expressed in Doper, resulting in the up-regulation of pathways related to lipid oxidation, glycerophospholipid metabolism, and amino acid anabolism. Tan sheep highly express the BSEP, LDLR, and ACHE genes, which up-regulate the pathways involved in bile transport and cholesterol homeostasis. Hindgut content analysis identified 200 differentially accumulated metabolites (DAMs). Purines, pyrimidines, bile acids, and fatty acid substances were more abundant in Dorper sheep. Based on combined gene and metabolite analyses, we have identified glycine, serine, and threonine metabolism, tryptophan metabolism, bile secretion, cholesterol metabolism, and neuroactive ligand-receptor interaction as key factors contributing to the differences among the breeds. CONCLUSIONS: This study indicates that different breeds of sheep exhibit unique breed characteristics through various physiological regulatory methods. Dorper sheep upregulate metabolic signals related to glycine, serine, and threonine, resulting in an increase in purine and pyrimidine substances. This, in turn, promotes the synthesis of amino acids and facilitates body development, resulting in a faster rate of weight gain. Tan sheep accelerate bile transport, reduce bile accumulation in the intestine, and upregulate cholesterol homeostasis signals in skeletal muscles. This promotes the accumulation of peripheral and intramuscular fat, resulting in improved meat quality. This work adopts a joint analysis method of multi-tissue transcriptome and gut metabolome, providing a successful case for analyzing the mechanisms underlying the formation of various traits.
Assuntos
Melhoramento Vegetal , Transcriptoma , Ovinos/genética , Animais , Metaboloma , Glicina , Serina , Treonina , ColesterolRESUMO
Previous research identified the expression of EDN3 in granulosa cells of preovulatory follicles in chickens. Notably, the expression level of EDN3 in Silky Fowl with low egg-laying performance was significantly higher than that in high-yield laying breed White Leghorn. Given the crucial role of granulosa cells in follicular development and maturation, it is very important to study the effect of EDN3 on the biological function of granular cells. In this study, an EDN3 overexpression plasmid was constructed and transfected into granular cells. The viability of these cells was detected using quantiative (qPCR), Cell Counting Kit-8 (CCK8), and 5-Ethynyl-2'-deoxyuridine (EdU) assays. Gonadal hormone synthesis was detected using enzyme-linked immunosorbent assay (ELISA) techniques. Finally, transcriptome sequencing was employed to identify differentially expressed genes. Result showed thatoverexpression of EDN3 was observed to promote cell viability. In addition, it significantly inhibits the expressions of LHR and cAMP-PKA signaling pathways. Cell transcriptome sequencing data displayed that EDN3 can upregulate energy metabolism and immune-related signaling pathways, whereas follicle maturation and the GnRH signaling pathway were downregulated. In conclusion, this study demonstrates that EDN3 can enhance granulosa cell viability and inhibit the expression of LHCGR, a process likely mediated through the cAMP-PKA signaling pathway. However, further evidence is required to substantiate the regulatory relationship between EDN3 and the cAMP-PKA signaling pathway.
RESUMO
Spermatogonial stem cells (SSCs) self-renew and contribute genetic information to the next generation. Pig is wildly used as a model animal for understanding reproduction mechanisms of human being. Inducing directional differentiation of porcine SSCs may be an important strategy in exploring the mechanisms of spermatogenesis and developing better treatment methods for male infertility. Here, we established an in-vitro culture model for porcine small seminiferous tubule segments, to induce SSCs to differentiate into single-tail haploid spermatozoa. The culture model subsequently enabled spermatozoa to express the sperm-specific protein acrosin and oocytes to develop to blastocyst stage after round spermatid injection. The addition of retinoic acid (RA) to the differentiation media promoted the efficiency of haploid differentiation. RT-PCR analysis indicated that RA stimulated the expression of Stra8 but reduced the expression of NANOS2 in spermatogonia. Genes involved in post-meiotic development, transition protein 1 (Tnp1) and protamine 1 (Prm1) were upregulated in the presence of RA. The addition of an RA receptor (RAR) inhibitor, BMS439, showed that RA enhanced the expression of cAMP responsive-element binding protein through RAR and promoted the formation of round spermatids. We established an efficient culture system for in-vitro differentiation of pig SSCs. Our study represents a model for human testis disease and toxicology screening. Molecular regulators of SSC differentiation revealed in this study might provide a therapeutic strategy for male infertility.
Assuntos
Diferenciação Celular , Haploidia , Espermatogônias/fisiologia , Espermatozoides/fisiologia , Suínos , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Masculino , Cultura Primária de Células/métodos , Cultura Primária de Células/veterinária , Espermatogênese/efeitos dos fármacos , Espermatogênese/fisiologia , Espermatogônias/citologia , Espermatogônias/efeitos dos fármacos , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Tretinoína/farmacologiaRESUMO
Transport stress causes not only physiological changes but also behavioral responses, including anxiety-like and depression-like behaviors in animals. The serotonergic system in the brain plays a pivotal role in processing anxiety. This study aimed to explore changes in concentrations of 5-hydroxytryptamine (serotonin), and the expression changes of tryptophan hydroxylase 2 (TPH2) mRNA and protein associated with anxiety-related behavioral responses under transport stress. A model of simulated transport stress was established in 40 adult male Sprague-Dawley rats, including a control group (n = 20) and a transport stress (TS) group (n = 20). The results showed that the rats in the TS group exhibited an increased feeding latency in the novelty-suppressed feeding test and a reduced frequency and dwelling time in the central area in the open-field test (OFT). Two hours following the final behavioral test, blood samples were collected. Creatine kinase (CK) activities and glucose and corticosterone concentrations in serum were significantly higher in the rats in the TS group than in the control group. Transport stress also significantly reduced the concentrations of 5-hydroxytryptamine in the hippocampus, striatum, and raphe nuclei and also reduced the expression levels of mRNA and protein for TPH2 in the raphe nuclei. Notably, the number of Fos-immunoreactive neurons was higher in the dorsal raphe nucleus under transport stress, whereas the number of 5-hydroxytryptamine-positive neurons was significantly lower. These findings are consistent with the hypothesis that the 5-hydroxytryptamine transmitter in the hippocampus, striatum, and raphe nuclei is involved in processing anxiety-related behavioral responses under transport stress. Lay summary Physiological and psychological stress responses were induced in a rat model of simulated transport stress. We examined whether serotonin in the brain may be involved in mediating behavioral responses following exposure to transport stress. Tissue concentrations of serotonin in rat brain regions, including the hippocampus, striatum, and raphe nuclei, were reduced following exposure to transport stress. Expression of tryptophan hydroxylase 2 mRNA and protein, which catalyses serotonin synthesis, as well as numbers of serotonin-immunoreactive neurons, were decreased in the brainstem raphe nuclei.
Assuntos
Ansiedade/metabolismo , Serotonina/metabolismo , Estresse Psicológico/metabolismo , Triptofano Hidroxilase/metabolismo , Animais , Encéfalo/metabolismo , Corticosterona/metabolismo , Masculino , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun , RNA Mensageiro/metabolismo , Núcleos da Rafe/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
ß-Catenin is an evolutionarily conserved molecule in the canonical Wnt signaling pathway, which controls decisive steps in embryogenesis and functions as a crucial effector in the development of hair follicles. However, the molecular mechanisms underlying wool production have not been fully elucidated. In this study, we investigated the effects of ovine ß-catenin on wool follicles of transgenic sheep produced by pronuclear microinjection with a skin-specific promoter of human keratin14 (k14). Both polymerase chain reaction and Southern blot analysis showed that the sheep carried the ovine ß-catenin gene and that the ß-catenin gene could be stably inherited. To study the molecular responses to high expression of ß-catenin, high-throughput RNA-seq technology was employed using three transgenic sheep and their wild-type siblings. These findings suggest that ß-catenin normally plays an important role in wool follicle development by activating the downstream genes of the Wnt pathway and enhancing the expression of keratin protein genes and keratin-associated protein genes.
Assuntos
Perfilação da Expressão Gênica/veterinária , Queratina-14/genética , Ovinos/genética , Lã/metabolismo , beta Catenina/genética , Animais , Animais Geneticamente Modificados/metabolismo , Feminino , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Microinjeções , Regiões Promotoras Genéticas , Análise de Sequência de RNA , Ovinos/metabolismo , Pele/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
The development of improved vaccines and vaccination strategies against Mycobacterium tuberculosis has been hindered by a limited understanding of the immune correlates of anti-tuberculosis protective immunity. Simple measurement of interferon-γ frequency or production per se does not provide adequate prediction of immune protection. In this study, we examined the relationship between T-cell immune responses and protective efficacy conferred by the heterologous vaccination strategy, bacillus Calmette-Guérin (BCG) prime-Ag85A DNA boost (B/D), in an early challenge mouse model of pulmonary tuberculosis. The results demonstrated that mice vaccinated with the B/D regimen had a significantly reduced bacillary load compared with BCG-vaccinated mice, and the reduction in colony-forming units was associated with decreased pathology and lower levels of inflammatory cytokines in the infected lungs. Further analysis of immunogenicity showed that the superior protection afforded by the B/D regimen was associated with significantly increased frequency of splenic interleukin-2 (IL-2) -producing CD4 T cells and increased IL-2 production when measured as integrated mean fluorescence intensity post-vaccination as well. These data suggest that measurement of elevated frequency of IL-2-producing CD4 T cells or IL-2 production in the spleens of vaccinated mice can predict vaccine efficacy, at least in the B/D strategy, and add to the accumulating body of evidence suggesting that BCG prime-boost strategies may be a useful approach to the control of M. tuberculosis infection.
Assuntos
Vacina BCG/imunologia , Linfócitos T CD4-Positivos/imunologia , Interleucina-2/biossíntese , Mycobacterium tuberculosis/imunologia , Baço/imunologia , Tuberculose/imunologia , Tuberculose/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Imunização Secundária , Mediadores da Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Contagem de Linfócitos , Camundongos , Baço/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Tuberculose/prevenção & controle , VacinaçãoRESUMO
Correction for 'Oral aged garlic (Allium sativum) alleviates ulcerative colitis in mice by improving gut homeostasis' by Deping Han et al., Food Funct., 2024, https://doi.org/10.1039/D4FO03105A.
RESUMO
Aged garlic, obtained by heating raw garlic (Allium sativum) under high temperature and controlled humidity for a period, possesses a wide range of bioactivities, but its role in ulcerative colitis and its mechanism are not fully elucidated. We investigated the bioactive constituents in aged garlic (AG) and explored the effect of oral AG delivery on DSS-induced murine colitis. The results revealed that the aging process up-regulated anti-oxidative, anti-inflammatory and anti-microbial compounds such as dihydrocaffeic acid, 5-acetylsalicylic acid, verticine, S-allyl-L-cysteine and D-fucose. Oral AG obviously alleviated colitis, reducing colon damage and enhancing anti-oxidative and anti-inflammatory effects. Escherichia coli and Streptococcus equinus dramatically were enriched in the colon of mice with colitis that were strongly associated with Parkinson's disease, bacterial invasion of epithelial cells, aerobactin biosynthesis, and heme biosynthesis, but a distinct AG-mediated alteration in the colon due to increasing abundance of Akkermansia muciniphila, Lactobacillus sp. L-YJ, Bifidobacterium breve, Blautia wexlerae, Desulfomicrobium sp. P100A, and Lentilactobacillus hilgardii was observed. Next, we demonstrated that colonic microbiome reconstruction by oral AG significantly increased the production of short-chain fatty acids such as acetic acid, propionic acid, isobutyric acid, and isovaleric acid. This study provides the first data indicating that oral AG ameliorates colonic inflammation in a gut microbiota-dependent manner. Our findings provide novel insights into the AG-mediated remission of colitis and AG as a functional food for modulating the gut microbiota to prevent and treat colitis.
Assuntos
Colite Ulcerativa , Alho , Microbioma Gastrointestinal , Animais , Camundongos , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/microbiologia , Alho/química , Microbioma Gastrointestinal/efeitos dos fármacos , Masculino , Homeostase/efeitos dos fármacos , Colo/microbiologia , Colo/metabolismo , Colo/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Extratos Vegetais/farmacologia , Extratos Vegetais/administração & dosagem , Modelos Animais de Doenças , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/administração & dosagem , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificaçãoRESUMO
The presence of a significant number of melanocytes in the ovary and follicular membrane of Silky Fowl suggests their potential involvement in follicle development. Currently, there is a lack of available data regarding to the isolation of primary melanocytes from adult chickens. To date, primary melanocytes and their in vitro culture system have been successfully conducted in the peritoneum of chicken embryos. Herein, melanocytes from silky fowl ovaries were isolated and identified. Silky Fowl ovaries were obtained by mixed digestion of 0.1% collagenase II and 0.25% trypsin-EDTA. Melanocytes could be further purified and cultured up to 5 generations in vitro. RNA-seq analysis was used to investigate whether there were differences in the functional status of melanocytes in different tissues and developmental stages. Consequently, differential gene expressions between peritoneal and ovarian melanocytes were compared. These findings demonstrated that the Silky Fowl ovary had higher expression levels of genes involved in the production of sexual hormones and melanogenesis, while those of melanocytes derived from the peritoneum were involved in amino acid metabolism, lipid synthesis, and overall metabolic rates. This suggests that the role of melanocytes is dependent on the origin tissue and developmental stage, and is tightly connected to the function of the specific source tissue from which the cells were derived. This study provides a method for isolating adult melanocytes and serve as a basis for further investigate the effect of SFOM on germ cells.
Assuntos
Galinhas , Ovário , Feminino , Embrião de Galinha , Animais , Galinhas/genética , Galinhas/metabolismo , Melanócitos/metabolismo , CarneRESUMO
Although an important role for mast cells in several viral infections has been demonstrated, its role in the invasion of highly pathogenic H5N1 influenza virus is unknown. In the present study, we demonstrate that mast cells were activated significantly by H5N1 virus (A/chicken/Henan/1/2004) infection both in vivo and in vitro. Mast cells could possibly intensify the lung injury that results from H5N1 infection by releasing proinflammatory mediators, including histamine, tryptase, and gamma interferon (IFN-γ). Lung lesions and apoptosis induced by H5N1 infection were reduced dramatically by treatment with ketotifen, which is a mast cell degranulation inhibitor. A combination of ketotifen and the neuraminidase inhibitor oseltamivir protected 100% of the mice from death postinfection. In conclusion, our data suggest that mast cells play a crucial role in the early stages of H5N1 influenza virus infection and provide a new approach to combat highly pathogenic influenza virus infection.
Assuntos
Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Humana/imunologia , Lesão Pulmonar/imunologia , Mastócitos/imunologia , Animais , Feminino , Humanos , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Humana/complicações , Influenza Humana/virologia , Lesão Pulmonar/etiologia , Lesão Pulmonar/virologia , Mastócitos/virologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
In this study, we compared two in vitro collagen production assays ([(3)H]-proline incorporation and Sirius Red) for their ability to determine the pattern shift from soluble to deposited collagen. The effect of the antifibrotic agent, triptolide (TPL), on collagen production was also studied. The results showed that: (1) 48 h after NIH 3T3 (murine embryo fibroblast) and HFL-1(human fetal lung fibroblast) were exposed to transforming growth factor-beta 1 (TGF-ß), there was an increase in soluble collagen in the culture medium; (2) on day 4, soluble collagen declined, whereas deposited collagen increased; (3) Sirius Red was easier to use than [(3)H]-proline incorporation and more consistently reflected the collagen pattern shift from soluble to deposited; (4) the in vitro Sirius Red assay took less time than the in vivo assay to determine the effect of TPL. Our results suggest that: (a) the newly synthesized soluble collagen can sensitively evaluate an agent's capacity for collagen production and (b) Sirius Red is more useful than [(3)H]-proline because it is easier to use, more convenient, less time consuming, and does not require radioactive material.
Assuntos
Compostos Azo , Bioensaio , Colágeno/metabolismo , Embrião de Mamíferos/metabolismo , Feto/metabolismo , Fibroblastos/metabolismo , Pulmão/metabolismo , Animais , Antineoplásicos Alquilantes/farmacologia , Células Cultivadas , Corantes , Diterpenos/farmacologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Feto/citologia , Feto/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Fenantrenos/farmacologia , Prolina/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Although glycoproteins possess a variety of functional and structural roles in intracellular and intercellular activities, the effect of ionizing radiation (IR) on glycosylation is largely unknown. To explore this effect, we established a sandwich assay in which PHA-L, a phytohaemagglutinin that agglutinates leukocytes, was used as a coating layer to capture glycoproteins containing complex oligosaccharides; the bound glycoproteins were then measured. C57BL/6 mice were exposed to 0, 3, 6, or 10 Gy, and the plasma was collected at 6, 12, 18, 24, 48, 72, or 168 h and then analyzed for galactose/N-acetylgalactosamine (Gal/GalNAc) containing proteins. We found that (1) the sandwich assay accurately measured the level of glycoproteins, (2) 6-12 h after IR, the amount of glycoproteins containing GalNAc increased, and (3) at 72 and 168 h, 10 Gy was associated with a decrease in Gal/GalNAc. These IR-induced alterations might relate to the release of glycoproteins into the blood and the damage of the proteins and genes that are related to the glycosylation process.
Assuntos
Acetilgalactosamina/sangue , Galactose/sangue , Glicoproteínas/sangue , Glicosilação/efeitos da radiação , Manose/sangue , Irradiação Corporal Total , Acetilgalactosamina/análogos & derivados , Animais , Ensaio de Imunoadsorção Enzimática , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fito-Hemaglutininas/metabolismoRESUMO
Various members of the fibroblast growth factor (FGF) family mitigate radiation-induced damage. We designed and synthesized the binding domain peptide of FGF-2 (FGF-P) with a dimer form resistant to peptidase and examined its mitigatory effect on murine bone marrow cells. NIH Swiss mice were exposed to different doses of total body irradiation (TBI) and treated with ten doses of 5 mg/kg FGF-P. We achieved the following results: (1) FGF-P stimulated the growth of bone marrow cells harvested from mice exposed to 3 Gy; (2) on day 25 after 6 Gy TBI, the number of leukocytes and granulocytes was higher in the FGF-P group than in the vehicle-alone group; (3) FGF-P significantly increased the number of pro-B and pre-B cells; and (4) FGF-P treatment in vivo increased the long-term hematopoietic stem cells (LT-HSC) in bone marrow. These data reveal the underlying mechanism by which FGF-P rescued a significant percentage of the exposed mice. The increase of LT-HSC in bone marrow leads to a concomitant increase of pro-B and pre-B cells followed by leukocytes and granulocytes, which in turn enhance immunity against infection.
Assuntos
Linfócitos B/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Fatores de Crescimento de Fibroblastos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Irradiação Corporal Total , Animais , Linfócitos B/patologia , Linfócitos B/efeitos da radiação , Medula Óssea/patologia , Células Cultivadas , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/efeitos da radiação , Masculino , CamundongosRESUMO
Amifostine is a first-line cytoprotective drug used to prevent radiotherapy-induced or chemotherapy-induced injuries. However, its mechanism of action is not well understood. In this study, freshly harvested bone marrow cells were treated with amifostine and analyzed with a series of mitochondrial indices. In vitro results showed that bone marrow cells treated with amifostine 0.5 h before irradiation (0.5 Gy) experienced several benefits, as compared to vehicle controls, including (1) reduced reactive oxygen species levels, which reduced the production of free radicals; (2) better preservation of mitochondria, as indicated by MitoTracker-positive staining and the increased intensity of staining; (3) reduced apoptosis, as demonstrated by Annexin V staining; and (4) a better proliferation rate, as illustrated by MTT assay. Our in vitro studies showed that amifostine-treated mice exhibited (1) higher ATP production; (2) reduced plasma IL-2 levels, suppressing the immune response triggered by radiotoxicity; and (3) enhanced radiation-induced production of granulocyte colony-stimulating factor. All of these processes benefit recovery from radiation-induced damage.
Assuntos
Amifostina/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Citocinas/metabolismo , Mitocôndrias/efeitos dos fármacos , Protetores contra Radiação/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Medula Óssea/crescimento & desenvolvimento , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Interleukin 11 (IL-11) is a multifunctional cytokine isolated from bone marrow (BM)-derived stromal cells that promotes hematopoiesis and prolongs the life span of lethally irradiated animals. However, the underlying mechanism for the protective effect of IL-11 on BM is unclear. In this study, we explored the effect of IL-11 on irradiated BM cells. Freshly harvested BM cells were pretreated with 20 ng/ml of recombinant IL-11 for 30 min, irradiated with a dose of 0.5 Gy, cultured for 24 h, and then subjected to several assays. In vitro data showed that, as compared to the vehicle controls, IL-11: (1) reduced the production of reactive oxygen species; (2) reduced the alteration of mitochondrial membrane potential; (3) increased MitoTracker staining, suggesting that the number of mitochondria and their functions were better maintained; and (4) reduced apoptosis of BM cells and enhanced BM cell proliferation. In vivo studies of mice pretreated with saline or 100 µg/kg of IL-11 at 12 and 2 h before 10-Gy total body irradiation (TBI) demonstrated that G-CSF and IL-6 were significantly upregulated, whereas IL-2 and IL-4 were reduced. We found that IL-11 protects mitochondrial functions, acts with G-CSF and IL-6 to stimulate the growth of radiation-damaged BM, and reduces the immune response to radiation injury.
Assuntos
Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Interleucina-11/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos da radiação , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos da radiação , Fator Estimulador de Colônias de Granulócitos/metabolismo , Interleucina-11/metabolismo , Interleucina-6/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos da radiação , Camundongos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Irradiação Corporal Total/métodosRESUMO
In this study, we investigated the response of irradiated bone marrow cells to granulocyte colony-stimulating factor (G-CSF). Freshly harvested bone marrow cells were treated with either saline (vehicle control) or 20 ng/ml of G-CSF. Thereafter, cells were separated into nonirradiated (no-IR) and irradiated (IR, 0.5 Gy) groups. IR cells exhibited a higher proliferation rate in response to G-CSF, as compared to the no-IR cells. Reduced levels of reactive oxygen species indicated that G-CSF-treated IR cells produced fewer free radicals, as compared to the no-IR cells. The G-CSF-treated IR cells also had a lower apoptotic rate than their no-IR counterparts. Furthermore, G-CSF-treated IR cells exhibited less alteration of mitochondrial membrane potential, as compared to the no-IR cells. Finally, the mitochondrial number increased in the G-CSF-treated IR cells. The radiation-induced increase in plasma IL-6 in vivo could be enhanced by the administration of G-CSF. The data suggest that radiation potentiates the response of bone marrow cells to G-CSF treatment.
Assuntos
Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Fator Estimulador de Colônias de Granulócitos/farmacologia , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos da radiação , Radicais Livres/metabolismo , Interleucina-6/sangue , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos da radiação , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismoRESUMO
AIMS: Emerging research indicates that γ-aminobutyric acid (GABA) provides substantial benefits during enteritis. Nevertheless, GABA signaling roles on enteric glial cells (EGCs) remain unknown. The study's objective was to evaluate the underlying mechanisms of GABA signaling on EGCs in vitro and in vivo. MAIN METHODS: We established LPS-induced mouse models and stimulated EGCs with LPS to mimic intestinal inflammation, and combined GABA, GABAA receptor (GABAAR) or GABAB receptor (GABABR) agonists to explore the exact mechanisms of GABA signaling. KEY FINDINGS: EGCs were immunopositive for GAD65, GAD67, GAT1, GABAARα1, GABAARα3, and GABABR1, indicating GABAergic and GABAceptive properties. GABA receptor activation significantly inhibited the high secretions of proinflammatory factors in EGCs upon LPS stimulation. Interestingly, we found that EGCs express immune-related molecules such as CD16, CD32, CD80, CD86, MHC II, iNOS, Arg1, and CD206, thus establishing their characterization of E1 and E2 phenotype. EGCs exposed to LPS mainly acted as E1 phenotype, whereas GABABR activation strongly promoted EGCs polarization into E2 phenotype. Transcriptome analysis of EGCs indicated that GABA, GABAAR or GABABR agonists treatment participated in various biological processes, however all of these treatments exhibit inhibitory effects on NF-κB pathway. Notably, in LPS-induced mice, activation of GABABR mitigated intestinal damage through modulating inflammatory factors expressions, strengthening sIgA and IgG levels, inhibiting NF-κB pathway and facilitating EGCs to transform into E2 phenotype. SIGNIFICANCE: These data demonstrate that the anti-inflammatory actions of GABA signaling system offer in enteritis via regulating EGCs-polarized function through impeding NF-κB pathway, thus providing potential targets for intestinal inflammatory diseases.
Assuntos
NF-kappa B , Receptores de GABA , Camundongos , Animais , NF-kappa B/metabolismo , Receptores de GABA/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Neuroglia/metabolismo , Ácido gama-Aminobutírico/metabolismo , Inflamação/metabolismoRESUMO
Introduction: Epstein-Barr virus (EBV) is a widely spread pathogen associated with lymphoproliferative diseases, B/ T/ NK cell lymphomas, nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). EBV lytic reactivations contribute to the genomic instability, inflammation and tumorigenesis of NPC, promoting cancer progression. Patients with NPC refractory to standard therapies show dismal survival. EBV gp350 is an envelope protein detectable in NPC specimens intracellularly and on the cell membrane of malignant cells, and is a potential viral antigen for T cell-directed immunotherapies. The potency of T cells engineered with a chimeric antigen receptor (CAR) targeting gp350 against EBV+ lymphoproliferative disease was previously shown. Methods: Here, we advanced towards preclinical and non-clinical developments of this virus-specific CAR-T cell immunotherapy against NPC. Different gp350CAR designs were inserted into a lentiviral vector (LV) backbone. Results: A construct expressing the scFv 7A1-anti-gp350 incorporating the CD8 transmembrane and CD28.CD3ζ signaling domain (ZT002) was selected. High titer ZT002 (~1x108 TU/ml) was manufactured in HEK 293T/17 suspension cells in serum free media as large-scale production under good manufacturing practices (GMP). A LV multiplicity of infection (MOI) of 1 resulted in high frequencies of functional gp350CAR+ T cells (>70%) at a low (<2) vector copy numbers in the genome. ZT002 was therefore used to establish gp350CAR-T batch run production methods. GMP upscaling and validation of T cell transduction and expansion in several runs resulted in average 3x109 gp350CAR-T cells per batch. >80% CD3+ gp350CAR-T cells bound to purified gp350 protein. In vitro cytotoxicity and cytokine secretion assays (IFN-γ and TNF-α) confirmed the specificity of gp350CAR-T cells against gp350+ NPC, GC and lymphoma cell targets. Immunocompromised B-NDG mice (NOD.CB17-PrkdcscidIl2rgtm1/Bcgen) were challenged s.c. with a EBV+ NPC C666.1 cell line expressing gp350 and then treated with escalating doses of gp350CAR-T cells or with non-transduced T cells. gp350CAR-T cells promoted antitumor responses, bio-distributed in several tissues, infiltrated in tumors and rejected gp350+ tumor cells. Discussion: These results support the use of gp350CAR-T cells generated with ZT002 as an Innovative New Drug to treat patients with solid and liquid EBV-associated malignancies.
Assuntos
Infecções por Vírus Epstein-Barr , Neoplasias Nasofaríngeas , Animais , Camundongos , Herpesvirus Humano 4 , Camundongos Endogâmicos NOD , Carcinoma Nasofaríngeo , Linfócitos T , Receptores de Antígenos Quiméricos/imunologiaRESUMO
BACKGROUND: It is not uncommon for some individuals to retain certain primitive characteristics even after domestication or long-term intensive selection. Wild ancestors or original varieties of animals typically possess strong adaptability to environmental preservation, a trait that is often lacking in highly artificially selected populations. In the case of the Merino population, a world-renowned fine wool sheep breed, a phenotype with primitive coarse wool characteristic has re-emerged. It is currently unclear whether this characteristic is detrimental to the production of fine wool or whether it is linked to the adaptability of sheep. The underlying genetic/epigenetic mechanisms behind this trait are also poorly understood. RESULTS: This study identified lambs with an ancestral-like coarse (ALC) wool type that emerged during the purebred breeding of Merino fine wool sheep. The presence of this primitive sheep characteristic resulted in better environmental adaptability in lambs, as well as improved fine wool yield in adulthood. Reciprocal cross experiments revealed that the ALC phenotype exhibited maternal genetic characteristics. Transcriptomic SNP analysis indicated that the ALC phenotype was localized to the imprinted Gtl2-miRNAs locus, and a significant correlation was found between the ALC wool type and a newly identified short Interstitial Telomeric Sequences (s-ITSs) at this locus. We further confirmed that a novel 38-nt small RNA transcribed from these s-ITSs, in combination with the previously reported 22-nt small RNAs cluster from the Gtl2-miRNAs locus, synergistically inhibited PI3K/AKT/Metabolic/Oxidative stress and subsequent apoptotic pathways in wool follicle stem cells, resulting in the ALC wool type. The necessity of Gtl2-miRNAs in controlling primary hair follicle morphogenesis, as well as the wool follicle type for ALC wool lambs, was verified using intergenic differentially methylated region-knockout mice. CONCLUSION: The ALC wool type of Merino sheep, which does not reduce wool quality but increases yield and adaptability, is regulated by epigenetic mechanisms in the imprinted Gtl2-miRNAs region on sheep chromosome 18, with the maternally expressed imprinted gene responsible for the ALC phenotype. This study highlights the significance of epigenetic regulation during embryonic and juvenile stages and emphasizes the advantages of early adaptation breeding for maternal parents in enhancing the overall performance of their offspring.