RESUMO
Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.
RESUMO
The p38 mitogen-activated protein kinase (p38 MAPK) is a multifunctional molecule that is involved in cellular response to various stressful stimuli. In the present study, the full-length cDNA sequence of p38 MAPK (Lcp38 MAPK) was identified from the large yellow croaker Larimichthys crocea, which encoded a polypeptide of 361 amino acid residues. The predicted Lcp38 MAPK protein contained a highly conserved Thr-Gly-Tyr (TGY) motif, a glutamate and aspartate (ED) site, a substrate binding site (Ala-Thr-Arg-Trp < ATRW>), and a serine/threonine kinase catalytic (S_TKc) domain characteristic of the MAPK family. The constitutive expression of Lcp38 MAPK was detected in most of the tissues examined with the strongest expression in intestine. Subcellular localization in LCK cells (kidney cell line from a L. crocea) revealed that Lcp38 MAPK existed in both the cytoplasm and cell nucleus. The expression of Lcp38 MAPK after temperature stress was tested in LCK cells. The results indicated that Lcp38 MAPK transcripts were significantly upregulated under both cold (10 °C) and heat stress (35 °C) (P < 0.05). Furthermore, the phosphorylation levels of p38 MAPK as well the transcriptional levels of heat shock protein 27 (HSP27) and caspase3 in LCK cells were significantly induced under thermal exposure (P < 0.05). However, the cold- and heat induced HSP27 and caspase3 expression was significantly suppressed by SB203580, a specific inhibitor of p38-MAPK (P < 0.05). These findings indicated that Lcp38 MAPK might be involved in the cellular stress response via HSP27 and caspase3 in large yellow croaker.
Assuntos
Perciformes , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas de Choque Térmico HSP27/metabolismo , Fosforilação , Temperatura , Perciformes/genética , Perciformes/metabolismoRESUMO
BACKGROUND: Up to 80% of pancreatic cancer patients suffer from cachexia. White adipose tissue (WAT) browning caused by the tumorigenicity and progression aggravates the cancer-associated cachexia (CAC). Cancer-initiated changes in the protein-38 mitogen-activated protein kinases (p38 MAPK) pathway are likely involved in the development of CAC. METHODS: p38 MAPK inhibitors, VCP979 or SB203580, were used in the in vitro and in vivo models of pancreatic cancer cachexia. Expression of uncoupling protein 1 (UCP1) in the p38 MARK pathway and the properties and level of white adipocytes were analyzed and correlated to browning, followed by immunohistochemistry and Western blotting validations. Changes in the volume and fat fraction of WAT in animals were monitored by magnetic resonance imaging (MRI). RESULTS: The size of white adipocytes was increased after being treated with the p38 MAPK inhibitors, along with increase in the MRI-measured volume and fat fraction of WAT. Comparing two p38 MAPK inhibitors, the p38α subunit-specific inhibitor VCP979 had a better therapeutic effect than SB203580, which targets both p38α and ß subunits. CONCLUSIONS: Blockade of p38 MAPK reduced the WAT browning that contributes to CAC. Thus, p38 MARK inhibitors can potentially be used as a therapy for treating CAC. Non-invasive MRI can also be applied to assess the progression and treatment responses of CAC.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno , Neoplasias Pancreáticas , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Caquexia/tratamento farmacológico , Caquexia/etiologia , Caquexia/metabolismo , Imageamento por Ressonância Magnética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Werner (WRN) expression is epigenetically downregulated in various tumors. It is imperative to understand differential repair process in WRN-proficient and WRN-deficient cancers to find pharmacological targets for radio-sensitization of WRN-deficient cancer. In the current investigation, we showed that pharmacological inhibition of CHK1 mediated homologous recombination repair (HRR), but not non-homologous end joining (NHEJ) repair, can causes hyper-radiosensitization of WRN-deficient cancers. This was confirmed in cancer cell lines of different tissue origin (osteosarcoma, colon adenocarcinoma and melanoma) with WRN silencing and overexpression. We established that WRN-depleted cells are dependent on a critical but compromised CHK1-mediated HRR-pathway for repairing ionizing radiation (IR) induced DSBs for their survival. Mechanistically, we unraveled a new finding that the MRE11, CTIP and WRN proteins are largely responsible for resections of late and persistent DSBs. In response to IR-treatment, MRE11 and CTIP-positively and WRN-negatively regulate p38-MAPK reactivation in a CHK1-dependent manner. A degradation resistant WRN protein, mutated at serine 1141, abrogates p38-MAPK activation. We also showed that CHK1-p38-MAPK axis plays important role in RAD51 mediated HRR in WRN-silenced cells. Like CHK1 inhibition, pharmacological-inhibition of p38-MAPK also hyper-radiosensitizes WRN-depleted cells by targeting HR-pathway. Combination treatment of CHK1-inhibitor (currently under various clinical trials) and IR exhibited a strong synergy against WRN-deficient melanoma tumor in vivo. Taken together, our findings suggest that pharmacological targeting of CHK1-p38-MAPK mediated HRR is an attractive strategy for enhancing therapeutic response of radiation treatment of cancer.
Assuntos
Reparo do DNA/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Radiossensibilizantes/administração & dosagem , Helicase da Síndrome de Werner/antagonistas & inibidores , Helicase da Síndrome de Werner/deficiência , Animais , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/metabolismo , Reparo do DNA/fisiologia , Inibidores Enzimáticos/administração & dosagem , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The p38 MAPK cascade is a key signaling pathway linked to a multitude of physiological functions and of central importance in inflammatory and autoimmune diseases. Although studied extensively, little is known about how conformation-specific inhibitors alter signaling outcomes. Here, we have explored the highly dynamic back pocket of p38 MAPK with allosteric urea fragments. However, screening against known off-targets showed that these fragments maintained the selectivity issues of their parent compound BIRB-796, while combination with the hinge-binding motif of VPC-00628 greatly enhanced inhibitor selectivity. Further efforts focused therefore on the exploration of the αC-out pocket of p38 MAPK, yielding compound 137 as a highly selective type-II inhibitor. Even though 137 is structurally related to a recent p38 type-II chemical probe, SR-318, the data presented here provide valuable insights into back-pocket interactions that are not addressed in SR-318 and it provides an alternative chemical tool with good cellular activity targeting also the p38 back pocket.
Assuntos
Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Regulação Alostérica , Sítio Alostérico , Animais , Linhagem Celular Tumoral , Fluorometria , Células HEK293 , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PH-797804 is a selective p38 MAPK inhibitor currently evaluated in clinical trials. This study described a validated UPLC-MS/MS combined with one-step protein precipitation extraction method for determination of PH-797804 in rat plasma. After protein precipitation with acetonitrile, the plasma sample was analyzed by a Waters Acquity UPLC BEH C18 column, with acetonitrile/0.1% formic acid (70:30) as the mobile phase. Mass spectrometric detection was conducted with a Waters TQ-S mass spectrometer via electrospray, positive-mode ionization, with target quantitative ion pairs of m/z 476.895â¯ââ¯126.860 for PH-797804, and 482.726â¯ââ¯269.707 for regorafenib (internal standard). The assay showed a good linearity over the range of 1.0-1600â¯ng/mL, with acceptable accuracy (RE from -7.8% to 8.5%) and precision (RSD within 8.4%) values. Recovery from plasma was 81.4-90.2% and matrix effect was negligible (93.3-95.4%). The validated method presented a quantification method of PH-797804 in detail for the first time and utilized for a pharmacokinetic study at three dose concentrations after oral administration in Wistar rats. The pharmacokinetic profiles of PH-797804 showed a linear relationship between drug concentration and dose, which provided dosage and safety information on further clinical studies.
Assuntos
Benzamidas/sangue , Benzamidas/farmacocinética , Cromatografia Líquida de Alta Pressão/métodos , Piridonas/sangue , Piridonas/farmacocinética , Espectrometria de Massas em Tandem/métodos , Animais , Benzamidas/química , Modelos Lineares , Masculino , Piridonas/química , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Diabetic cardiomyopathy, especially myocardial ischemia reperfusion (I/R) injury, is a major cause of morbidity and mortality in type 2 diabetic patients. The increasing of basal p38 MAP Kinase (p38 MAPK) activation is a major factor that aggravates cardiac death on diabetic cardiomyopathy. In addition, metformin also shows cardio-protective effects on myocardial ischemia/reperfusion injury. In this study, we investigated the effect of the combination between metformin and p38 MAPK inhibitor (SB203580) in diabetic rats subjected to I/R injury. H9c2 cells were induced into a hyperglycemic condition and treated with metformin, SB203580 or the combination of metformin and SB203580. In addition, cells in both the presence and absence of drug treatment were subjected to simulated ischemia/reperfusion injury. Cell viability and cellular reactive oxygen species (ROS) were determined. Moreover, the Goto-Kakizaki (GK) rats were treated with metformin, SB203580, and the combination of metformin and SB203580 for 4 weeks. Diabetic parameters and cardiac functions were assessed. Finally, rat hearts were induced ischemia/reperfusion injury for the purpose of infarct size analysis and determination of signal transduction. A high-glucose condition did not reduce cell viability but significantly increased ROS production and significantly decreased cell viability after induced sI/R. Treatment using drugs was shown to reduce ROS generation and cardiac cell death. The GK rats displayed diabetic phenotype by increasing diabetic parameters and these parameters were significantly decreased when treated with drugs. Treatment with metformin or SB203580 could significantly reduce the infarct size. Interestingly, the combination of metformin and SB203580 could enhance cardio-protective ability. Myocardial I/R injury significantly increased p38 MAPK phosphorylation, Bax/Bcl-2 ratio and caspase-3 level. Treatment with drugs significantly decreased the p38 MAPK phosphorylation, Bax/Bcl-2 ratio, caspase-3 level and increased Akt phosphorylation. In conclusion, using the combination of metformin and SB203580 shows positive cardio-protective effects on diabetic ischemic cardiomyopathy.
RESUMO
BACKGROUND: Many inflammatory responses including chemotaxis, production of nitric oxide, and modulation of pro-inflammatory cytokines in immunological cells are mediated by p38MAPK. Due to its pivotal role, p38MAPK has been extensively explored as a molecular target for inhibition of chronic inflammation; however, it has not been successful so far due to serious toxicity issues. Among several downstream substrates of p38, mitogen-activated protein kinase-activated protein kinase 2 (MK2) has been reported to be a direct and essential downstream component in regulation of innate immune and inflammatory responses. Thus, in this study, we aimed to understand relative molecular differences between p38 and MK2 kinase inhibition in terms of a comparative anti-inflammatory potential along with molecular regulation of toxicity biomarkers such as Phospho c-Jun N-Terminal Kinase (pJNK), caspase-3, and hepatic enzyme levels in relevant human cells in vitro. RESULTS: Both p38 and MK2 inhibitors attenuated lipopolysaccharide-induced pro-inflammatory biomarkers expression. In addition, both these kinase inhibitors inhibited release of Th1 and Th17 cytokines in phytohemagglutinin-induced cells with MK2 inhibitor showing a better potency for inhibition of Th1 cytokine release, interferon-γ. In the mechanistic differentiation studies, p38 inhibitors displayed an increase in pJNK and caspase-3 activity in U937 cells and elevation in aspartate transaminase enzyme in HepG2 cells, whereas MK2 inhibitor did not show such adverse toxic effects. CONCLUSION: Taken together, inhibition of MK2 kinase can be a relatively preferred strategy as an anti-inflammatory therapy over direct inhibition of p38 kinase in p38MAPK pathway.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Biomarcadores/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Células Hep G2 , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Imidazóis/farmacologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos , MAP Quinase Quinase 4/metabolismo , Naftalenos/farmacologia , Óxido Nítrico/metabolismo , Pirazóis/farmacologia , Piridazinas/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Células THP-1 , Células U937RESUMO
BACKGROUND: Lung ischemia-reperfusion injury (LIRI) is a pathologic process that is observed in several clinical conditions, and p38 mitogen-activated protein kinase (MAPK) is involved. Diabetes mellitus (DM) results in an increased incidence of ischemia-induced organ damage. The aims of this study were to examine the effects of DM on LIRI in a rat model of DM and to explore the possible mechanisms in relation to the p38 MAPK pathway. METHODS: Forty rats were randomly divided into the following five groups (n = 8 each): a control + sham group, a control + IR group (CIR), a DM + sham group, a DM + IR group (DIR), and a DM + IR + SB203580 group. The control and streptozotocin-induced diabetic rats underwent a sham operation or left hilum occlusion for 90 min followed by reperfusion for 4 h. SB203580 was used to inhibit the p38 MAPK pathway. The pulmonary oxygenation index, inflammatory cytokines in the serum, lung edema, histopathology, oxidant stress, apoptosis, and phosphorylated/total-p38 MAPK protein levels were measured. RESULTS: The DIR group displayed greater concentrations of tumor necrosis factor-α, interleukin-6, and intercellular adhesion molecule-1 and increases in the wet weight-to-dry weight ratio, lung injury scores, malondialdehyde levels, and cellular apoptosis, and these effects were accompanied by lower pulmonary oxygenation compared with the CIR group (P < 0.05). In the DIR group, the expression levels of p38 MAPK protein were significantly upregulated compared with those of the CIR group. Additionally, all of these alterations were attenuated in the DM + IR + SB203580 group compared with the DIR group. CONCLUSIONS: Diabetes exacerbates LIRI by activating the p38 MAPK pathway.
Assuntos
Diabetes Mellitus Experimental/complicações , Lesão Pulmonar/enzimologia , Traumatismo por Reperfusão/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Biomarcadores/metabolismo , Diabetes Mellitus Experimental/enzimologia , Pulmão/irrigação sanguínea , Pulmão/enzimologia , Pulmão/patologia , Lesão Pulmonar/complicações , Lesão Pulmonar/patologia , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/patologiaRESUMO
BACKGROUND: Hyperphosphorylation and aggregation of tau protein are the pathological hallmarks of Alzheimer's disease and related tauopathies. We previously demonstrated that the microglial activation induces tau hyperphosphorylation and cognitive impairment via activation of p38 mitogen-activated protein kinase (p38 MAPK) in the hTau mouse model of tauopathy that was deficient for microglial fractalkine receptor CX3CR1. METHOD: We report an isoform-selective, brain-permeable, and orally bioavailable small molecule inhibitor of p38α MAPK (MW181) and its effects on tau phosphorylation in vitro and in hTau mice. RESULTS: First, pretreatment of mouse primary cortical neurons with MW181 completely blocked inflammation-induced p38α MAPK activation and AT8 (pS199/pS202) site tau phosphorylation, with the maximum effect peaking at 60-90 min after stimulation. Second, treatment of old (~20 months of age) hTau mice with MW181 (1 mg/kg body weight; 14 days via oral gavage) significantly reduced p38α MAPK activation compared with vehicle-administered hTau mice. This also resulted in a significant reduction in AT180 (pT231) site tau phosphorylation and Sarkosyl-insoluble tau aggregates. Third, MW181 treatment significantly increased synaptophysin protein expression and resulted in improved working memory. Fourth, MW181 administration reduced phosphorylated MAPK-activated protein kinase 2 (pMK2) and phosphorylated activating transcription factor 2 (pATF2), which are known substrates of p38α MAPK. Finally, MW181 reduced the expression of interferon-γ and interleukin-1ß. CONCLUSIONS: Taken together, these studies support p38α MAPK as a valid therapeutic target for the treatment of tauopathies.
Assuntos
Fator 2 Ativador da Transcrição/efeitos dos fármacos , Interferon gama/efeitos dos fármacos , Interleucina-1beta/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Memória de Curto Prazo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Piridazinas/farmacologia , Piridinas/farmacologia , Tauopatias/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas tau/metabolismo , Animais , Comportamento Animal , Córtex Cerebral/efeitos dos fármacos , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Piridazinas/administração & dosagem , Piridinas/administração & dosagem , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas tau/efeitos dos fármacosRESUMO
BACKGROUND: Systemic inflammation and steroid resistance are the hallmarks of COPD. We examined the impact of p38 inhibitor (SB203580) in in vitro assays of systemic inflammation using pulmonary cells and patients' sera. OBJECTIVE AND METHODS: Data from 66 COPD patients and 15 age-/sex-matched healthy controls were compared. Interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and CCL5 were measured in serum samples and culture media from peripheral blood mononuclear cells. The impact of sera on IL-10 and CCL5 expression in alveolar macrophage cell line (MH-S) was examined. The in vitro effects of SB203580 on lipopolysaccharide-induced inflammation were investigated. RESULTS: Peripheral blood mononuclear cells from Global initiative for chronic Obstructive Lung Disease (GOLD) D patients produced more CCL5 and TNF-α, and less IL-10 compared to GOLD A-C patients. SB203580 treatment suppressed CCL5 and TNF-α and stimulated IL-10 production; however, the effect of SB203580 on IL-10 was lower in the COPD group. Culture of MH-S cells with COPD serum showed a significant increase in CCL5 and a significant decrease in IL-10 compared to healthy serum. This effect was not suppressed with SB203580 treatment. CONCLUSION: COPD serum has a potent proinflammatory effect on pulmonary cells. Inhibition of p38 phoshorylation had a limited effect in restoring impaired lymphocyte function and suppressing inflammation induced by COPD serum, implying important p38-independent inflammatory mechanisms in COPD.
Assuntos
Anti-Inflamatórios/farmacologia , Citocinas/sangue , Imidazóis/farmacologia , Mediadores da Inflamação/sangue , Macrófagos Alveolares/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Doença Pulmonar Obstrutiva Crônica/sangue , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Idoso , Estudos de Casos e Controles , Linhagem Celular , Quimiocina CCL5/sangue , Feminino , Humanos , Interleucina-10/sangue , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/imunologia , Masculino , Pessoa de Meia-Idade , Fosforilação , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/imunologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/sangue , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Perfluorooctanoic acid (PFOA) is correlated with male reproductive dysfunction in animals and humans, but the underlying mechanisms for this remain unknown. To explore the potential reproductive toxicity of PFOA, we studied blood-testis barrier (BTB) damage using in vivo and in vitro models. Male mice were gavage-administered PFOA (0-20 mg/kg/d) for 28 consecutive days, and breeding capacity and permeability of the Sertoli cell-based BTB were estimated. Primary Sertoli cells (SCs) were exposed to PFOA (0-500 µM) for 48 h, and transepithelial electrical resistance (TER) was assessed. Furthermore, BTB-associated protein expression, TNFα content, and phosphorylation and protein levels of the mitogen-activated protein kinase (MAPK) pathway were detected. An apparent decrease in mated and pregnant females per male mouse as well as litter weight was observed. Marked BTB damage was evidenced by increased red biotin fluorescence in the lumen tubular of the testes and the decrease in TER in SCs in vitro. The protein levels of claudin-11, connexin-43, N-cadherin, ß-catenin, and occludin were significantly decreased in the testes and also in the SCs in vitro except for N-cadherin and ß-catenin. TNFα content showed a dose-dependent increase in the testes and a dose- and time-dependent increase in the SCs, with the p-p38/p38 MAPK ratio also increasing in testes and SCs after PFOA exposure. Moreover, PFOA altered expressions of claudin-11, connexin-43, TNFα, and p-p38 MAPK were recovered 48 h after PFOA removal in the SCs. The SCs appeared to be target to PFOA, and the disruption of the BTB may be crucial to PFOA-induced reproductive dysfunction in mice.
Assuntos
Barreira Hematotesticular/efeitos dos fármacos , Caprilatos/toxicidade , Fluorocarbonos/toxicidade , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Barreira Hematotesticular/fisiologia , Feminino , Fertilidade/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Gravidez , Células de Sertoli/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/imunologiaRESUMO
Each year, despite optimal use of recommended acute and secondary prevention therapies, 4%-5% of patients with acute coronary syndrome (ACS) experience relapse of ACS or other cardiovascular events including stroke, heart failure, or sudden cardiac death after the index ACS. The sudden atherosclerotic plaque rupture leading to an ACS event is often accompanied by inflammation, which is thought to be a key pathogenic pathway to these excess cardiovascular events. Losmapimod is a novel, oral p38 mitogen-activated protein kinase (MAPK) inhibitor that targets MAPKs activated in macrophages, myocardium, and endothelial cells that occur as a part of global coronary vascular inflammation following plaque rupture. This review aims to 1) discuss the pathophysiological pathways through which p38 MAPKs may play key roles in initiation and progression of inflammatory disease and how losmapimod is thought to counteract these p38 MAPKs, and 2) to describe the efficacy and safety data for losmapimod obtained from preclinical studies and randomized controlled trials that support the hypothesis that it has promise as a treatment for patients with ACS.
Assuntos
Síndrome Coronariana Aguda/tratamento farmacológico , Anti-Inflamatórios/uso terapêutico , Fármacos Cardiovasculares/uso terapêutico , Ciclopropanos/uso terapêutico , Proteínas de Membrana/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Síndrome Coronariana Aguda/enzimologia , Síndrome Coronariana Aguda/fisiopatologia , Animais , Anti-Inflamatórios/efeitos adversos , Fármacos Cardiovasculares/efeitos adversos , Ciclopropanos/efeitos adversos , Ativação Enzimática , Humanos , Proteínas de Membrana/metabolismo , Inibidores de Proteínas Quinases/efeitos adversos , Piridinas/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
Autologous stem cells are a promising cell source for cartilage regeneration; however, cell replicative senescence and joint posttraumatic inflammation provide challenges in bringing this treatment modality to fruition. In this study, we hypothesized that preconditioning with p38 MAPK inhibitor (sb203580) would recharge decellularized extracellular matrix (dECM) expanded human synovium-derived stem cell (hSDSC) chondrogenesis in an inflammatory environment. We found that preconditioning with sb203580 greatly enhanced dECM expanded hSDSC proliferation and chondrogenic potential while supplementation with sb203580 in an induction medium dramatically retarded hSDSC chondrogenic differentiation, even for dECM expanded cells. We also found that sb203580 preconditioning enhanced matrix-expanded hSDSC chondrogenic capacity even in an interleukin-1 (IL-1) induced inflammatory environment. Non-detectable expression of HLA-DR in the hSDSCs grown on allogeneic dECM indicates the feasibility of commercial preparation of these dECMs from healthy, young donors for patients who need autologous transplantation. Our study indicated that p38 MAPK inhibitor has a distinctive priming effect on dECM mediated stem cell cartilage regeneration. Combined rejuvenation with sb203580 and dECM expansion can precondition hSDSCs' resurfacing capacity for osteoarthritic patients with cartilage defects.
Assuntos
Células-Tronco Adultas/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/farmacologia , Condrogênese/efeitos dos fármacos , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Membrana Sinovial/citologia , Adulto , Cartilagem/patologia , Cartilagem/fisiologia , Células Cultivadas , Meios de Cultura/farmacologia , Avaliação Pré-Clínica de Medicamentos , Matriz Extracelular , Feminino , Antígenos HLA-DR/análise , Humanos , Técnicas In Vitro , Inflamação , Interleucina-1beta/farmacologia , Masculino , Osteoartrite/tratamento farmacológico , Regeneração/efeitos dos fármacos , Rejuvenescimento , Transplante Autólogo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
BACKGROUND: A six month study of the p38 MAPK inhibitor, losmapimod, suggested a trend in reducing COPD exacerbations with the 15 mg twice daily dose. OBJECTIVE AND METHODS: Using data from this study which evaluated the efficacy of twice daily losmapimod, 2.5 mg, 7.5 mg, and 15 mg, versus placebo in patients with moderate-to-severe COPD, we analysed the effect of losmapimod in reducing the rate of moderate/severe exacerbations in patient subgroups with ≤2% and >2% blood eosinophils at baseline. Lung function, fibrinogen and hsCRP were also evaluated. RESULTS: In the ≤2% eosinophil subgroup, there was an exposure-related reduction in the rate of moderate/severe exacerbations with losmapimod relative to placebo (losmapimod 15 mg: 55% reduction; losmapimod 7.5 mg: 29%; losmapimod 2.5 mg: 10%); with the 15 mg dose reaching statistical significance (15 mg/placebo mean rate ratio [95% CI]: 0.45 [0.22; 0.90]). There was also an improvement in lung function with 15 mg losmapimod over Weeks 1-12. No improvement in the rate of moderate/severe exacerbations or post-bronchodilator FEV1 was observed for subjects treated with Losmapimod compared to placebo in the patient subgroup with blood eosinophils >2% at baseline. Transient reductions in fibrinogen and hsCRP were observed with losmapimod 7.5 mg and 15 mg in both eosinophil subgroups. CONCLUSIONS: These findings indicate eosinophil-related heterogeneity within COPD and suggest that losmapimod could be a potential therapy to reduce exacerbations in COPD patients with eosinophil levels ≤2%. This needs to be explored further in a prospectively designed study with pre-specified criteria for blood eosinophil subgroups in COPD patients.
Assuntos
Ciclopropanos/administração & dosagem , Eosinófilos/patologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Piridinas/administração & dosagem , Idoso , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Seguimentos , Volume Expiratório Forçado , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Índice de Gravidade de Doença , Resultado do Tratamento , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
OBJECTIVE: To investigated whether CBS3830, a highly selectively inhibitor of p38MAPK, could ameliorate inflammation and intimal hyperplasia in arterialized vein grafts (AVGs). METHODS: Sixty male Sprague-Dawley rats underwent a reversed right jugular vein to common carotid artery interposition graft and were randomly treatment with vehicle (control) or single-dose (3 mg/kg, preoperative) or double-dose (3 mg/kg, preoperative and 4 d postoperative) CBS3830. Twenty rats underwent sham operation. The levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) were determined by ELISA. Vein grafts were analyzed by intimal/medial morphometry, proliferating cell nuclear antigen (PCNA) expression, and p38MAPK phosphorylation. RESULTS: TNF-α, IL-1ß, and IL-6 gradually increased then slowly decreased in AVG rats. However, at 4 d and 7 d, TNF-α levels decreased by 37.5% and 29.5% (p = 0.003, 0.05, respectively) in the single-dose CBS3830 group, and by 37.6% and 32.5%, respectively (both p = 0.003) in the double-dose group compared with those of control. IL-1ß levels significantly reduced at 4 d and 14 d in both dosage groups. IL-6 levels significantly reduced at 7 d in both groups. Intima and medial thickening were significantly reduced in both dosage treated groups at 7, 14, and 28 d (all p = 0.000) compared to the controls. Further study showed CBS3830 inhibited p38MAPK phosphorylation and decreased PCNA expression. CONCLUSIONS: CBS3830 significantly decreases inflammation and intimal hyperplasia in AVGs.
Assuntos
Anti-Inflamatórios/farmacologia , Proliferação de Células/efeitos dos fármacos , Dibenzocicloeptenos/farmacologia , Veias Jugulares/transplante , Túnica Íntima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Oclusão de Enxerto Vascular/enzimologia , Oclusão de Enxerto Vascular/imunologia , Oclusão de Enxerto Vascular/patologia , Oclusão de Enxerto Vascular/prevenção & controle , Hiperplasia , Imunidade Inata/efeitos dos fármacos , Veias Jugulares/enzimologia , Veias Jugulares/imunologia , Veias Jugulares/patologia , Masculino , Antígeno Nuclear de Célula em Proliferação/biossíntese , Ratos Sprague-Dawley , Túnica Íntima/enzimologia , Túnica Íntima/imunologia , Túnica Íntima/patologiaRESUMO
OBJECTIVE: Following bypass surgery vein grafts undergo a remodelling process that can lead to restenosis and ultimately vein graft failure. Signalling through mitogen activated protein kinases (MAPKs) is a key mechanism involved in vein graft failure. Here, we investigated whether CBS3830 (c-a-i-r biosciences GmbH, Tübingen, Germany), a new highly selectively inhibitor of p38 MAPK, has a significant effect on inhibiting intimal, medial and adventitial hyperplasia. METHODS: Sixty specific pathogen free Sprague Dawley male rats were randomly divided into three groups. The control group with a reversed right jugular vein, which is common to carotid artery interposition graft, was compared with sham-operated, and CBS3830 treated animals. Intimal, medial and adventitia morphometric examinations and expression of proliferating cell nuclear antigen (PCNA) were analysed after one, two and four weeks for vein grafts. RESULTS: Intimal, medial and adventitia thickening in CBS3830 group were significantly lower than in the control group at each time point. Moreover, CBS3830 significantly reduced the phosphorylation of p38 MAPK and PCNA expression compared to the control. CONCLUSION: On the basis of the present work, intima, media and adventitia of saphenous vein grafts undergo vascular remodelling after surgery. The new, highly selective p38 MAPK inhibitor, CBS3830, ameliorates intimal, medial, and adventitial remodelling by varying degrees.
Assuntos
Ponte de Artéria Coronária , Oclusão de Enxerto Vascular/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Veia Safena/enzimologia , Túnica Íntima/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Túnica Adventícia/enzimologia , Túnica Adventícia/patologia , Túnica Adventícia/fisiopatologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Oclusão de Enxerto Vascular/enzimologia , Oclusão de Enxerto Vascular/patologia , Oclusão de Enxerto Vascular/fisiopatologia , Masculino , Fosforilação/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/biossíntese , Ratos , Ratos Sprague-Dawley , Veia Safena/patologia , Veia Safena/fisiopatologia , Túnica Íntima/patologia , Túnica Íntima/fisiopatologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Objective To observe the change of renal function and cell apoptosis after injecting SB203580 before and after reperfusion, and investigate the protective role of p38 MAPK inhibitor SB203580 for ischemic/reperfused kidney in rats. Methods p38 MAPK inhibitor SB203580 was injected by tail vein into rats with ischemic kidney before and after reperfusion. The plasma levels of creatine and BUN were measured at various time points. The apoptotic rate in the renal tissue at various time points was determined using TUNEL. Results Administering SB203580 before reperfusion could decrease renal cell apoptotic rate, and renal function damage. Administering SB203580 after reperfusion had not obvious effect on the renal function and cell apoptosis. Conclusion Administering p38 MAPK inhibitor before reperfusion can attenuate post-ischemic renal fuction damage and cell apoptosis.