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This study used constitutive CD226 gene knockout (KO) mice as a model to investigate the functions and mechanisms of CD226 in megakaryocyte (MK) maturation and platelet activation. Although CD226 deficiency did not cause MK polyploidization or platelet granule abnormalities, increased MK counts were detected in the femora bone marrow (BM) and spleen of CD226 KO mice. Particularly, CD226 KO mice have a more extensive membrane system in MKs and platelets than wild-type (WT) mice. We also demonstrated that CD226 KO mice displayed increased platelet counts, shortened bleeding time, and enhanced platelet aggregation. CD226 KO platelets had an increased mature platelet ratio compared to the control platelets. In addition, the observed reduction in bleeding time may be due to decreased nitric oxide (NO) production in the platelets. Platelet-specific CD226-deficient mice showed similar increased MK counts, shortened bleeding time, enhanced platelet aggregation, and decreased NO production in platelets. Furthermore, we performed middle cerebral artery occlusion-reperfusion surgery on WT and CD226 KO mice to explore the potential effect of CD226 on acute ischemia-reperfusion injury; the results revealed that CD226 deficiency led to significantly increased infarct area. Thus, CD226 is a promising candidate for the treatment of thrombotic disorders.
Assuntos
Antígenos de Diferenciação de Linfócitos T/sangue , Megacariócitos/citologia , Megacariócitos/fisiologia , Ativação Plaquetária/fisiologia , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Plaquetas/fisiologia , Plaquetas/ultraestrutura , Isquemia Encefálica/sangue , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Feminino , Integrina beta3/sangue , Masculino , Megacariócitos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Ativação Plaquetária/genética , Adesividade Plaquetária/genética , Adesividade Plaquetária/fisiologia , Agregação Plaquetária/genética , Agregação Plaquetária/fisiologia , Contagem de Plaquetas , Trombopoese/genética , Trombopoese/fisiologiaRESUMO
BACKGROUND: Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a collagen receptor belonging to the immunoglobulin superfamily. Although previous studies have evaluated the biological role of LAIR in solid tumors, the precise mechanisms underlying the functions of LAIR-1 as a regulator of tumor biological functions remain unclear. METHODS: LAIR-1 expression was evaluated by immunohistochemical analysis using an osteosarcoma (OS) tissue microarray. Wound healing and transwell migration assays were performed to evaluate tumor cell migration. Quantitative real-time polymerase chain reaction (qPCR) and western blotting were conducted to detect the expression of epithelial-mesenchymal transition (EMT)-related molecules. RNA-sequencing (RNA-seq) was conducted to evaluate the mRNA expression profiles after overexpressing LAIR-1 in OS cells. Glucose transporter (Glut)1 expression in OS cells was evaluated by western blotting. RESULTS: LAIR-1 expression was significantly different between the T1 and T2 stages of OS tumors, and it inhibited OS cell migration. LAIR-1 expression was inversely correlated with the expression of Twist1, an EMT-associated transcription factor, via the Forkhead box O1 signal transduction pathway. Furthermore, RNA-seq and qPCR demonstrated that the expression of EMT energy metabolism-related molecules was significantly reduced after LAIR-1 overexpression. CONCLUSIONS: LAIR-1 overexpression decreased the expression of Glut1 and inhibited the expression of EMT-related molecules in OS cells. These findings provide new insights into the molecular mechanism underlying OS progression.
Assuntos
Neoplasias Ósseas/patologia , Metabolismo Energético , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Transportador de Glucose Tipo 1/metabolismo , Osteossarcoma/patologia , Receptores Imunológicos/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Masculino , Osteossarcoma/genética , Osteossarcoma/metabolismo , Prognóstico , Receptores Imunológicos/genética , Transdução de Sinais , Taxa de Sobrevida , Adulto JovemRESUMO
Formation of macrophage-derived foam cells may mark the initial stages of atherosclerosis. We investigated the association between the expression of the leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) in macrophages and foam cell formation. A foam cell model was established by incubating THP-1-derived macrophages and bone marrow macrophages (BMMs) with oxidized low-density lipoprotein (ox-LDL). The role of LAIR-1 in foam cell formation was evaluated via Oil Red O staining and Dil-ox-LDL fluorescence intensities. Peroxisome proliferator-activated receptor gamma (PPARγ), cholesterol metabolism-related genes, and the role of LAIR-1 in activating classically activated (M1) and alternatively activated (M2) macrophages were evaluated by qPCR. Additionally, activation of protein-tyrosine phosphatase-1 (SHP-1) and cAMP-response element binding protein (CREB) were detected by western blotting. Results indicated that silencing LAIR-1 in macrophages modulated the SHP-1/CREB/PPARγ pathway, thereby promoting M2 macrophage polarization and increasing foam cell formation. Therefore, Inhibition of LAIR-1 in macrophages may promote foam cell formation and atherosclerosis.
Assuntos
Células Espumosas/metabolismo , Macrófagos/metabolismo , PPAR gama/metabolismo , Receptores Imunológicos/metabolismo , Células THP-1/metabolismo , Animais , Aterosclerose/metabolismo , Linhagem Celular , Colesterol/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Humanos , Metabolismo dos Lipídeos/fisiologia , Lipoproteínas LDL/metabolismo , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Células RAW 264.7 , Transdução de Sinais/fisiologiaRESUMO
A salamander is an ideal animal for studying the spinal locomotor network mechanism of vertebrates from an evolutionary perspective since it represents the transition from an aquatic to a terrestrial animal. However, little is known about the spinal locomotor network of a salamander. A spinal locomotor network model is a useful tool for exploring the working mechanism of the spinal networks of salamanders. A new spinal locomotor network model for a salamander is built for a three-dimensional (3D) biomechanical model of the salamander using a novel locomotion-controlled neural network model. Based on recent experimental data on the spinal circuitry and observational results of gaits of vertebrates, we assume that different interneuron sets recruited for mediating the frequency of spinal circuits are also related to the generation of different gaits. The spinal locomotor networks of salamanders are divided into low-frequency networks for walking and high-frequency networks for swimming. Additionally, a new topological structure between the body networks and limb networks is built, which only uses the body networks to coordinate the motion of limbs. There are no direct synaptic connections among limb networks. These techniques differ from existing salamander spinal locomotor network models. A simulation is performed and analyzed to validate the properties of the new spinal locomotor networks of salamanders. The simulation results show that the new spinal locomotor networks can generate a forward walking gait, a backward walking gait, a swimming gait, and a turning gait during swimming and walking. These gaits can be switched smoothly by changing external inputs from the brainstem. These properties are consistent with those of a real salamander. However, it is still difficult for the new spinal locomotor networks to generate highly efficient turning during walking, 3D swimming, nonrhythmic movements, and so on. New experimental data are required for further validation.
Assuntos
Locomoção/fisiologia , Modelos Neurológicos , Vias Neurais/fisiologia , Neurônios/fisiologia , Medula Espinal/fisiologia , Animais , Fenômenos Biomecânicos , Modelos Teóricos , Rede Nervosa/fisiologia , Natação , Urodelos/fisiologiaRESUMO
Platelet-rich plasma (PRP) is a platelet-based application used to treat osteoarthritis (OA) clinically. The co-stimulatory molecule CD226 is expressed in T cells, NK cells, and also platelets. However, exact effects of CD226 on platelets and whether its expression level influences PRP efficacy are largely unknown. Here, CD226fl/flPF4-Cre mice were obtained from mating CD226 fl/fl mice with PF4-Cre mice. Blood samples and washed platelets were collected from the mice eyeballs to undergo routine blood tests and transmission electron microscopy. Differentially expressed proteins were detected by iTRAQ-based proteomics analysis. Animal OA models were established through surgical destabilization of the medial meniscus (DMM) for C57BL/6 wildtype mice, followed by PRP injection to evaluate the effects of platelet CD226 on PRP efficacy. The results showed that deletion of platelet CD226 increased the number of megakaryocytes (MKs) in bone marrow (BM) but reduced MKs in spleen, combined with significantly decreased platelet amounts, α-granule secretion, and reduced immature platelets; indicating that absence of platelet CD226 may disrupt MK/platelet homeostasis and arrested platelet release from MKs. Sequencing analysis showed abnormal ribosomal functions and much downregulated proteins in the absence of platelet CD226. Autophagy-related proteins were also reduced in the CD226-absent MKs/platelets. Moreover, deletion of platelet CD226 diminished the protective effects of PRP on DMM-induced cartilage lesions in mice, and PDGF restored it. Therefore, deficiency of platelet CD226 inhibited platelet maturation, secretion, and normal ribosomal functions, which may lead to depressed PRP efficacy on OA, suggesting that CD226 is required to regulate platelet growth, functions, and its application.
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BACKGROUND: Osteosarcoma (OS) is a highly malignant and aggressive bone tumor. This study was performed to explore the mechanisms of HuR (human antigen R) in the progression of OS. METHODS: HuR expression levels in OS tissues and cells were detected by immunohistochemistry and western blotting. HuR siRNA was transfected into SJSA-1 OS cells to downregulate HuR expression, and then cell proliferation, migration, and epithelial-mesenchymal transition (EMT) were evaluated. RNA immunoprecipitation was performed to determine the association of the long non-coding RNA (lncRNA) XIST and argonaute RISC catalytic component (AGO) 2 with HuR. Fluorescence in situ hybridization analysis was performed to detect the expression of lncRNA XIST. Western blotting and immunofluorescence assays were performed to observe AGO2 expression after HuR or/and lncRNA XIST knockdown. RESULTS: Knockdown of HuR repressed OS cell migration and EMT. AGO2 was identified as a target of HuR and silencing of HuR decreased AGO2 expression. The lncRNA XIST was associated with HuR-mediated AGO2 suppression. Moreover, knockdown of AGO2 significantly inhibited cell proliferation, migration, and EMT in OS. CONCLUSION: Our findings indicate that HuR knockdown suppresses OS cell EMT by regulating lncRNA XIST/AGO2 signaling.
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In this study, we observed that deletion of CD226 on regulatory T cells (Tregs) precedes renal fibrosis in a mouse unilateral ureteral obstruction (UUO) model. First, we generated Treg-specific CD226 gene knockout mice (CD226fl/fl Foxp3YFP-Cre ). Next, CD226fl/fl Foxp3YFP-Cre mice and Foxp3YFP-Cre control mice were subjected to UUO surgery. Pathologic analysis and Sirius red and Masson's trichrome staining showed that the kidneys of CD226fl/fl Foxp3YFP-Cre mice following UUO showed much more severe interstitial fibrosis than Foxp3YFP-Cre control mice at days 10 and 20. Additionally, CD226fl/fl Foxp3YFP-Cre mice showed increased fibronectin expression, as demonstrated by immunohistochemistry (IHC) staining. Although Treg cell-restricted CD226 deficiency showed increased Foxp3+ expression, expression of the cell surface functional molecule CD103 was significantly reduced, indicating impaired homeostasis in the Tregs of CD226fl/fl Foxp3YFP-Cre mice. To better understand CD226 function, RNA sequencing (RNA-Seq) analysis was conducted in Tregs isolated from CD226fl/fl Foxp3YFP-Cre and Foxp3YFP-Cre mice. RNA-Seq data showed that the helper T cell (Th) 2-related cytokines IL-4 and IL-10 were significantly up-regulated in CD226 deficient Tregs. In addition, mRNA analysis of kidney samples from the mice following UUO by qPCR also showed increased IL-4 and IL-10 expression in CD226fl/fl Foxp3YFP-Cre mice, as well as elevated TGF-ß1 levels, indicating that CD226 deficiency in Tregs resulted in the acquisition of the ability to produce Th2 cytokines. Finally, we found that microRNA-340 (miR-340), which was down-regulated in Tregs isolated from CD226fl/fl Foxp3YFP-Cre mice, directly regulated IL-4 gene expression in vitro. These data suggest that the promotion of CD226 signaling on Tregs is a therapeutic target for renal disease.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Citocinas/metabolismo , Rim/patologia , MicroRNAs/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th2/metabolismo , Regulação para Cima , Animais , Sequência de Bases , Sítios de Ligação , Regulação para Baixo , Fibrose , Fatores de Transcrição Forkhead/metabolismo , Homeostase , Inflamação/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Fenótipo , RNA-Seq , Células Th1 , Obstrução Ureteral/patologiaRESUMO
Intervertebral disc degeneration (IDD) induces serious back, neck and radicular pain. Recently, moxibustion has been suggested as an effective treatment for IDD. Thus, our study aims to investigate the molecular mechanism of moxibustion in IDD. A rat model of IDD was established by moxibustion treatment. Nucleus pulposus (NP) cells isolated from IDD rats or IDD rats treated with moxibustion were transfected with plasmids harboring overexpressed hypoxia-inducible factor-1 alpha (HIF-1α) to understand the role of treatment on cell autophagy and apoptosis. To investigate the mechanism of moxibustion in IDD, aggrecan, cyclo-oxygenase 2 (COX-2), HIF-1α and vascular endothelial growth factor (VEGF) expression in NP cells was measured. The expression of aggrecan and COX-2 was elevated by moxibustion treatment. Moxibustion induced autophagy and suppressed apoptosis of NP cells from IDD rats. Compared with IDD rats, the expression of light chain 3 (LC3) II/I, Beclin-1, B-cell lymphoma-2 (Bcl-2) and HIF-1α was regulated significantly after moxibustion treatment, while the expression of cleaved-caspase-3, Bcl-2 associated protein X and VEGF was downregulated. In general, moxibustion may be beneficial to IDD by enhancing autophagy and reducing apoptosis of NP cells via the HIF-1α/VEGF pathway.
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Objective To investigate the effect of CD226 knockout (KO) on radiation-induced liver injury in mice. Methods Wild-type (WT) and CD226KO mice were randomly divided into naive group and irradiated group. The mice in the irradiated group were exposed to an 8 Gy of 60Co γ-radiation. The survival rate was observed and body mass loss was measured. The pathological changes in the liver and colon were examined by HE staining, and the mRNA levels of inflammatory factors, including inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), IL-6, IL-12p40, tumor necrosis factor α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were detected by real-time quantitative PCR. Results Compared with the irradiated WT mice, the irradiated CD226KO mice had increased body mass loss and decreased survival rate. In the irradiated CD226KO mice, the liver injury was more serious, the liver index (liver mass/body mass) was reduced, and the expressive levels of iNOS, IL-1ß, IL-6, IL-12p40, TNF-α and MCP-1 were enhanced; whereas, the colitis was less serious, and the expressive levels of inflammatory factors were down-regulated. Conclusion The deficiency of CD226 can decrease the survival rate of irradiated mice, which is associated with the aggravated liver injury.
Assuntos
Fígado , Animais , Antígenos de Diferenciação de Linfócitos T , Interleucina-1beta , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfaRESUMO
This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N-4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA-binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Megacariócitos/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Subunidade p45 do Fator de Transcrição NF-E2/genética , Subunidade p45 do Fator de Transcrição NF-E2/imunologia , Selectina-P/genética , Selectina-P/imunologia , Ploidias , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/imunologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
CD226 is a co-stimulatory adhesion molecule found on immune and endothelial cells. Here, we evaluated a possible role for CD226 in inhibiting glucose uptake in isolated human umbilical vein endothelial cells (HUVECs) and in wild-type (WT) and CD226 knockout (KO) mice with high-fat diet (HFD)-induced type 2 diabetes (T2DM). CD226 expression increased under hyperglycemic conditions in the presence of TNF-α. Furthermore, CD226 knockdown improved glucose uptake in endothelial cells, and CD226 KO mice exhibited increased glucose tolerance. Levels of soluble CD226 in plasma were higher in T2DM patients following an oral glucose tolerance test (OGTT) than under fasting conditions. Our results indicate that low-grade inflammation coupled with elevated blood glucose increases CD226 expression, resulting in decreased endothelial cell glucose uptake in T2DM.
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Antígenos de Diferenciação de Linfócitos T/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Hiperglicemia/metabolismo , Animais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Dieta Hiperlipídica , Células Endoteliais , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To observe analgesic effect of scalp acupuncture on labor. METHODS: Seventy primiparae with term pregnancy and monocyesis were randomly divided into scalp acupuncture group treated by acupuncture at the Shengzhi area of scalp, and control group by no treatment. Pain grades before and after scalp acupuncture were evaluated with the pain 4-grade rating criteria stipulated by WHO, and the active stage and the second birth process, the Apgar scores of new-born and postpartum hemorrage amount were compared between the two groups. RESULTS: The labor pain with 1 to approximately 2 grades was found in 33 cases in the scalp acupuncture group, and 2 cases in the control group with very significant difference between the two groups (P < 0.01); the active stage was (130.70 +/- 74.16 ) min and the second birth process was (40.70 +/- 21.65) min in the scalp acupuncture group, and (166.15 +/- 62.65) min and (53.30 +/- 26.93) min in the control group, respectively, with significant differences between the two groups (all P < 0.05); and there were no significant differences in Apgar score of new-born and postpartum hemorrhage amount. CONCLUSION: Scalp acupuncture has a better analgesic effect in vaginal delivery with no adverse effect on the mother and infant.