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1.
Biomedicines ; 12(3)2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38540147

RESUMO

Circulating exosomes derived from polymicrobial sepsis contain various non-coding RNAs and proteins. Isobaric tags for a relative or absolute quantitation proteomic analysis of the exosomal content revealed 70 dysregulated proteins in the circulating exosomes from septic mice. Next-generation sequencing was used to profile the long non-coding RNA expression in primary cultured macrophages treated with exosomes obtained from the blood of septic C57BL/6 mice, and it was discovered that the nuclear factor-kappa B (NF-κB)/miR-17-92a-1 cluster host gene (MIR17HG) pathways were activated in the macrophages. The inhibition of MIR17HG expression by RNA interference resulted in significantly decreased cell viability. RNA pull-down assays of MIR17HG revealed that ten protein targets bind to MIR17HG. Interaction networks of proteins pulled down by MIR17HG were constructed using GeneMANIA, and their functions were mainly involved in ribonucleoprotein granules, type I interferons, the regulation of organelle assembly, the biosynthesis of acetyl coenzyme A, as a signal transducer and activator of transcription (STAT) protein phosphorylation, and mRNA splicing. Furthermore, RNA interference inhibited MIR17HG expression, resulting in significantly decreased cell survival. In conclusion, this work discovered considerable MIR17HG overexpression in macrophages treated with circulating exosomes from sepsis-affected animals. This study's findings assist us in comprehending the role of exosomes in modulating inflammatory responses and mediating pathogenic pathways in macrophages during sepsis.

2.
Biomed Pharmacother ; 153: 113481, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36076501

RESUMO

BACKGROUND: Platelet-rich plasma (PRP) is beneficial for clinical applications in various medical fields. Although commercial PRP preparation kits are already available in the market, most of these kits employ centrifugation. METHODS: We used a new cationic copolymer coating on a polyurethane (PU) sponge to promote platelet separation from the blood. This copolymer showed no cytotoxicity against cell viability or hemolysis. We further evaluated the efficiency of the new PRP preparation device by comparing it with that of a commercially available kit (RegenKit-THT). RESULTS: We demonstrated that PRP obtained using copolymer device contains high concentrations of platelets and angiogenic growth factors (epidermal growth factor, vascular endothelial growth factor-A, growth differentiation factor 2, and interleukin-8). The separated PRP also displayed beneficial effects on cell migration, angiogenesis, and matrix metalloproteinase gene expression. CONCLUSION: Based on these results, we developed a cationic copolymer-coated PU sponge as a PRP preparation device without the need for any centrifugation.


Assuntos
Plasma Rico em Plaquetas , Fator A de Crescimento do Endotélio Vascular , Plaquetas , Centrifugação/métodos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Plasma Rico em Plaquetas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
3.
Int J Mol Sci ; 22(17)2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34502537

RESUMO

Macrophages emerge in the milieu around innervated neurons after nerve injuries. Following nerve injury, autophagy is induced in macrophages and affects the regulation of inflammatory responses. It is closely linked to neuroinflammation, while the immunosuppressive drug tacrolimus (FK506) enhances nerve regeneration following nerve crush injury and nerve allotransplantation with additional neuroprotective and neurotrophic functions. The combined use of FK506 and adipose-derived stem cells (ADSCs) was employed in cell therapy for organ transplantation and vascularized composite allotransplantation. This study aimed to investigate the topical application of exosomes secreted by ADSCs following FK506 treatment (ADSC-F-exo) to the injured nerve in a mouse model of sciatic nerve crush injury. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ) were used to profile the potential exosomal proteins involved in autophagy. Immunohistochemical analysis revealed that nerve crush injuries significantly induced autophagy in the dorsal root ganglia and dorsal horn of the spinal segments. Locally applied ADSC-F-exo significantly reduced autophagy of macrophages in the spinal segments after nerve crush injury. Proteomic analysis showed that of the 22 abundant exosomal proteins detected in ADSC-F-exo, heat shock protein family A member 8 (HSPA8) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) are involved in exosome-mediated autophagy reduction.


Assuntos
Autofagia/efeitos dos fármacos , Lesões por Esmagamento/complicações , Exossomos/metabolismo , Macrófagos/efeitos dos fármacos , Traumatismos da Coluna Vertebral/metabolismo , Células-Tronco/efeitos dos fármacos , Tacrolimo/farmacologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Células Cultivadas , Cromatografia Líquida/métodos , Exossomos/ultraestrutura , Imunossupressores/farmacologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mapas de Interação de Proteínas , Proteoma/metabolismo , Proteômica/métodos , Traumatismos da Coluna Vertebral/etiologia , Células-Tronco/metabolismo , Espectrometria de Massas em Tandem/métodos
4.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445251

RESUMO

Exosomes secreted by adipose-derived stem cells (ADSC-exo) reportedly improve nerve regeneration after peripheral nerve injury. Herein, we investigated whether pretreatment of ADSCs with FK506, an immunosuppressive drug that enhances nerve regeneration, could secret exosomes (ADSC-F-exo) that further augment nerve regeneration. Designed exosomes were topically applied to injured nerve in a mouse model of sciatic nerve crush injury to assess the nerve regeneration efficacy. Outcomes were determined by histomorphometric analysis of semi-thin nerve sections stained with toluidine blue, mouse neurogenesis PCR array, and neurotrophin expression in distal nerve segments. Isobaric tags for relative and absolute quantitation (iTRAQ) were used to profile potential exosomal proteins facilitating nerve regeneration. We observed that locally applied ADSC-exo and ADSC-F-exo significantly enhanced nerve regeneration after nerve crush injury. Pretreatment of ADSCs with FK506 failed to produce exosomes possessing more potent molecules for enhanced nerve regeneration. Proteomic analysis revealed that of 192 exosomal proteins detected in both ADSC-exo and ADSC-F-exo, histone deacetylases (HDACs), amyloid-beta A4 protein (APP), and integrin beta-1 (ITGB1) might be involved in enhancing nerve regeneration.


Assuntos
Tecido Adiposo/metabolismo , Exossomos , Regeneração Nervosa , Traumatismos dos Nervos Periféricos/terapia , Nervos Periféricos/fisiologia , Células-Tronco/metabolismo , Tacrolimo/farmacologia , Animais , Exossomos/metabolismo , Exossomos/transplante , Camundongos , Traumatismos dos Nervos Periféricos/metabolismo
5.
J Cell Mol Med ; 25(15): 7436-7450, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34235869

RESUMO

Exosomes are secreted into the extracellular space by most cell types and contain various molecular constituents, which play roles in many biological processes. Adipose-derived mesenchymal stem cells (ADSCs) can differentiate into a variety of cell types and secrete a series of paracrine factors through exosomes. ADSC-derived exosomes have shown diagnostic and therapeutic potential in many clinical diseases. The molecular components are critical for their mechanisms. Several methods have been developed for exosome purification, including ultracentrifugation, ultrafiltration, density gradient purification, size-based isolation, polymer precipitation and immuno-affinity purification. Thus, we employed four methods to isolate exosomes from the hADSC culture medium, including ultracentrifugation, size exclusion chromatography, ExoQuick-TC precipitation and ExoQuick-TC ULTRA isolation. Following exosome isolation, we performed quantitative proteomic analysis of the exosome proteins using isobaric tags for relative and absolute quantification (iTRAQ) labelling, combined with 2D-LC-MS/MS. There were 599 universal and 138 stably expressed proteins in hADSC-derived exosomes. We proved that these proteins were potential hADSC-derived exosomes markers, including CD109, CD166, HSPA4, TRAP1, RAB2A, RAB11B and RAB14. From the quantitative proteomic analysis, we demonstrated that hADSC-derived exosome protein expression varied, with lipopolysaccharide (LPS) treatment, in the different isolation methods. Pathway analysis and proliferation, migration and endothelial tube formation assays showed varying effects in cells stimulated with hADSC-derived exosomes from different isolation methods. Our study revealed that different isolation methods might introduce variations in the protein composition in exosomes, which reflects their effects on biological function. The pros and cons of these methods are important points to consider for downstream research applications.


Assuntos
Fracionamento Celular/métodos , Exossomos/química , Células-Tronco Mesenquimais/química , Proteoma/química , Proteômica/métodos , Adipócitos/química , Células Cultivadas , Exossomos/metabolismo , Células Endoteliais da Veia Umbilical Humana/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Redes e Vias Metabólicas
6.
Int J Med Sci ; 18(4): 1058-1066, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33456364

RESUMO

The heterogeneity of exosome populations presents a great challenge to their study. The current study was designed to investigate the potential heterogeneity miRNA contents in circulating exosomes purified via different exosomal markers. In this study, exosomes from the serum of C57BL/6 mice after cecum ligation and perforation (CLP) or sham operation were isolated by precipitation using ExoQuick-TC and affinity purified with anti-Rab5b, anti-CD9, anti-CD31, and anti-CD44 antibodies using the Exo-Flow Exosome Capture kit to collect exosome subpopulations. RNA extracted from the exosomes isolated by ExoQuick-TC were profiled by next-generation sequencing (NGS). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was also employed to determine the expression profiles of four representative exosomal miRNAs (mmu-miR-486-5p, mmu-miR-10a-5p, mmu-miR-143-3p, and mmu-miR-25-3p) selected from the NGS analysis. The results revealed that the expression patterns of these miRNAs in exosomes isolated by ExoQuick-TC as determined by RT-qPCR and NGS were similar, showing upregulation of mmu-miR-10a-5p and mmu-miR-143-3p but downregulation of mmu-miR-25-3p and mmu-miR-486-5p following CLP when compared to the levels in exosomes from sham control mice. However, their expression levels in the antibody-captured exosome subpopulations varied. The miRNAs in the exosomes captured by anti-Rab5b or anti-CD9 antibodies were more similar to those isolated by ExoQuick-TC than to those captured by anti-CD44 antibodies. However, there were no significant differences in these four miRNAs in CD31-captured exosomes. This study demonstrated that purification with different exosomal markers allows the collection of different exosome subpopulations with various miRNA contents. The results of this study demonstrate the heterogeneity of circulating exosomes and suggest the importance of stratifying exosome subpopulations when using circulating exosomes as biomarkers or investigating exosome function. In addition, this study also emphasized the necessity of using a consistent exosome marker across different samples as detecting biomarkers.


Assuntos
MicroRNA Circulante/análise , Exossomos/metabolismo , Sepse/diagnóstico , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , MicroRNA Circulante/sangue , MicroRNA Circulante/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Sepse/sangue , Sepse/genética
7.
PLoS One ; 14(7): e0220398, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31348811

RESUMO

OBJECTIVE: To establish the composition of bacteria in mice following cecum ligation and puncture (CLP) through metagenomic analysis and investigate the role of TLRs on the composition of bacteria. METHODS: Total DNA extraction was done from the ascites, blood, and fecal samples from C57BL/6 mice sacrificed at 0, 4, 8, and 16 h, as well as from Tlr2-/-, Tlr4-/-, Tlr5-/-, and NF-κB-/-mice sacrificed at 16 h following CLP. Amplification of the V3-V4 regions of the bacterial 16S rRNA genes by PCR and the Illumina MiSeq sequencer was used for deep sequencing. Hierarchical clustering of the isolates was performed with Ward's method using Euclidean distances. The relative abundance according to operational taxonomic unit (OTU) number or taxa was used to compare the richness among subgroups in the experiments. RESULTS: There were 18 taxa that had significantly different abundances among the different samples of the C57BL/6 mice at 16 h following CLP. Various dynamic changes in the infectious bacteria inside the peritoneal cavity after CLP were found. While knockout of Tlr5 and NF-κB impaired the ability of bacterial clearance inside the peritoneal cavity for some kinds of bacteria found in the C57BL/6 mice, the knockout of Tlr4 enhanced clearance for other kinds of bacteria, and they presented excessive abundance in the peritoneal cavity despite their scarce abundance in the stool. CONCLUSION: NF-κB and TLRs are involved in bacterial clearance and in the expression pattern of the bacterial abundance inside the peritoneal cavity during polymicrobial infection.


Assuntos
Bactérias/classificação , Metagenômica/métodos , Cavidade Peritoneal/microbiologia , Receptores Toll-Like/genética , Animais , Ascite/microbiologia , Bactérias/genética , Sangue/microbiologia , DNA Bacteriano , DNA Ribossômico/genética , Fezes/microbiologia , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Filogenia , RNA Ribossômico 16S/genética
8.
Nutrients ; 10(10)2018 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-30304787

RESUMO

Background: This study aimed at assessing the effect of a low-fat diet (LFD) in obese mice lacking toll⁻like receptors (Tlr) and understanding the expression and regulation of microRNAs during weight reduction. Methods: C57BL/6, Tlr5-/-, Tlr2-/- and Tlr4-/- mice were used in this study. A group of mice were fed with a high-fat diet (HFD) (58% kcal) for 12 weeks to induce obesity (diet-induced obesity, DIO). Another group that had been fed with HFD for eight weeks (obese mice) were switched to a low-fat diet (LFD) (10.5% kcal) for the next four weeks to reduce their body weight. The control mice were fed with a standard AIN-76A diet for the entire 12 weeks. The body weight of the mice was measured weekly. At the end of the experiment, epididymal fat weight and adipocyte size were measured. The differentially expressed miRNAs in the fat tissue was determined by next-generation sequencing with real-time quantitative reverse transcription polymerase chain reaction (RT⁻qPCR). Target prediction and functional annotation of miRNAs were performed using miRSystem database. Results: Switching to LFD significantly reduced the body weight and epididymal fat mass in the HFD-fed C57BL/6 and Tlr5-/- mice but not in Tlr2-/- and Tlr4-/- mice. Weight reduction significantly decreased the size of adipocytes in C57BL/6 but not in the Tlr knockout mice. In Tlr2-/- and Tlr4-/- mice, feeding with HFD and the subsequent weight reduction resulted in an aberrant miRNA expression in the epididymal fat tissue unlike in C57BL/6 and Tlr5-/-. However, target prediction and functional annotation by miRSystem database revealed that all the top 10 Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathways of the dysregulated miRNAs during weight reduction in the C57BL/6 mice were also found in the regulated pathways of Tlr5-/-, Tlr2-/-, and Tlr4-/- strains. However, among these pathways, gene sets involved in arginine and proline metabolism and glutathione metabolism were mainly involved in the Tlr knockout mice but not in the C57BL/6 mice. Conclusions: In this study, we demonstrated that feeding of LFD leads to significant body weight reduction in C57BL/6 and Tlr5-/- mice, but not in Tlr2-/- and Tlr4-/- mice. Significant reduction in the size of adipocytes of epididymal fat was only found in C57BL/6, but not in Tlr5-/-, Tlr2-/-, and Tlr4-/- mice. The dysregulated miRNAs in Tlr2-/- and Tlr4-/- mice were different from those in C57BL/6 and Tlr5-/- strains. Among those miRNA-regulated pathways, arginine and proline metabolism as well as glutathione metabolism may have important roles in the Tlr knockout mice rather than in C57BL/6 mice.


Assuntos
Tecido Adiposo/metabolismo , Dieta com Restrição de Gorduras , MicroRNAs/metabolismo , Obesidade/genética , Receptores Toll-Like/genética , Adipócitos/patologia , Animais , Dieta Hiperlipídica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Obesidade/etiologia , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor 5 Toll-Like/genética , Redução de Peso
9.
Oncotarget ; 8(46): 80741-80756, 2017 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-29113341

RESUMO

BACKGROUND: Toll-like receptors (TLRs) are involved in the initiation of Schwann cell activation and subsequent recruitment of macrophages for clearance of degenerated myelin and neuronal debris after nerve injury. The present study was designed to investigate the regenerative outcome and expression of myelination-related factors in Tlr-knockout mice following a sciatic nerve crush injury. MATERIALS AND METHODS: A standard sciatic nerve crush injury, induced by applying constant pressure to the nerve with a No. 5 jeweler's forceps for 30 s, was performed in C57BL/6, Tlr2-/- , Tlr3-/- , Tlr4-/- , Tlr5-/- , and Tlr7-/- mice. Quantitative histomorphometric analysis of toluidine blue-stained nerve specimens and walking track analysis were performed to evaluate nerve regeneration outcomes. PCR Arrays were used to detect the expression of neurogenesis-related genes of dorsal root ganglia as well as of myelination-related genes of the distal nerve segments. RESULTS: Worse nerve regeneration after nerve crush injury was found in all Tlr-knockout mice than in C57BL/6 mice. Delayed expression of myelin genes and a different expression pattern of myelination-related neurotrophin genes and transcription factors were found in Tlr-knockout mice in comparison to C57BL/6 mice. In these TLR-mediated pathways, insulin-like growth factor 2 and brain-derived neurotrophic factor, as well as early growth response 2 and N-myc downstream-regulated gene 1, were significantly decreased in the early and late stages, respectively, of nerve regeneration after a crush injury. CONCLUSIONS: Knockout of Tlr genes decreases the expression of myelination-related factors and impairs nerve regeneration after a sciatic nerve crush injury.

10.
Mediators Inflamm ; 2015: 852126, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26681840

RESUMO

BACKGROUND: This study aims to investigate the effect of feeding low-fat diet (LFD) to diet-induced obesity (DIO) mice lacking TLR5 (TLR5(-/-)), which have a tendency to develop glucose intolerance with increased adiposity, compared to that in C57BL/6 mice. RESULTS: TLR5(-/-) and C57BL/6 male mice were divided into three subgroups: (1) control, mice were fed a standard AIN-76A (fat: 11.5 kcal%) diet for 12 weeks; (2) DIO, mice were fed a 58 kcal% high-fat diet (HFD) for 12 weeks; and (3) diet, mice were fed a HFD for 8 weeks to induce obesity and then switched to a 10.5 kcal% LFD for 4 weeks. The glucose intolerance in DIO TLR5(-/-) mice was more significant than that in DIO C57BL/6 mice and was not attenuated by a switch to the LFD. Weight-reduction with LFD had significantly decreased the epididymal fat mass in C57BL/6 mice but not in TLR5(-/-) mice. In addition, the LFD-fed TLR5(-/-) mice showed significantly higher expression of ghrelin in the serum and resistin in the epididymal fat than that in C57BL/6 mice. CONCLUSIONS: This study demonstrated that TLR5 gene knockout impairs some effects of weight-reduction in DIO.


Assuntos
Dieta com Restrição de Gorduras , Obesidade/dietoterapia , Obesidade/imunologia , Receptor 5 Toll-Like/deficiência , Adiposidade , Animais , Citocinas/sangue , Dieta Hiperlipídica/efeitos adversos , Grelina/sangue , Intolerância à Glucose/etiologia , Intolerância à Glucose/imunologia , Intolerância à Glucose/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Resistina/metabolismo , Receptor 5 Toll-Like/genética , Aumento de Peso , Redução de Peso
11.
Dis Markers ; 2015: 863192, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26435568

RESUMO

BACKGROUND: This study aimed to establish the expression profile of circulating microRNAs (miRNAs) during nerve allotransplantation in the presence and absence of FK506 immunosuppression. METHODS: A 1 cm BALB/c donor sciatic nerve graft was transplanted into the sciatic nerve gaps created in recipient C57BL/6 mice with or without daily FK506 immunosuppression [1 mg/(kg·d)]. At 3, 7, and 14 d after nerve allotransplantation, serum samples were collected for miRNA expression analysis by Illumina small RNA deep sequencing. RESULTS: Sequence analysis showed that the dominant size of circulating small RNAs after nerve allotransplantation was 22 nucleotides, followed by 23-nucleotide sequences. Nine upregulated circulating miRNAs (let-7e-5p, miR-101a-3p, miR-151-5p, miR-181a-5p, miR-204-5p, miR-340-5p, miR-381-3p, miR-411-5p, miR-9-5p, and miR-219-2-3p) were identified at 3 d, but none was identified at 7 or 14 d. Among them, miR-9-5p had the highest fold-change of >50-fold, followed by miR-340-5p with 38.8-fold. The presence of these nine miRNAs was not significant at 7 and 14 d after nerve allotransplantation with or without immunosuppression, showing that these miRNAs are not ideal biomarkers for monitoring rejection of deep-buried nerve allografts, a response usually observed later. CONCLUSIONS: We identified nine upregulated circulating miRNAs, which may have a biological function, particularly during the early stages after nerve allotransplantation under FK506 immunosuppression.


Assuntos
Aloenxertos/transplante , Imunossupressores/farmacologia , MicroRNAs/sangue , Nervo Isquiático/transplante , Tacrolimo/farmacologia , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Imunossupressores/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Tacrolimo/efeitos adversos , Regulação para Cima/efeitos dos fármacos
12.
BMC Genomics ; 16: 699, 2015 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-26377847

RESUMO

BACKGROUND: To examine the circulating microRNA (miRNA) expression profile in a mouse model of diet-induced obesity (DIO) with subsequent weight reduction achieved via low-fat diet (LFD) feeding. RESULTS: Eighteen C57BL/6NCrl male mice were divided into three subgroups: (1) control, mice were fed a standard AIN-76A (fat: 11.5 kcal %) diet for 12 weeks; (2) DIO, mice were fed a 58 kcal % high-fat diet (HFD) for 12 weeks; and (3) DIO + LFD, mice were fed a HFD for 8 weeks to induce obesity and then switched to a 10.5 kcal % LFD for 4 weeks. A switch to LFD feeding led to decreases in body weight, adiposity, and blood glucose levels in DIO mice. Microarray analysis of miRNA using The Mouse & Rat miRNA OneArray® v4 system revealed significant alterations in the expression of miRNAs in DIO and DIO + LFD mice. Notably, 23 circulating miRNAs (mmu-miR-16, mmu-let-7i, mmu-miR-26a, mmu-miR-17, mmu-miR-107, mmu-miR-195, mmu-miR-20a, mmu-miR-25, mmu-miR-15b, mmu-miR-15a, mmu-let-7b, mmu-let-7a, mmu-let-7c, mmu-miR-103, mmu-let-7f, mmu-miR-106a, mmu-miR-106b, mmu-miR-93, mmu-miR-23b, mmu-miR-21, mmu-miR-30b, mmu-miR-221, and mmu-miR-19b) were significantly downregulated in DIO mice but upregulated in DIO + LFD mice. Target prediction and function annotation of associated genes revealed that these genes were predominantly involved in metabolic, insulin signaling, and adipocytokine signaling pathways that directly link the pathophysiological changes associated with obesity and weight reduction. CONCLUSIONS: These results imply that obesity-related reductions in the expression of circulating miRNAs could be reversed through changes in metabolism associated with weight reduction achieved through LFD feeding.


Assuntos
Dieta com Restrição de Gorduras , Regulação da Expressão Gênica , MicroRNAs/genética , Obesidade/genética , Redução de Peso/genética , Adiposidade/genética , Animais , Biomarcadores , Glicemia , Peso Corporal , Análise por Conglomerados , Biologia Computacional , Citocinas/sangue , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Mediadores da Inflamação/sangue , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/sangue , Anotação de Sequência Molecular , Obesidade/sangue
13.
Int J Med Sci ; 12(8): 650-4, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26283885

RESUMO

INTRODUCTION: The balance between regulatory T cells (Tregs) and effector T help cells (Th cells) is critical for the control of adaptive immune response during nerve transplantation. However, whether the homeostasis of immune regulation between Tregs and Th cells requires toll-like receptor (TLR) signaling is unclear. The aim of this study is to profile the distribution of spleen Tregs and Th cells in a mouse model of nerve xenografting in the TLR2 and NF-κB gene knockout mice. METHODS: The sciatic nerve was taken from a SD rat or an allogeneic mouse and transplanted to a right back leg of recipient C57BL/6, TLR2(-/-), or NF-κB(-/-) mice by subcutaneous transplantation. After 7 days, the T lymphocytes were then isolated from spleen, stained with phenotyping kits, and analyzed by flow cytometry. RESULTS: The results showed that Tregs were decreased after nerve xenografting in the recipient C57BL/6 mouse. In addition, nerve xenografting also increased the Th1 and Th17 but not the Th2 cell populations. In contrast, amelioration of the Tregs elimination was found in TLR2(-/-) and NF-κB(-/-) mice after transplantation of the nerve xenograft. Moreover, the mice lacking TLR2 or NF-κB showed attenuation of the increase in Th1 and Th17 cells after nerve xenografting. CONCLUSIONS: TLR signaling is involved in T cell population regulation during tissue transplantation. Knock-out of TLR2 and NF-κB prevented Tregs elimination and inhibited Th1- and Th17-driven immune response after nerve xenografting. This study highlighted the potential of inhibiting TLR signaling to modulate T cell-mediated immune regulation to facilitate tolerance to nerve transplantation.


Assuntos
NF-kappa B/metabolismo , Neurônios/transplante , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Reguladores/citologia , Receptor 2 Toll-Like/genética , Animais , Citometria de Fluxo , Xenoenxertos , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/transplante , Transdução de Sinais , Células Th17/citologia , Receptor 2 Toll-Like/metabolismo
14.
J Biomed Sci ; 22: 40, 2015 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-26059504

RESUMO

BACKGROUND: The NF-κB signaling pathway plays a role in local and remote tissue damage following ischemia-reperfusion (I/R) injury to skeletal muscles. Evidence suggests that exosomes can act as intercellular communicators by transporting active proteins to remote cells and may play a role in regulating inflammatory processes. This study aimed to profile the exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury. RESULTS: To investigate the potential changes in protein expression mediated by NF-κB in secreted exosomes in the serum following I/R injury, the levels of circulating exosomal proteomes in C57BL/6 and NF-κB(-/-) mice were compared using two dimensional differential in-gel electrophoresis (2-DE), liquid chromatography tandem mass spectrometry (LC-MS/MS), and proteomic analysis. In C57BL/6 mice, the levels of circulating exosomal proteins, including complement component C3 prepropeptide, PK-120 precursor, alpha-amylase one precursor, beta-enolase isoform 1, and adenylosuccinate synthetase isozyme 1, increased following I/R injury. However, in the NF-κB(-/-) mice, the expression of the following was upregulated in the exosomes: protease, serine 1; glyceraldehyde-3-phosphate dehydrogenase-like isoform 1; glyceraldehyde-3-phosphate dehydrogenase; and pregnancy zone protein. In contrast, the expression of apolipoprotein B, complement component C3 prepropeptide, and immunoglobulin kappa light chain variable region was downregulated in NF-κB(-/-) mice. Bioinformatic annotation using the Protein Analysis Through Evolutionary Relationships (PANTHER) database revealed that the expression of the exosomal proteins that participate in metabolic processes and in biological regulation was lower in NF-κB(-/-) mice than in C57BL/6 mice, whereas the expression of proteins that participate in the response to stimuli, in cellular processes, and in the immune system was higher. CONCLUSIONS: The data presented in this study suggest that NF-κB might regulate exosomal protein expression at a remote site via circulation following I/R injury.


Assuntos
Músculo Esquelético/metabolismo , NF-kappa B/deficiência , Proteoma , Traumatismo por Reperfusão/fisiopatologia , Transdução de Sinais , Animais , Exossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética
15.
Biomed Res Int ; 2015: 410721, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25692136

RESUMO

BACKGROUND: The aim of this study was to profile TLR4/NF-κB-responsive microRNAs (miRNAs) and their potential target genes in the skeletal muscles of mice following ischemia-reperfusion injury. METHODS: Thigh skeletal muscles of C57BL/6, Tlr4(-/-), and NF-κB(-/-) mice isolated based on femoral artery perfusion were subjected to ischemia for 2 h and reperfusion for 0 h, 4 h, 1 d, and 7 d. The muscle specimens were analyzed with miRNA arrays. Immunoprecipitation with an argonaute 2- (Ago2-) specific monoclonal antibody followed by whole genome microarray was performed to identify mRNA associated with the RNA-silencing machinery. The potential targets of each upregulated miRNA were identified by combined analysis involving the bioinformatics algorithm miRanda and whole genome expression. RESULTS: Three TLR4/NF-κB-responsive miRNAs (miR-15a, miR-744, and miR-1196) were significantly upregulated in the muscles following ischemia-reperfusion injury. The combined in silico and whole genome microarray approaches identified 5, 4, and 20 potential target genes for miR-15a, miR-744, and miR-1196, respectively. Among the 3 genes (Zbed4, Lrsam1, and Ddx21) regulated by at least 2 of the 3 upregulated miRNAs, Lrsam1 and Ddx21 are known to be associated with the innate immunity pathway. CONCLUSIONS: This study profiled TLR4/NF-κB-responsive miRNAs and their potential target genes in mouse skeletal muscle subjected to ischemia-reperfusion injury.


Assuntos
Regulação da Expressão Gênica , MicroRNAs/biossíntese , Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , NF-kappa B/metabolismo , Traumatismo por Reperfusão/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Camundongos , Camundongos Knockout , MicroRNAs/genética , Proteínas Musculares/genética , Músculo Esquelético/patologia , NF-kappa B/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Receptor 4 Toll-Like/genética
16.
J Biomed Sci ; 22: 1, 2015 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-25563241

RESUMO

BACKGROUND: We profiled the expression of circulating microRNAs (miRNAs) in mice using Illumina small RNA deep sequencing in order to identify the miRNAs that may potentially be used as biomarkers to distinguish between gram-negative and gram-positive bacterial infections. RESULTS: Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) were used to induce bacterial infection in mice at a concentration of 1 × 10(8) bacteria/100 µL of phosphate buffered saline (PBS). Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14 + Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer. Following exposure to gram-negative bacteria alone, no differentially expressed miRNA was found in the injection, cut, or skin graft models. Exposure to mixed bacteria induced a similar expression pattern of the circulating miRNAs to that induced by gram-positive bacterial infection. Upon gram-positive bacterial infection, 9 miRNAs (mir-193b-3p, mir-133a-1-3p, mir-133a-2-3p, mir-133a-1-5p, mir-133b-3p, mir-434-3p, mir-127-3p, mir-676-3p, mir-215-5p) showed upregulation greater than 4-fold with a p-value < 0.01. Among them, mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p presented the most common miRNA targets expressed in the mice exposed to gram-positive bacterial infection. CONCLUSIONS: This study identified mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p as potential circulating miRNAs for gram-positive bacterial infections.


Assuntos
Infecções por Escherichia coli/genética , Escherichia coli/fisiologia , MicroRNAs/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/fisiologia , Animais , Infecções por Escherichia coli/microbiologia , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Infecções Estafilocócicas/microbiologia
17.
Toxicol Sci ; 140(2): 315-26, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24863965

RESUMO

This aim of this study was to explore the role of miRNA-146a (miR-146a) and its target genes in endothelial cells. We demonstrated that lipopolysaccharide (LPS) induced the upregulation of miR-146a in human umbilical vein endothelial cells (HUVECs), and that the induction was blocked by silencing toll-like receptors, the adaptor molecule MyD88, and the nonspecific NF-κB inhibitor BAY 11-7082. In addition, knockdown of miR-146a by transfection of the locked nucleic acid antimiR-146a significantly inhibited LPS-induced cell migration and tube formation. A combined analysis of bioinformatics miRanda algorithms and a whole genome expression microarray of immunoprecipitated Ago2 ribonucleoprotein complexes identified 14 potential target genes. Subsequent transfection with the miR-146a precursor pre-miR-146a into HUVECs validated that CARD10 was the target gene of the miR-146a, both at the mRNA and protein levels. Silencing CARD10 inhibited p65 nuclear translocation in the cells receiving LPS stimulation and increased angiogenesis. Therefore, miR-146a may play a role in regulating the angiogenesis in HUVECs by downregulating CARD10, which acts in a negative feedback regulation loop to inhibit the activation of NF-κB that normally impairs angiogenesis.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , Neovascularização Fisiológica/efeitos dos fármacos , Sequência de Bases , Western Blotting , Primers do DNA , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase
18.
J Biomed Sci ; 21: 20, 2014 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-24618279

RESUMO

BACKGROUND: Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model. RESULTS: The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 µg LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-µg LPS injection in Tlr4-/- mice. When the PI3K inhibitor LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4-/- mice was 30%. In the Tlr4-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 µg LPS led to a significant induction in O2⁻ detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4-/- mice. Addition of LY294002 only significantly increased the O2⁻ level in the lung and liver of the Tlr4-/- mice but not in the C57BL/6 mice following 500-µg LPS injection. In addition, the serum IL-1ß and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4-/- mice. Notably, IL-1ß and IL-2 were significantly increased in Tlr4-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by LY294002 prior to LPS injection. CONCLUSIONS: In this study, we demonstrate that innate resistance to LPS toxicity in Tlr4-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O2⁻ and inflammatory cytokines.


Assuntos
Lipopolissacarídeos/toxicidade , Fosfatidilinositol 3-Quinases/metabolismo , Receptor 4 Toll-Like/genética , Animais , Humanos , Interleucina-1beta/biossíntese , Camundongos , NF-kappa B/metabolismo , Oxigênio/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositóis/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/biossíntese , Transdução de Sinais/efeitos dos fármacos
19.
PLoS One ; 8(10): e77936, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24205035

RESUMO

The levels of circulating microRNAs (miRNAs) in mice with experimental sepsis induced by cecal ligation and puncture (CLP) were determined using whole blood samples obtained from C57BL/6 mice at 4, 8, and 24 h after CLP; miRNA expression analysis was performed in these samples using an miRNA array. Microarray analysis revealed upregulation of 10 miRNA targets (miR-16, miR-17, miR-20a, miR-20b, miR-26a, miR-26b, miR-106a, miR-106b, miR-195, and miR-451). The expression of these miRNA targets in the whole blood, serum, and white blood cells (WBCs) of CLP mice was quantified using quantitative real-time PCR; these values were compared to those in sham-operated C57BL/6 mice, and the results indicated that these miRNA targets were significantly up-regulated in the whole blood and serum but not in the WBCs. In addition, the levels of these 10 miRNA targets in the serum of Tlr2-/-, Tlr4-/-, and NF-κB-/- mice at 8 h after CLP did not decrease significantly., which indicated that the transcription of these miRNAs was not directly mediated by the TLR2/NF-κB or TLR4/NF-κB pathway, and pathways induced by exposure to the gram-positive or gram-negative bacteria. Immunoprecipitation with the Argonaute 2 ribonucleoprotein complex revealed significantly increased expression of the 10 miRNA targets in the serum of mice after CLP, and the levels of 6 (miR-16, miR-17, miR-20a, miR-20b, miR-26a, and miR-26b) of these 10 miRNA targets increased significantly in exosomes isolated using ExoQuick precipitation solution. In this study, we identified circulating miRNAs that were up-regulated after CLP and determined the increase in the levels of these miRNAs, and our results suggest that circulating Ago2 complexes and exosomes may be responsible for the stability of miRNAs in the serum.


Assuntos
Biomarcadores/metabolismo , Ceco/lesões , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , MicroRNAs/sangue , Sepse/metabolismo , Ferimentos Perfurantes/genética , Animais , Western Blotting , Ceco/metabolismo , Imunoprecipitação , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Análise em Microsséries , NF-kappa B/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/etiologia , Sepse/patologia , Receptor 4 Toll-Like/fisiologia
20.
J Biomed Sci ; 20: 64, 2013 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-24011263

RESUMO

BACKGROUND: The lack of noninvasive biomarkers of rejection remains a challenge in the accurate monitoring of deeply buried nerve allografts and precludes optimization of therapeutic intervention. This study aimed to establish the expression profile of circulating microRNAs (miRNAs) during nerve allotransplantation with or without immunosuppression. RESULTS: Balb/c mice were randomized into 3 experimental groups, that is, (1) untreated isograft (Balb/c → Balb/c), (2) untreated allograft (C57BL/6 → Balb/c), and (3) allograft (C57BL/6 → Balb/c) with FK506 immunosuppression. A 1-cm Balb/c or C57BL/6 donor sciatic nerve graft was transplanted into sciatic nerve gaps created in recipient mice. At 1, 3, 7, 10, and 14 d after nerve transplantation, nerve grafts, whole blood, and sera were obtained for miRNA expression analysis with an miRNA array and subsequent validation with quantitative real-time PCR (qRT-PCR). Three circulating miRNAs (miR-320, miR-762, and miR-423-5p) were identified in the whole blood and serum of the mice receiving an allograft with FK506 immunosuppression, within 2 weeks after nerve allotransplantation. However, these 3 circulating miRNAs were not expressed in the nerve grafts. The expression of all these 3 upregulated circulating miRNAs significantly decreased at 2, 4, and 6 d after discontinuation of FK506 immunosuppression. In the nerve graft, miR-125-3b and miR-672 were significantly upregulated in the mice that received an allograft with FK506 only at 7 d after nerve allotransplantation. CONCLUSIONS: We identified the circulating miR-320, miR-762, and miR-423-5p as potential biomarkers for monitoring the immunosuppression status of the nerve allograft. However, further research is required to investigate the mechanism behind the dysregulation of these markers and to evaluate their prognostic value in nerve allotransplantation.


Assuntos
Aloenxertos/transplante , MicroRNAs/genética , Nervo Isquiático/transplante , Aloenxertos/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/sangue , Reação em Cadeia da Polimerase em Tempo Real , Nervo Isquiático/metabolismo , Transplante Homólogo
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