RESUMO
Vascular smooth muscle cells (VSMCs) can transdifferentiate into macrophage-like cells in the context of sustained inflammatory injury, which drives vascular hyperplasia and atherosclerotic complications. Using single-cell RNA sequencing, we identify that macrophage-like VSMCs are the key cell population in mouse neointimal hyperplasia. Sex-determining region Y (SRY)-related HMG-box gene 10 (Sox10) upregulation is associated with macrophage-like VSMC accumulation and pyroptosis in vitro and in the neointimal hyperplasia of mice. Tumor necrosis factor α (TNF-α)-induced Sox10 lactylation in a phosphorylation-dependent manner by PI3K/AKT signaling drives transcriptional programs of VSMC transdifferentiation, contributing to pyroptosis. The regulator of G protein signaling 5 (RGS5) interacts with AKT and blocks PI3K/AKT signaling and Sox10 phosphorylation at S24. Sox10 silencing mitigates vascular inflammation and forestalls neointimal hyperplasia in RGS5 knockout mice. Collectively, this study shows that Sox10 is a regulator of vascular inflammation and a potential control point in inflammation-related vascular disease.
Assuntos
Músculo Liso Vascular , Proteínas Proto-Oncogênicas c-akt , Camundongos , Animais , Hiperplasia/patologia , Músculo Liso Vascular/metabolismo , Proliferação de Células/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piroptose , Fosfatidilinositol 3-Quinases/metabolismo , Transdiferenciação Celular , Neointima/metabolismo , Neointima/patologia , Camundongos Knockout , Inflamação/patologia , Miócitos de Músculo Liso/metabolismo , Células Cultivadas , Movimento Celular , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismoRESUMO
The congenital intellectual disability (ID)-causing gene mutations remain largely unclear, although many genetic variations might relate to ID. We screened gene mutations in Chinese Han children suffering from severe ID and found a single-nucleotide polymorphism (SNP) in the 5'-untranslated region (5'-UTR) of fibroblast growth factor 13 (FGF13) mRNA (NM_001139500.1:c.-32c>G) shared by three male children. In both HEK293 cells and patient-derived induced pluripotent stem cells, this SNP reduced the translation of FGF13, which stabilizes microtubules in developing neurons. Mice carrying the homologous point mutation in 5'-UTR of Fgf13 showed delayed neuronal migration during cortical development, and weakened learning and memory. Furthermore, this SNP reduced the interaction between FGF13 5'-UTR and polypyrimidine-tract-binding protein 2 (PTBP2), which was required for FGF13 translation in cortical neurons. Thus, this 5'-UTR SNP of FGF13 interferes with the translational process of FGF13 and causes deficits in brain development and cognitive functions.
Assuntos
Regiões 5' não Traduzidas/genética , Fatores de Crescimento de Fibroblastos/genética , Deficiência Intelectual/genética , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Adolescente , Animais , Criança , Pré-Escolar , Fatores de Crescimento de Fibroblastos/metabolismo , Células HEK293 , Humanos , Aprendizagem , Masculino , Memória , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Objective: Vascular smooth muscle cells (VSMCs) undergo the phenotypic changes from contractile to synthetic state during vascular remodeling after ischemia. SIRT1 protects against stress-induced vascular remodeling via maintaining VSMC differentiated phenotype. However, the effect of smooth muscle SIRT1 on the functions of endothelial cells (ECs) has not been well clarified. Here, we explored the role of smooth muscle SIRT1 in endothelial angiogenesis after ischemia and the underlying mechanisms. Methods: We performed a femoral artery ligation model using VSMC specific human SIRT1 transgenic (SIRT1-Tg) and knockout (KO) mice. Angiogenesis was assessed in in vivo by quantification of the total number of capillaries, wound healing and matrigel plug assays, and in vitro ECs by tube formation, proliferation and migration assays. The interaction of HIF1α with circRNA was examined by using RNA immunoprecipitation, RNA pull-down and in situ hybridization assays. Results: The blood flow recovery was significantly attenuated in SIRT1-Tg mice, and markedly improved in SIRT1-Tg mice treated with SIRT1 inhibitor EX527 and in SIRT1-KO mice. The density of capillaries significantly decreased in the ischemic gastrocnemius of SIRT1-Tg mice compared with SIRT1-KO and WT mice, with reduced expression of VEGFA, which resulted in decreased number of arterioles. We identified that the phenotypic switching of SIRT1-Tg VSMCs was attenuated in response to hypoxia, with high levels of contractile proteins and reduced expression of the synthetic markers and NG2, compared with SIRT1-KO and WT VSMCs. Mechanistically, SIRT1-Tg VSMCs inhibited endothelial angiogenic activity induced by hypoxia via the exosome cZFP609. The cZFP609 was delivered into ECs, and detained HIF1α in the cytoplasm via its interaction with HIF1α, thereby inhibiting VEGFA expression and endothelial angiogenic functions. Meantime, the high cZFP609 expression was observed in the plasma of the patients with atherosclerotic or diabetic lower extremity peripheral artery disease, associated with reduced ankle-brachial index. Knockdown of cZFP609 improved blood flow recovery after hindlimb ischemia in SIRT1-Tg mice. Conclusions: Our findings demonstrate that SIRT1 may impair the plasticity of VSMCs. cZFP609 mediates VSMCs to reprogram endothelial functions, and serves as a valuable indicator to assess the prognosis and clinical outcomes of ischemic diseases.
Assuntos
Células Endoteliais , Isquemia , Miócitos de Músculo Liso , Neovascularização Fisiológica , Sirtuína 1/fisiologia , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Artéria Femoral/fisiologia , Fêmur/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/metabolismo , Isquemia/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fluxo Sanguíneo Regional , Transativadores/metabolismoRESUMO
Systemic inflammatory response markers are associated with poor survival in many types of malignances. This study aimed to evaluate the prognostic value of preoperative neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), lymphocyte-monocyte ratio (LMR), and C-reactive protein (CRP) in patients with non-small cell lung cancer (NSCLC).We retrospectively evaluated 254 NSCLC patients who underwent radical surgery between January 2012 and April 2014 in the Sichuan Provincial Cancer Hospital. The cut-off values of NLR, PLR, LMR, and CRP were determined according to the receiver operating characteristic curve, and the correlation of NLR, PLR, LMR, and CRP with prognosis was analyzed based on the cut-off value.The cut-off value for NLR, PLR, LMR, and CRP were 3.18, 122, 4.04, and 8.8, respectively. Univariate analysis showed that age (Pâ=â.022), tumor-node-metastasis (TNM) stage (Pâ<â.001), T stage (Pâ=â.001), and N stage (Pâ<â.001) were significantly correlated with disease-free survival (DFS), while age (Pâ=â.011), TNM stage (Pâ<â.001), T stage (Pâ=â.008), N stage (Pâ<â.001), and PLR (Pâ=â.001) were significantly correlated with overall survival (OS). In multivariate analysis, age (hazard ratio [HR]: 1.564, 95% confidence interval [CI]: 1.087-2.252, Pâ=â.016) and TNM stage (HR: 1.704, 95% CI: 1.061-2.735, Pâ=â.027) remained independent risk factors affecting DFS, while age (HR: 1.721, 95% CI: 1.153-2.567, Pâ=â.008), TNM stage (HR: 2.198, 95% CI: 1.263-3.824, Pâ=â.005), and PLR (HR: 1.850, 95% CI: 1.246-2.746, Pâ=â.002) were independent risk factors affecting OS.The preoperative PLR is superior to NLR, LMR, and CRP as a biomarker for evaluating the prognosis of patients undergoing curative surgery for NSCLC.
Assuntos
Plaquetas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Inflamação/metabolismo , Neoplasias Pulmonares/patologia , Linfócitos/metabolismo , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Proteína C-Reativa/metabolismo , Carcinoma Pulmonar de Células não Pequenas/sangue , Feminino , Humanos , Neoplasias Pulmonares/sangue , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Neutrófilos/metabolismo , Análise de SobrevidaRESUMO
Vesicular stomatitis virus (VSV) has shown promise in cancer treatment. However, it achieved limited effects against lung cancer. Lung cancer has intrinsic mechanisms that render resistance to VSV. In this study, we attempted to explore the expression of the anti-apoptotic factor Livin in lung adenocarcinoma and its possible relationship to VSV vulnerability. We found VSV induced apoptosis in a time- and dose-dependent manner, with the concomitant change in the expression of Livin. We elevated the expression of Livin both transiently and stably, and the cells became insensitive to VSV treatment. We further found the BIR domain of Livin was mainly responsible for its modulation effects. This finding suggested a possible interaction with the second mitochondria-derived activator of caspase (SMAC). The knock-down of SMAC also inhibited apoptosis by VSV. The relationship was confirmed by the co-immunoprecipitation. Finally, we knocked down the endogenous Livin, and the knock-down sensitized cells to VSV treatment. Our results suggested the important role of Livin and its partner molecule in the process of VSV treatment.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma/terapia , Proteínas Inibidoras de Apoptose/genética , Neoplasias Pulmonares/terapia , Proteínas de Neoplasias/genética , Terapia Viral Oncolítica , Vírus da Estomatite Vesicular Indiana/genética , Adenocarcinoma/genética , Adenocarcinoma/virologia , Adenocarcinoma de Pulmão , Apoptose/genética , Caspase 3/genética , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/virologiaRESUMO
Xingnaojing (XNJ), is a standardized Chinese herbal medicine product derived from An Gong Niu Huang Pill. It may be involved in neuroprotection in a number of neurological disorders. Exposure to anesthetic agents during the brain growth spurt may trigger widespread neuroapoptosis in the developing brain. Thus the present study aimed to identify whether there was a neuroprotective effect of XNJ on anesthesiainduced neuroapoptosis. Sevendayold rats received treatment with 2.1% sevoflurane for 6 h. Rat pups were injected intraperitoneally with 1 or 10 ml/kg XNJ at 0.2, 24 and 48 h prior to sevoflurane exposure. The striata of neonatal rats were collected following administration of anesthesia. Western blotting and immunohistochemistry were used to analyze the expression of activated caspase 3, Bax and phosphorylated protein kinase B (pAKT) in the striatum. It was found that activated caspase 3 and Bax expression were upregulated in the striatum following sevoflurane treatment. Preconditioning with XNJ attenuated the neuronal apoptosis induced by sevoflurane in a dosedependent manner. Anesthesia reduced the expression of pAKT (phosphorylated at sites Thr308 and Ser473) and phosphorylated extracellularregulated protein kinase (pERK) in the striatum. Pretreatment with XNJ reversed the reduction in pAKT, but not pERK expression. These data suggest that XNJ has an antiapoptotic effect against sevofluraneinduced cell loss in the striatum. It thus holds promise as a safe and effective neuroprotective agent. The action of XNJ on pAKT may make a significant contribution to its neuroprotective effect.
Assuntos
Apoptose/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Animais , Animais Recém-Nascidos , Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Corpo Estriado/patologia , Medicamentos de Ervas Chinesas/administração & dosagem , Feminino , Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Éteres Metílicos/efeitos adversos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Pré-Medicação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Sevoflurano , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Platelet dysfunction and thrombocytopenia are common responses to sepsis, but how sepsis changes platelet function is not completely understood. This is due, in part, to our lack of understanding of how sepsis alters platelet protein patterns. The aim of the present study, accordingly, was to investigate the response of the platelet proteome to sepsis. We applied proteomic technology to analyze platelet samples of rats with sepsis. Rats were divided into two groups: 1) sham surgery and 2) sepsis induced by cecal ligation and puncture (CLP) surgery. Platelet samples were collected from surviving rats 12 and 24h after surgery, and platelet proteins were separated by two-dimensional electrophoresis (2-DE). Differentially expressed proteins were identified by mass spectrometry (MS). In the CLP group, there were 20 spots that were statistically significantly different at 12h. Of these spots, 16 spots were increased and four spots were decreased. At 24h, there were six spots increased in the CLP group. Of the 26 spots, 12 proteins associated with platelet activation, acute phase proteins, cytoskeleton structure, and energy production were identified. Of interest, alpha-1-antitrypsin precursor (AAT) and ATP synthase beta subunit (ATPB) were both increased at 12 and 24h of sepsis by 2-DE and immunoblotting. By providing the platelet profiles, our results demonstrate that this proteomic approach brings us closer to understanding how platelet dysfunction develops after sepsis.
Assuntos
Plaquetas/metabolismo , Perfilação da Expressão Gênica , Ativação Plaquetária/genética , Proteoma/genética , Proteômica , Sepse/genética , Trombocitopenia/genética , Animais , Plaquetas/citologia , Ceco/cirurgia , Modelos Animais de Doenças , Eletroforese em Gel Bidimensional , Masculino , Espectrometria de Massas , Contagem de Plaquetas , Ratos , Ratos Sprague-Dawley , Sepse/complicações , Sepse/metabolismo , Trombocitopenia/complicações , Trombocitopenia/metabolismoRESUMO
Triptolide is a potent immunosuppressive drug capable of inhibiting T cell activation and proliferation. Recent studies show that T cells play an important role in neuropathic pain following nerve injury in rats. In this study, we investigated the effect of triptolide on T cell activation and development of neuropathic pain. Neuropathic pain by chronic constriction injury (CCI) was induced by loose ligation of the sciatic nerve in Sprague-Dawley rats. Triptolide (5 or 10 µg/kg) or vehicle (DMSO) was administered intrathecally after surgery for 7 days (n=8 per group). The right hind paw withdrawal threshold to von Frey filament stimuli and withdrawal latency to radiant heat were determined before and after the surgery (days 0 to 7). NF-κB activation and pro-inflammatory cytokine (TNF-α and IL-2) expression were determined by ELISA, Western blot, and real time-PCR. CCI of the sciatic nerve induced mechanical allodynia and thermal hyperalgesia in these rats. Intrathecal triptolide (5 and 10 µg/kg) suppressed the development of allodynia and thermal hyperalgesia. It also inhibited CCI-induced inflammation and T cell activation, by decreasing spinal cord TNF-α, IL-2 and NF-κB p65 levels. Motor dysfunction was not observed after triptolide treatment. In the present study, we demonstrated the suppressive effect of triptolide on the development of neuropathic pain. Therefore, triptolide could be a promising immunosuppressive agent in the treatment of neuropathic pain. Further studies are required to examine the safety of intrathecal triptolide for clinical application.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Dor Crônica/tratamento farmacológico , Diterpenos/administração & dosagem , Neuralgia/tratamento farmacológico , Fenantrenos/administração & dosagem , Animais , Compostos de Epóxi/administração & dosagem , Hiperalgesia/tratamento farmacológico , Interleucina-2/imunologia , Interleucina-2/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ratos , Traumatismos da Medula Espinal/tratamento farmacológico , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Excitatory synaptic transmission is modulated by inhibitory neurotransmitters and neuromodulators. We found that the synaptic transmission of somatic sensory afferents can be rapidly regulated by a presynaptically secreted protein, follistatin-like 1 (FSTL1), which serves as a direct activator of Na(+),K(+)-ATPase (NKA). The FSTL1 protein is highly expressed in small-diameter neurons of the dorsal root ganglion (DRG). It is transported to axon terminals via small translucent vesicles and secreted in both spontaneous and depolarization-induced manners. Biochemical assays showed that FSTL1 binds to the α1 subunit of NKA and elevates NKA activity. Extracellular FSTL1 induced membrane hyperpolarization in cultured cells and inhibited afferent synaptic transmission in spinal cord slices by activating NKA. Genetic deletion of FSTL1 in small DRG neurons of mice resulted in enhanced afferent synaptic transmission and sensory hypersensitivity, which could be reduced by intrathecally applied FSTL1 protein. Thus, FSTL1-dependent activation of NKA regulates the threshold of somatic sensation.
Assuntos
Proteínas Relacionadas à Folistatina/metabolismo , Células Receptoras Sensoriais/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Transmissão Sináptica/fisiologia , Análise de Variância , Animais , Northern Blotting , Western Blotting , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas Relacionadas à Folistatina/genética , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , RatosAssuntos
Carcinoma de Células Escamosas/cirurgia , Neoplasias Uretrais/cirurgia , Adenocarcinoma/radioterapia , Adenocarcinoma/secundário , Adenocarcinoma/cirurgia , Adulto , Idoso , Carcinoma de Células Escamosas/radioterapia , Carcinoma de Células Escamosas/secundário , Terapia Combinada , Feminino , Seguimentos , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias Uretrais/patologia , Neoplasias Uretrais/radioterapiaRESUMO
Interferon-gamma (IFN-gamma) is a macrophage-activating cytokine that serves critical functions in innate and adaptive immunity and is thought to be mediated by the Jak-Stat signaling pathway. The present study establishes for the first time that cyclic adenosine monophosphate, protein kinase A, and cAMP response element-binding protein (cAMP/PKA/CREB) are coregulators of the IFN-gamma signaling pathway. Experimental data indicate that exogenous IFN-gamma stimulated cAMP accumulation and PKA activation in time-dependent and dose-dependent manners in murine peritoneal macrophages. Moreover, IFN-gamma stimulated CREB phosphorylation and CREB DNA binding, which could be significantly attenuated by PKA inhibition with H89. It appears that a novel cAMP/PKA/CREB signaling pathway is activated by IFN-gamma in macrophages, suggesting that an alternate signaling pathway exists in macrophages in response to IFN-gamma.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Interferon gama/fisiologia , Macrófagos Peritoneais/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/antagonistas & inibidores , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Relação Dose-Resposta a Droga , Ativação Enzimática , Feminino , Interferon gama/farmacologia , Isoquinolinas/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Sulfonamidas/farmacologiaRESUMO
cAMP mediated signaling may play a suppressive role in immune response. We previously found that the cAMP-elevators (CTx and 8-Br-cAMP) inhibited IL-12, IL-la, IL-6 gene expression, but increased the transcriptional levels of IL-10 and IL-1Ra in LPS-treated murine peritoneal macrophages. The present study examined a possible molecular mechanism involved in cAMP elevators-induced inhibition of IL-12 p40 expression in response to LPS. Our data demonstrated that cAMP elevators downregulated IL-12 p40 mRNA expression and IL-12 p70 production in murine peritoneal macrophages. Subsequent studies revealed that cAMP-elevators blocked phosphorylation of p38 MAPK, but did not affect the activity of NF-kappaB binding to IL-12 promoter (-136/-112). This is the first report that cAMP elevators inhibit LPS-induced IL-12 production by a mechanism that is associated, at least in part, with p38-dependent inhibition by cAMP signaling pathways.