RESUMEN
Tissue-engineered corneal epithelium transplantation is effective treatment for severe limbal stem cell deficiency (LSCD), while epithelial terminal differentiation, tans-differentiation, and insufficient stem cell during construction affect the quality of tissue-engineered corneal epithelium. In this study, we applied SB203580 in the culture medium to downregulate the p38 mitogen-activated protein kinase (MAPK) signaling pathway during construction of tissue-engineered corneal epithelium. With application of SB203580, tissue-engineered corneal epithelium showed enhanced strength and condensed structure. The expression of progenitor cell markers ATP-binding cassette sub-family G member 2, tumor protein p63, keratin 14, and Wnt family member 7A was increased, differentiation markers keratin 12, paired box 6, keratin 10, and keratin 13 and trans-differentiation markers actin alpha 2, smooth muscle and snail family transcriptional repressor 1 was decreased, while cell junction markers claudin 1 and cadherin 1 was increased in the tissue-engineered corneal epithelium. The Wnt/catenin beta 1 signaling pathway was upregulated in the epithelium after p38 MAPK inhibition. Transplantation of tissue-engineered corneal epithelium treated with SB203580 to rabbit LSCD model showed faster wound healing and improved epithelial quality. We conclude that downregulation of p38 MAPK signaling pathway helps maintain the stemness and prevent terminal differentiation and abnormal differentiation of corneal epithelial cells during epithelium construction process, and thus can improve the quality of tissue-engineered corneal epithelium. Impact statement Downregulation of p38 MAPK signaling pathway helps maintain the self-renewal of limbal stem cells and prevents terminal differentiation and abnormal differentiation of corneal epithelial cells. Small molecules modulating p38 MAPK signaling pathway ameliorate tissue-engineered corneal epithelium.
Asunto(s)
Epitelio Corneal , Limbo de la Córnea , Animales , Conejos , Limbo de la Córnea/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Regulación hacia Abajo , Transducción de SeñalRESUMEN
One undescribed lignan, one new natural product, along with fourteen known compounds, were isolated from the roots of Ficus hirta. The structures of the isolates were elucidated by comprehensive spectroscopic technologies, including UV, IR, HRESIMS, and NMR. The absolute configuration of 1 was determined by comparison of experimental and calculated ECD data. The cytotoxicity of all the compounds against HeLa and HepG2 cell lines was evaluated and compound 7 showed considerable cytotoxic effect towards HepG2 cells. Also, the apoptotic effect of 7 on HepG2 cells and the effect of 7 on the key proteins (p-JNK and p-p38) in MAPK (Mitogen-activated protein kinases) pathways were studied by flow cytometry and western blotting experiment. As a result, compound 7 induced the apoptosis of HepG2 cells, and dose-dependently increased the phosphorylation of JNK and p38. Thus, 7 might trigger HepG2 cells apoptosis via JNK/p38 MAPK signaling pathway.
Asunto(s)
Antineoplásicos , Ficus , Lignanos , Antineoplásicos/farmacología , Apoptosis , Ficus/química , Células Hep G2 , Humanos , Lignanos/análisis , Lignanos/farmacología , Raíces de Plantas/química , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/farmacologíaRESUMEN
Mitogen-activated protein kinase kinase 4 (MAP2K4) is a tumor suppressor in many cancers. However, its roles and action mechanisms in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Here, we analyzed MAP2K4 and its downstream kinases (c-Jun N-terminal kinase (JNK) and p38) using immunohistochemical staining and their prognostic significances using univariate and multivariate Cox proportional hazards regression analysis in our PDAC cohort. Then, we validated MAP2K4/JNK/p38 mRNA levels and prognostic significances using The Cancer Genome Atlas (TCGA) database. Finally, we evaluated the effects of MAP2K4 on the proliferation and invasion of PDAC cells. MAP2K4, JNK, and p38 proteins were expressed in 97.3 % (72/74), 95.6 % (65/68), and 88.6 % (62/70) of the samples, respectively, and their levels in tumor tissues were significantly higher than those in normal ducts. MAP2K4 protein expression was lower in male patients (p = 0.028). In our PDAC cohort, advanced TNM stage, low MAP2K4, and high JNK protein levels were significant prognostic factors for poor overall survival (OS) based on a univariate survival analysis (p = 0.006, p < 0.001, and p = 0.004, respectively). N stage and MAP2K4 and JNK protein levels were independent prognostic factors for OS based on multivariate analysis. We then built a prognosis prediction nomogram combining the standard TNM staging system with MAP2K4 and JNK expression that had a Harrell's C-index of 0.645. The new prognosis prediction model effectively stratified the resected patients with PDAC, from both our cohort and TCGA database, into low- and high-risk groups. Finally, MAP2K4 overexpression inhibited pancreatic cancer cell proliferation and migration in vitro. This study shows that reduced protein and mRNA levels of MAP2K4 found in PDAC patients, coupled to in vitro effects observed, support the tumor suppressor role of MAP2K4 in PDAC. Importantly, combining MAP2K4 and JNK expression with the TNM staging system results in a better prediction of postoperative survival of patients with PDAC.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma Ductal Pancreático/enzimología , Técnicas de Apoyo para la Decisión , Proteínas Quinasas JNK Activadas por Mitógenos/análisis , MAP Quinasa Quinasa 4/análisis , Nomogramas , Neoplasias Pancreáticas/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/cirugía , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , MAP Quinasa Quinasa 4/genética , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/cirugía , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Resultado del Tratamiento , Proteínas Quinasas p38 Activadas por Mitógenos/análisisRESUMEN
Our previous studies have shown that sulbactam can play a neuroprotection role in hippocampal neurons by upregulating the expression and function of glial glutamate transporter-1 (GLT-1) during ischemic insult. Here, using rat global cerebral ischemia model, we studied in vivo the role of p38 mitogen-activated protein kinases (MAPK) in the sulbactam-induced GLT-1 upregulation and neuroprotection against ischemia. The hippocampal CA1 field was selected as observing target. The expressions of phosphorylated-p38 MAPK and GLT-1 were assayed with western blot analysis and immunohistochemistry. The condition of delayed neuronal death (DND) was assayed with neuropathological evaluation under thionin staining. It was shown that administration of sulbactam protected CA1 hippocampal neurons against ischemic insult accompanied with significantly upregulation in the expressions of phosphorylated-p38 MAPK and GLT-1. The time course analysis showed that sulbactam activated p38 MAPK before the GLT-1 upregulation in either normal or global cerebral ischemic rats. Furthermore, inhibiting p38 MAPK activation by SB203580 blocked the GLT-1 upregulation and neuroprotection induced by sulbactam. The above results suggested that p38 MAPK, at least partly, participated in the sulbactam-induced brain tolerance to ischemia mediated by GLT-1 upregulation in rats.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Transportador 2 de Aminoácidos Excitadores/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Sulbactam/uso terapéutico , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Activación Enzimática/efectos de los fármacos , Transportador 2 de Aminoácidos Excitadores/análisis , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Masculino , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Wistar , Sulbactam/farmacología , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/análisisRESUMEN
Osteoclasts are multinucleated bone-resorbing cells derived from monocyte/macrophage progenitor cells. Excessive formation and resorbing activities of osteoclasts are involved in the bone-destructive pathologies of rheumatoid arthritis and osteoporosis. Recently, it has been found that nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor for anti-oxidative stress genes, functions in osteoclastogenesis. Dimethyl fumarate (DMF) is a potent activator of Nrf2 and has been shown to inhibit osteoclastogenesis. Here, we investigated the mechanisms of this inhibition by examining the activation of several signalling pathways during the differentiation of bone marrow-derived macrophages into osteoclasts. DMF inhibited the differentiation of osteoclasts in a dose-dependent manner and suppressed the bone-resorbing activity of osteoclasts. DMF treatment decreased the expression of nuclear factor of activated T-cells cytoplasmic-1, and significantly decreased phosphorylation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in osteoclasts. We also found that DMF inhibited the extracellular release of high mobility group box 1, associated with an up-regulation of heme oxygenase-1, likely mediated through Nrf2 activation. Our results indicate that DMF inhibits osteoclast differentiation through multiple pathways.
Asunto(s)
Dimetilfumarato/farmacología , Proteína HMGB1/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Factores de Transcripción NFATC/metabolismo , Osteogénesis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Animales , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína HMGB1/análisis , Masculino , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: The P38-protein is known to be expressed in colorectal adenomas (CRA). Expression in low- and high-grade tubular adenomas is decreased when compared to adenocarcinomas and increased with regard to normal mucosa. We aimed to study P38 expression in human CRAs and the relationships to cell proliferation Ki67-protein, stem-phenotype CD133-protein and, to mTOR-protein (AKT pathway). METHODS: The immunohistochemical expression of P38 was evaluated in CRAs on tissue microarrays. Data were analyzed with the Kendall-rank-correlation test. RESULTS: Nuclear P38 correlated to low-grade dysplasia (Kendall P<0.01/tau=-0.254) and to decreased adenoma size (P<0.01/tau=-0.267). Nuclear P38 also correlated to cytoplasmic or membrane mTOR (P<0.01/tau=-0.223 and P<0.01/tau=-0.340) and to cytoplasmic CD133 (P<0.01/0.293). An inverse relationship was observed to Ki67 (P<0.00/ tau=-0.110). CONCLUSIONS: Our results suggest an interference of P38 with initial steps of colorectal adenomagenesis. The correlation to mTOR suggests a biological crosstalk between the MAPK- and AKT-signaling-pathways in colorectal adenomagenesis at P38 level.
Asunto(s)
Antígeno AC133 , Adenoma , Proliferación Celular , Neoplasias Colorrectales , Antígeno Ki-67 , Serina-Treonina Quinasas TOR , Proteínas Quinasas p38 Activadas por Mitógenos , Antígeno AC133/fisiología , Adenoma/química , Adenoma/genética , Adenoma/metabolismo , Adenoma/patología , Neoplasias Colorrectales/química , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Humanos , Antígeno Ki-67/fisiología , Masculino , Fenotipo , Transducción de Señal , Serina-Treonina Quinasas TOR/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/biosíntesisRESUMEN
Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.
Asunto(s)
Ameloblastos/citología , Células Madre Pluripotentes Inducidas/citología , Transducción de Señal/fisiología , Proteína Smad1/fisiología , Receptores de Activinas/análisis , Receptores de Activinas/fisiología , Western Blotting , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/análisis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Medio de Cultivo Libre de Suero , Técnica del Anticuerpo Fluorescente , Expresión Génica , Sistema de Señalización de MAP Quinasas/fisiología , Fosforilación , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Smad1/análisis , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.
Asunto(s)
Transducción de Señal/fisiología , Proteína Smad1/fisiología , Células Madre Pluripotentes Inducidas/citología , Ameloblastos/citología , Fosforilación , Factores de Tiempo , Expresión Génica , Diferenciación Celular/fisiología , Diferenciación Celular/genética , Células Cultivadas , Western Blotting , Técnica del Anticuerpo Fluorescente , Medio de Cultivo Libre de Suero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sistema de Señalización de MAP Quinasas/fisiología , Receptores de Activinas/análisis , Receptores de Activinas/fisiología , Interferencia de ARN , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/análisis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/fisiología , Proteína Smad1/análisisRESUMEN
Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.
Asunto(s)
Transducción de Señal/fisiología , Proteína Smad1/fisiología , Células Madre Pluripotentes Inducidas/citología , Ameloblastos/citología , Fosforilación , Factores de Tiempo , Expresión Génica , Diferenciación Celular/fisiología , Diferenciación Celular/genética , Células Cultivadas , Western Blotting , Técnica del Anticuerpo Fluorescente , Medio de Cultivo Libre de Suero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sistema de Señalización de MAP Quinasas/fisiología , Receptores de Activinas/análisis , Receptores de Activinas/fisiología , Interferencia de ARN , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/análisis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/fisiología , Proteína Smad1/análisisRESUMEN
Gut microbiota has been proposed as an important environmental factor which can intervene and modulate central nervous system autoimmunity. Here, we altered the composition of gut flora with Clostridium butyricum and norfloxacin in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We found that appropriate C. butyricum (5.0 × 106 CFU/mL intragastrically daily, staring at weaning period of age) and norfloxacin (5 mg/kg intragastrically daily, 1 week prior to EAE induction) treatment could both ameliorate EAE although there are obvious differences in gut microbiota composition between these two interventions. C. butyricum increased while norfloxacin decreased the abundance and diversity of the gut microbiota in EAE mice, and both of the treatments decreased firmicutes/bacteroidetes ratio. In the genus level, C. butyricum treatment increased the abundance of Prevotella while Akkermansia and Allobaculum increased in norfloxacin treatment. Moreover, both interventions reduced Desulfovibroneceae and Ruminococcus species. Although there was discrepancy in the gut microbiota composition with the two interventions, C. butyricum and norfloxacin treatment both reduced Th17 response and increased Treg response in the gastrointestinal tract and extra-gastrointestinal organ systems in EAE mice. And the reduced activity of p38 mitogen-activated kinase and c-Jun N-terminal kinase signaling in spinal cord could be observed in the two interventions. The results suggested that manipulation of gut microbiota interventions should take factors such as timing, duration, and dosage into consideration. The discrepancy in the gut microbiota composition and the similar protective T cells response of C. butyricum and norfloxacin implies that achieving intestinal microecology balance by promoting and/or inhibiting the gut microbiota contribute to the well-being of immune response in EAE mice.
Asunto(s)
Clostridium butyricum , Encefalomielitis Autoinmune Experimental/terapia , Microbioma Gastrointestinal/inmunología , Norfloxacino/uso terapéutico , Probióticos/uso terapéutico , Animales , Bacteroidaceae/efectos de los fármacos , Bacteroidaceae/aislamiento & purificación , Clostridium butyricum/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Firmicutes/efectos de los fármacos , Firmicutes/aislamiento & purificación , Microbioma Gastrointestinal/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/análisis , Recuento de Linfocitos , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/análisis , Norfloxacino/farmacología , Distribución Aleatoria , Transducción de Señal/efectos de los fármacos , Organismos Libres de Patógenos Específicos , Médula Espinal/enzimología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/análisisRESUMEN
Monocytes from HIV-infected patients produce increased levels of inflammatory cytokines, which are associated with chronic immune activation and AIDS progression. Chronic immune activation is often not restored even in patients showing viral suppression under ART. Therefore, new therapeutic strategies to control inflammation and modulate immune activation are required. Hydroxypropyl-beta-cyclodextrin (HP-BCD) is a cholesterol-sequestering agent that has been reported to be safe for human use in numerous pharmaceutical applications and that has been shown to inactivate HIV in vitro and to control SIV infection in vivo Since cellular cholesterol content or metabolism has been related to altered cellular activation, we evaluated whether HP-BCD treatment could modulate monocyte response to inflammatory stimuli. Treatment of monocytes isolated from HIV-positive and HIV-negative donors with HP-BCD inhibited the expression of CD36 and TNF-α after LPS stimulation, independent of raft disruption. Accordingly, HP-BCD-treated cells showed significant reduction of TNF-α and IL-10 secretion, which was associated with lower mRNA expression. LPS-induced p38MAPK phosphorylation was dampened by HP-BCD treatment, indicating this pathway as a target for HP-BCD-mediated anti-inflammatory response. The expression of HLA-DR was also reduced in monocytes and dendritic cells treated with HP-BCD, which could hinder T cell activation by these cells. Our data suggest that, besides its well-known antiviral activity, HP-BCD could have an immunomodulatory effect, leading to decreased inflammatory responses mediated by antigen-presenting cells, which may impact HIV pathogenesis and AIDS progression.IMPORTANCE Chronic immune activation is a hallmark of HIV infection and is often not controlled even in patients under antiretroviral therapy. Indeed, chronic diseases with inflammatory pathogenesis are being reported as major causes of death for HIV-infected persons. Hydroxypropyl-beta cyclodextrin (HP-BCD) is a cholesterol-sequestering drug that inhibits HIV replication and infectivity in vitro and in vivo Recent studies have demonstrated the importance of cholesterol metabolism and content in different inflammatory conditions; therefore, we investigated the potential of HP-BCD as an immunomodulatory drug, regulating the activation of cells from HIV-infected patients. Treatment of monocytes with HP-BCD inhibited the expression and secretion of receptors and mediators that are usually enhanced in HIV patients. Furthermore, we investigated the molecular mechanisms associated with the immunomodulatory effect of HP-BCD. Our results indicate that, besides reducing viral replication, HP-BCD treatment may contribute to modulation of chronic immune activation associated with AIDS.
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2-Hidroxipropil-beta-Ciclodextrina/farmacología , Antiinflamatorios/farmacología , Inmunosupresores/farmacología , Monocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Adulto , Anciano , Antígenos CD36/análisis , Células Cultivadas , Femenino , Infecciones por VIH/patología , Antígenos HLA-DR/análisis , Humanos , Interleucina-10/análisis , Lipopolisacáridos/inmunología , Masculino , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/análisisRESUMEN
This study aimed to explore whether mechanical stretch aggravated aortic dissection through regulating MAPK pathway, MMP-9, and inflammation factors. We first established aortic dissection model rats. Mechanical stretch (3 g) was exerted on vascular ring of aortic dissection which was also treated by inhibitors of MAPK pathway (SB203580, SP600125, and U0126). HE and Masson staining showed that aortic dissection severity with 3 g tension was worse than that without tension (0 g); after the treatments of diverse inhibitors, the fracture and breakage of the elastic fibers decreased. The expression of MMP-9, TNF-α, IL-1ß) p38/p-p38, JNK1/p-JNK1, and ERK1/2/p-ERK1/2 were determined by immunohistochemical analysis, RT-PCR, and western blot. No matter whether tension was exerted or inhibitors were added, there was no change in the expression of p38, JNK1, and ERK1/2. However, compared to the 0 g group, the expression of MMP-9, TNF-α, IL-1ß, p-p38, p-JNK1, and p-ERK1/2 was significantly upregulated in the 3 g group (P < 0.05). In both 0 g and 3 g groups, the expression of MMP-9, TNF-α, IL-1ß, p-p38, p-JNK1, and p-ERK1/2 was remarkably downregulated after inhibitors treatment (P < 0.05). In conclusion, mechanical stretch aggravated aortic dissection by regulating the MAPK pathway and the consequent expression of MMP-9 and inflammation factors.
Asunto(s)
Disección Aórtica/etiología , Inflamación/complicaciones , Sistema de Señalización de MAP Quinasas/fisiología , Metaloproteinasa 9 de la Matriz/fisiología , Animales , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/análisis , Interleucina-1beta/análisis , Proteínas Quinasas JNK Activadas por Mitógenos/análisis , Masculino , Metaloproteinasa 9 de la Matriz/análisis , Ratas , Ratas Sprague-Dawley , Estrés Mecánico , Factor de Necrosis Tumoral alfa/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/análisisRESUMEN
OBJECTIVE: To investigate whether exogenous agmatine inhibits lipopolysaccharide (LPS)-induced activation and dysfunction of human umbilical vein endothelial cells (HUVECs) by modulating nuclear factor-κB (NF-κB) and MAPK signal pathways and the production of reactive oxygen species (ROS). METHODS: Cultured HUVECs were treated with agmatine at the optimized concentration of 1.0 mmolγL, LPS (10 µgγmL), and LPS + agmatine, with or without pretreatment with the inhibitors of NF-κB (PDTC), p38 (SB203580), and ERK (PD98059) for 1 h. The levels of soluble vascular cell adhesion molecule 1 (VCAM-1), soluble intercellular adhesion molecule 1 (sICAM-1), soluble E-selectin and monocyte chemoattractant protein 1 (MCP-1) in the supernatant were determined using ELISA, and their mRNA expressions, along with heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO-1), were assessed using real-time PCR. ROS production in the cells was determined using 2, 7-dichlorofluoresce in diacetate (DCFH-DA) as the fluorescence probe. The protein expressions of VCAM-1, ICAM-1, p65, phospho-p65 (p-p65), IκBα, p-IκBα, ERK, p-ERK, p38, p-p38, JNK, and p-JNK were detected using Western blotting. RESULTS: LPS stimulation for 6 and 24 h significantly increased the levels of sVCAM-1, sICAM-1, sE-selectin and MCP-1 in the supernatant, intracellular ROS production, and the mRNA expressions of these molecules (P<0.05). Intervention with 1 mmolγL agmatine, similar with pretreatment with p38, ERK and NF-κB inhibitors, obviously inhibited such effects of LPS in HUVECs (P<0.05). Agmatine significantly up-regulated the mRNA expression of HO-1 (P<0.05), inhibited LPS-induced phosphorylation of p38, ERK, nuclear p65 and cytoplasmic IκBα, and up-regulated the protein expression of cytoplasmic IκBα. CONCLUSION: Agmatine inhibits LPS-induced activation and dysfunction of HUVECs by modulating NF-κB and MAPK signal pathways to down-regulate the expressions of adhesion molecules and chemokines and by up-regulating the expression of HO-1 to reduce ROS production.
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Agmatina/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Lipopolisacáridos/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/metabolismo , Células Cultivadas , Quimiocina CCL2/análisis , Selectina E/análisis , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/análisis , Especies Reactivas de Oxígeno/metabolismo , Molécula 1 de Adhesión Celular Vascular/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/análisisRESUMEN
PURPOSE: To evaluate the effects of 1,25 dihydroxy vitamin D3 (1,25(OH)2D3) on the content of triglyceride (TG), as well as on the gene and protein expressions of adiponectin receptor 2 (AdipoR2), p38 mitogen-activated protein kinase (P38MAPK), and lipoprotein lipase (LPL) in the liver of rats with type 2 diabetes mellitus (T2DM) so as to provide theoretical basis for exploring the mechanism by which 1,25(OH)2D3 regulates TG. METHODS: Wistar rats were divided into four groups (n=25), with different treatments and detected the gene and protein expressions of AdipoR2, p38MAPK, and LPL in the liver tissue by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Meanwhile, the content of TG in the liver tissue was detected by the Enzyme-linked immunosorbent assay. RESULTS: The expression of AdipoR2, p38MAPK, LPL gene and protein in the liver of VitD intervention group was significantly higher than that in T2DM group (P <0.05), while the TG content was significantly lower than that in T2DM group (P <0.05). CONCLUSION: 1,25(OH)2D3 can decrease the content of TG in the liver, and its mechanism may be achieved by upregulating the expressions of AdipoR2, p38MAPK, and LPL in the liver.
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Calcitriol/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Triglicéridos/sangre , Animales , Glucemia/análisis , Western Blotting , Peso Corporal , Diabetes Mellitus Tipo 2/prevención & control , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Lipoproteína Lipasa/análisis , Lipoproteína Lipasa/efectos de los fármacos , Masculino , Ratas Wistar , Receptores de Adiponectina/análisis , Receptores de Adiponectina/efectos de los fármacos , Valores de Referencia , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacosRESUMEN
Abstract Purpose: To evaluate the effects of 1,25 dihydroxy vitamin D3 (1,25(OH)2D3) on the content of triglyceride (TG), as well as on the gene and protein expressions of adiponectin receptor 2 (AdipoR2), p38 mitogen-activated protein kinase (P38MAPK), and lipoprotein lipase (LPL) in the liver of rats with type 2 diabetes mellitus (T2DM) so as to provide theoretical basis for exploring the mechanism by which 1,25(OH)2D3 regulates TG. Methods: Wistar rats were divided into four groups (n=25), with different treatments and detected the gene and protein expressions of AdipoR2, p38MAPK, and LPL in the liver tissue by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Meanwhile, the content of TG in the liver tissue was detected by the Enzyme-linked immunosorbent assay. Results: The expression of AdipoR2, p38MAPK, LPL gene and protein in the liver of VitD intervention group was significantly higher than that in T2DM group (P <0.05), while the TG content was significantly lower than that in T2DM group (P <0.05). Conclusion: 1,25(OH)2D3 can decrease the content of TG in the liver, and its mechanism may be achieved by upregulating the expressions of AdipoR2, p38MAPK, and LPL in the liver.
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Animales , Masculino , Triglicéridos/sangre , Calcitriol/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Valores de Referencia , Glucemia/análisis , Peso Corporal , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Regulación hacia Arriba , Western Blotting , Reproducibilidad de los Resultados , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Diabetes Mellitus Tipo 2/prevención & control , Receptores de Adiponectina/análisis , Receptores de Adiponectina/efectos de los fármacos , Lipoproteína Lipasa/análisis , Lipoproteína Lipasa/efectos de los fármacosRESUMEN
Berberine has multiple clinical applications, including the treatment of tumors, diabetes, cardiovascular diseases, hyperlipidemia, inflammation, bacterial and viral infections, cerebral ischemic injuries, mental diseases, Alzheimer's disease and osteoporosis. In the present study, the neuroprotective effect of berberine agonist rescue learning and memory in severe traumatic brain injury (TBI), and the possible mechanism underlying these observations was explored. The protective effect of berberine agonist significantly recovered learning and memory skills, attenuated brain edema and inhibited matrix metalloproteinase3 and 9 protein expression in mice with severe TBI. Berberine agonist significantly reduced inflammation, oxidative stress and apoptosis levels in mice with severe TBI. Berberine agonist promoted choline acetyltransferase activity and inhibited the activity of acetylcholinesterase. Collectively, results of the present study revealed that the neuroprotective effect of berberine agonist rescues learning and memory skills in severe TBI.
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Berberina/uso terapéutico , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Memoria/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Sirtuina 1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/fisiopatología , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/fisiopatología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Sirtuina 1/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/análisisRESUMEN
Caspase recruitment domain-containing protein 9 (Card9) is located upstream of the nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) inflammatory pathways. This study investigated the therapeutic effect and potential mechanism of pioglitazone in rats with severe acute pancreatitis (SAP). SAP was induced by a retrograde infusion of 5.0% sodium taurocholate into the biliopancreatic duct of Sprague Dawley rats (n=54), which were then treated with pioglitazone. Blood and pancreatic tissues were harvested at 3, 6, and 12 h after SAP induction. Pancreatic pathological damage was evaluated by hematoxylin and eosin staining. Serum amylase, serum pro-inflammatory cytokines, and pancreatic myeloperoxidase (MPO) activities were determined by enzyme-linked immunosorbent assay. The expression of Card9 mRNA and protein in pancreatic tissues was detected by real-time polymerase chain reaction and western blotting. Pioglitazone had a therapeutic effect in treating rats with SAP by decreasing the level of amylase activity, ameliorating pancreatic histological damage, decreasing serum pro-inflammatory cytokine levels and tissue MPO activity, and downregulating the expression of NF-κB, p38MAPK, and Card9 mRNAs and proteins (P<0.05). The present study demonstrated that the inhibition of Card9 expression could reduce the severity of SAP. Card9 has a role in the pathogenic mechanism of SAP.
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Antiinflamatorios/farmacología , Pancreatitis/tratamiento farmacológico , Pancreatitis/patología , Tiazolidinedionas/farmacología , Amilasas/sangre , Amilasas/efectos de los fármacos , Animales , Antiinflamatorios/uso terapéutico , Western Blotting , Proteínas Adaptadoras de Señalización CARD/análisis , Proteínas Adaptadoras de Señalización CARD/efectos de los fármacos , Citocinas/sangre , Citocinas/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Masculino , FN-kappa B/análisis , Peroxidasa/análisis , Pioglitazona , Distribución Aleatoria , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Índice de Severidad de la Enfermedad , Tiazolidinedionas/uso terapéutico , Factores de Tiempo , Resultado del Tratamiento , Proteínas Quinasas p38 Activadas por Mitógenos/análisisRESUMEN
BACKGROUND: Neuromediators produced by enteric nervous system regulate inflammatory processes via interacting with enteric immune system. Role of γ-aminobutyric acid (GABA), which is also a neuromediator, has been implicated in autoimmune diseases like multiple sclerosis, type 1 diabetes, and rheumatoid arthritis, where they modulate the immune responses. However, its role in ulcerative colitis (UC) has not been defined. AIMS: This study was carried out to investigate the role of GABA and its signaling components in pathogenesis of UC. METHODS: Peripheral blood, colon mucosal biopsy, and fecal specimens were collected from UC and control groups. Quantification of GABA was done using ELISA. Expression of GABAergic signal system components was analyzed through RT-PCR analysis. Enumeration of GABA-producing bacteria was done by qPCR analysis. Activity of p38 MAPK and expression of proinflammatory cytokines were determined by immunohistochemistry and RT-PCR analysis, respectively. RESULTS: GABA levels were significantly reduced in patients with UC as compared to control group when measured in serum and colon biopsy. Altered expression of GABAergic signal system was observed in UC patients. Reduced abundance of selected GABA-producing bacteria was detected in stool samples of UC patients as compared to control. p38 MAPK activity and expression of its downstream effector cytokines were found to be increased in UC patients as compared to control. CONCLUSIONS: Reduced levels of GABA were observed in patients with UC, and this leads to hyperactivation of p38 MAPK and overexpression of downstream effector cytokines suggesting a role of GABA in pathogenesis of UC.
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Colitis Ulcerosa/metabolismo , Colon/química , Mucosa Intestinal/química , Receptores de GABA/análisis , Ácido gamma-Aminobutírico/análisis , Adulto , Bacterias/química , Bacterias/genética , Biopsia , Estudios de Casos y Controles , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Colon/microbiología , Colon/patología , Citocinas/análisis , Citocinas/genética , Regulación hacia Abajo , Heces/química , Heces/microbiología , Femenino , Humanos , Mediadores de Inflamación/análisis , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Receptores de GABA/genética , Transducción de Señal , Adulto Joven , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Objective: To study the effect of plumbagin on epithelial-mesenchymal transition (EMT) and underlying mechanisms in human tongue squamous cell carcinoma (TSCC) cells. Methods: Methyl thiazolyl tetrazolium assay was apllied to examine the proliferation inhibition effect and half maximal inhibitory concentration (IC(50)) of plumbagin (0.1, 1.0, 5.0, 10.0, 20.0 µmol/L) in 12, 24, 48 h in TSCC cells. Transwell assay was used to count the number of transmembrane cells and scratch test was performed to examine cells mobility. The flow cytometry was applied to measure intracellular reactive oxygen species (ROS) level in control group, plumbagin group (1.0 µmol/L, 24 h) and glutathione (GSH)+plumbagin group. The expression of E-cadherin, vimentin, Slug, p38 mitogen activated protein kinases (p38MAPK) and phospho-p38MAPK (p-p38MAPK) proteins were determined by Western blotting. The expression of E-cadherin, vimentin and Slug were detected by Western blotting in control group, plumbagin group, activator combined group (p38MAPK activator+plumbagin) and inhibitor combined group (p38MAPK inhibitor+plumbagin). Results: After the treatment of plumbagin for 12, 24, and 48 h, the IC(50) of TSCC cells were 10.3, 3.1, 1.5 µmol/L. After treated by 1.0 µmol/L plumbagin for 24 h, the number of transmembrane cells were significantly reduced ([50±13], P<0.05) in comparison to control group (204±6), as well as the cells mobility ([18.2±2.3]%, P<0.05) in comparison to control group ([49.3±1.2]%). Compared to control group (2.32±0.52), the ROS level was increased in plumbagin group (902.20±10.69), while compared to plumbagin group, the ROS level was reduced in GSH combined group (2.18±0.15). In comparison to control group, the expression of E-cadherin was up-regulated (P<0.05), and vimentin, Slug, p-p38MAPK/p38MAPK were down-regulated in plumbagin group (P<0.05). In comparison to plumbagin group, the expression of E-cadherin was down-regulated (P<0.05), and vimentin, Slug, p-p38MAPK/p38MAPK were up-regulated in GSH combined group (P<0.05). Treatment of cells with p38MAPK activator could decrease the expression of E-cadherin significantly (P<0.05) and increase the expression of vimentin (P<0.05) and Slug (P<0.05) in comparison to plumbagin group. Treatment of cells with p38MAPK inhibitor could increase the expression of E-cadherin significantly (P<0.05) and decrease the expression of vimentin (P<0.05) and Slug (P<0.05) in comparison to plumbagin group. Conclusions: Plumbagin inhibits EMT via ROS/p38MAPK-mediated pathway in human TSCC cells.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Transición Epitelial-Mesenquimal/efectos de los fármacos , Naftoquinonas/farmacología , Neoplasias de la Lengua/tratamiento farmacológico , Antígenos CD , Cadherinas/análisis , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias de la Lengua/química , Neoplasias de la Lengua/patología , Regulación hacia Arriba , Vimentina/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
BACKGROUND: Beta defensins are secreted from ovine oviduct epithelial cells (OOECs) in response to microbial infection, and are potential alternatives to antibiotic agents in the treatment of microorganism infection, particularly given the abuse of antibiotic agents and the increasing number of drug-resistant bacteria. The aberrant expression of defensins may result in disorders involving organ and oviduct inflammation, such as salpingitis. METHODS: In the present study, we investigated the effects of LPS on the mRNA expression levels of sheep ß-defensin-1 (SBD-1) in ovine oviduct epithelial cells. The OOECs in vitro culturing system were established and treated with different concentrations of LPS for indicated time. In addition, MAPK inhibitors and TLR4 antibodies were pretreated to investigate the potential mechanism which involves in LPS regulating SBD-1 expression. RESULTS: LPS markedly upregulated SBD-1 expression in a concentration- and time-dependent manner. Treatment with 100 ng/mL LPS resulted in the phosphorylation of JNK, ERK and P38 MAPK. Interestingly, the LPS stimulated SBD-1 expression was attenuated by pretreatment with the P38 MAPK inhibitors SB203580 and SB202190 but not the JNK inhibitor SP600125, while the ERK inhibitor PD98059 had a minor effect. Furthermore, treatment with a Toll-like receptor 4 (TLR4) neutralizing antibody significantly decreased P38 MAPK phosphorylation and LPS induced SBD-1 expression. CONCLUSIONS: Together, these findings suggest that SBD-1 is upregulated by LPS via the TLR4 receptor, mainly through the P38 MAPK signaling pathway in ovine oviduct epithelial cells to protect the ovine oviduct epithelium from pathogen invasion.