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1.
Int J Mol Sci ; 22(18)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34576119

RESUMO

Although apoptosis of keratinocytes has been relatively well studied, there is a lack of information comparing potentially proapoptotic treatments for healthy and diseased skin cells. Psoriasis is a chronic autoimmune-mediated skin disease manifested by patches of hyperproliferative keratinocytes that do not undergo apoptosis. UVB phototherapy is commonly used to treat psoriasis, although this has undesirable side effects, and is often combined with anti-inflammatory compounds. The aim of this study was to analyze if cannabidiol (CBD), a phytocannabinoid that has anti-inflammatory and antioxidant properties, may modify the proapoptotic effects of UVB irradiation in vitro by influencing apoptotic signaling pathways in donor psoriatic and healthy human keratinocytes obtained from the skin of five volunteers in each group. While CBD alone did not have any major effects on keratinocytes, the UVB treatment activated the extrinsic apoptotic pathway, with enhanced caspase 8 expression in both healthy and psoriatic keratinocytes. However, endoplasmic reticulum (ER) stress, characterized by increased expression of caspase 2, was observed in psoriatic cells after UVB irradiation. Furthermore, decreased p-AKT expression combined with increased 15-d-PGJ2 level and p-p38 expression was observed in psoriatic keratinocytes, which may promote both apoptosis and necrosis. Application of CBD partially attenuated these effects of UVB irradiation both in healthy and psoriatic keratinocytes, reducing the levels of 15-d-PGJ2, p-p38 and caspase 8 while increasing Bcl2 expression. However, CBD increased p-AKT only in UVB-treated healthy cells. Therefore, the reduction of apoptotic signaling pathways by CBD, observed mainly in healthy keratinocytes, suggests the need for further research into the possible beneficial effects of CBD.


Assuntos
Apoptose/efeitos dos fármacos , Canabidiol/farmacologia , Queratinócitos/citologia , Queratinócitos/efeitos da radiação , Psoríase/patologia , Raios Ultravioleta , Biomarcadores/metabolismo , Linhagem Celular , Dinoprostona/farmacologia , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Queratinócitos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação
2.
FASEB J ; 35(10): e21941, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34559928

RESUMO

Thromboxane receptor (TP) mediates nasal obstruction, a typical symptom of allergic rhinitis. Since it has been reported that several types of eicosanoids, such as non-enzymatic oxidation product of arachidonic acid isoprostane, act as a TP ligand, there is a possibility that some other eicosanoids contribute to the TP-mediated nasal obstruction. The aim of this study is to investigate the mechanisms of TP-mediated nasal obstruction. Intranasal challenges of ovalbumin (OVA) induced nasal obstruction in mice. Pharmacological blockade of TP receptor but not thromboxane A2 synthase inhibited OVA-induced nasal obstruction. Simultaneous analysis of eicosanoids in nasal lavage fluid and the responses in trans-endothelial resistance suggested that 8-iso-prostaglandin E2 (PGE2 ) can be a candidate for TP ligand. Intranasal challenge of 8-iso-PGE2 induced vascular hyperpermeability and nasal obstruction in TP receptor-dependent manner. Wholemount immunostaining of nasal septum mucosa revealed that 8-iso-PGE2 increased plasma leakage accompanied by distention of venous sinusoids. This study shows that 8-iso-PGE2 is a contributor in TP-mediated nasal obstruction in mice.


Assuntos
Dinoprostona/análogos & derivados , Modelos Animais de Doenças , Isoprostanos/farmacologia , Obstrução Nasal/induzido quimicamente , Obstrução Nasal/complicações , Receptores de Tromboxanos/metabolismo , Rinite Alérgica/complicações , Rinite Alérgica/metabolismo , Administração Intranasal , Animais , Permeabilidade Capilar/efeitos dos fármacos , Dinoprostona/administração & dosagem , Dinoprostona/farmacologia , Feminino , Isoprostanos/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais/efeitos dos fármacos
3.
PLoS One ; 16(8): e0255335, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34347801

RESUMO

The SARS-CoV-2 coronavirus has led to a pandemic with millions of people affected. The present study finds that risk-factors for severe COVID-19 disease courses, i.e. male sex, older age and sedentary life style are associated with higher prostaglandin E2 (PGE2) serum levels in blood samples from unaffected subjects. In COVID-19 patients, PGE2 blood levels are markedly elevated and correlate positively with disease severity. SARS-CoV-2 induces PGE2 generation and secretion in infected lung epithelial cells by upregulating cyclo-oxygenase (COX)-2 and reducing the PG-degrading enzyme 15-hydroxyprostaglandin-dehydrogenase. Also living human precision cut lung slices (PCLS) infected with SARS-CoV-2 display upregulated COX-2. Regular exercise in aged individuals lowers PGE2 serum levels, which leads to increased Paired-Box-Protein-Pax-5 (PAX5) expression, a master regulator of B-cell survival, proliferation and differentiation also towards long lived memory B-cells, in human pre-B-cell lines. Moreover, PGE2 levels in serum of COVID-19 patients lowers the expression of PAX5 in human pre-B-cell lines. The PGE2 inhibitor Taxifolin reduces SARS-CoV-2-induced PGE2 production. In conclusion, SARS-CoV-2, male sex, old age, and sedentary life style increase PGE2 levels, which may reduce the early anti-viral defense as well as the development of immunity promoting severe disease courses and multiple infections. Regular exercise and Taxifolin treatment may reduce these risks and prevent severe disease courses.


Assuntos
COVID-19/patologia , Dinoprostona/sangue , Imunidade , Adolescente , Adulto , Animais , COVID-19/sangue , COVID-19/imunologia , Estudos de Casos e Controles , Células Cultivadas , Chlorocebus aethiops , Dinoprostona/farmacologia , Dinoprostona/fisiologia , Progressão da Doença , Feminino , Humanos , Imunidade/efeitos dos fármacos , Imunidade/fisiologia , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Células Vero , Adulto Jovem
4.
PLoS One ; 16(7): e0253957, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228760

RESUMO

BACKGROUND: As uterine rupture may affect as many as 11/1000 women with 1 prior cesarean birth and 5/10.000 women with unscarred uterus undergoing labor induction, we intended to estimate the prevalence of such rare outcome when PGE2 is used for cervical ripening and labor induction. METHODS: We searched MEDLINE, ClinicalTrials.gov and the Cochrane library up to September 1st 2020. Retrospective and prospective cohort studies, as well as randomized controlled trials (RCTs) on singleton viable pregnancies receiving PGE2 for cervical ripening and labor induction were reviewed. Prevalence of uterine rupture was meta-analyzed with Freeman-Tukey double arcsine transformation among women with 1 prior low transverse cesarean section and women with unscarred uterus. RESULTS: We reviewed 956 full text articles to include 69 studies. The pooled prevalence rate of uterine rupture is estimated to range between 2 and 9 out of 1000 women with 1 prior low transverse cesarean (5/1000; 95%CI 2-9/1000, 122/9000). The prevalence of uterine rupture among women with unscarred uterus is extremely low, reaching at most 0.7/100.000 (<1/100.000.000; 95%CI <1/100.000.000-0.7/100.000, 8/17.684). CONCLUSIONS: Uterine rupture is a rare event during cervical ripening and labor induction with PGE2.


Assuntos
Cesárea , Dinoprostona/farmacologia , Trabalho de Parto Induzido , Ruptura Uterina/epidemiologia , Útero/patologia , Adulto , Feminino , Humanos , Gravidez , Prevalência , Viés de Publicação , Útero/efeitos dos fármacos
5.
Sci Rep ; 11(1): 13559, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193890

RESUMO

Prostaglandin E2 (PGE2), an active lipid compound derived from arachidonic acid, regulates different stages of the immune response of the host during several pathologies such as chronic infections or cancer. In fact, manipulation of PGE2 levels was proposed as an approach for countering the Type I IFN signature of tuberculosis (TB). However, very limited information regarding the PGE2 pathway in patients with active TB is currently available. In the present work, we demonstrated that PGE2 exerts a potent immunosuppressive action during the immune response of the human host against Mycobacterium tuberculosis (Mtb) infection. Actually, we showed that PGE2 significantly reduced the surface expression of several immunological receptors, the lymphoproliferation and the production of proinflammatory cytokines. In addition, PGE2 promoted autophagy in monocytes and neutrophils cultured with Mtb antigens. These results suggest that PGE2 might be attenuating the excessive inflammatory immune response caused by Mtb, emerging as an attractive therapeutic target. Taken together, our findings contribute to the knowledge of the role of PGE2 in the human host resistance to Mtb and highlight the potential of this lipid mediator as a tool to improve anti-TB treatment.


Assuntos
Dinoprostona/farmacologia , Imunossupressores/farmacologia , Monócitos/imunologia , Mycobacterium tuberculosis/imunologia , Neutrófilos/imunologia , Tuberculose/imunologia , Adulto , Dinoprostona/imunologia , Feminino , Humanos , Imunossupressores/imunologia , Masculino
6.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071686

RESUMO

Prostaglandins are a group of lipids that produce diverse physiological and pathological effects. Among them, prostaglandin E2 (PGE2) stands out for the wide variety of functions in which it participates. To date, there is little information about the influence of PGE2 on gap junctional intercellular communication (GJIC) in any type of tissue, including epithelia. In this work, we set out to determine whether PGE2 influences GJIC in epithelial cells (MDCK cells). To this end, we performed dye (Lucifer yellow) transfer assays to compare GJIC of MDCK cells treated with PGE2 and untreated cells. Our results indicated that (1) PGE2 induces a statistically significant increase in GJIC from 100 nM and from 15 min after its addition to the medium, (2) such effect does not require the synthesis of new mRNA or proteins subunits but rather trafficking of subunits already synthesized, and (3) such effect is mediated by the E2 receptor, which, in turn, triggers a signaling pathway that includes activation of adenylyl cyclase and protein kinase A (PKA). These results widen the knowledge regarding modulation of gap junctional intercellular communication by prostaglandins.


Assuntos
Comunicação Celular/efeitos dos fármacos , Dinoprostona/farmacologia , Células Epiteliais/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cães , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Junções Comunicantes/metabolismo , Células Madin Darby de Rim Canino , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
7.
Cells ; 10(4)2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805551

RESUMO

The primary means of intestinal absorption of nutrients by villus cells is via Na-dependent nutrient co-transporters located in the brush border membrane (BBM). These secondary active co-transport processes require a favorable transcellular Na gradient that is provided by Na-K-ATPase. In chronic enteritis, malabsorption of essential nutrients is partially due to inhibition of villus Na-K-ATPase activity mediated by specific immune inflammatory mediators that are known to be elevated in the inflamed mucosa. However, how Prostaglandin E2 (PGE2), a specific mediator of nutrient malabsorption in the villus BBM, may mediate the inhibition of Na-K-ATPase is not known. Therefore, this study aimed to determine the effect of PGE2 on Na-K-ATPase in villus cells and define its mechanism of action. In vitro, in IEC-18 cells, PGE2 treatment significantly reduced Na-K-ATPase activity, accompanied by a significant increase in the intracellular levels of cyclic Adenosine Monophosphate (cAMP). The treatment with cAMP analog 8-Bromo-cAMP mimicked the PGE2-mediated effect on Na-K-ATPase activity, while Rp-cAMP (PKA inhibitor) pretreatment reversed the same. The mechanism of inhibition of PGE2 was secondary to a transcriptional reduction in the Na-K-ATPase α1 and ß1 subunit genes, which was reversed by the Rp-cAMP pretreatment. Thus, the PGE2-mediated activation of the PKA pathway mediates the transcriptional inhibition of Na-K-ATPase activity in vitro.


Assuntos
Dinoprostona/farmacologia , Células Epiteliais/enzimologia , Intestinos/citologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , Espaço Intracelular/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Xantonas/farmacologia
8.
PLoS Med ; 18(2): e1003448, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33571294

RESUMO

BACKGROUND: Prolonged pregnancies are a frequent indication for induction of labour. When the cervix is unfavourable, cervical ripening before oxytocin administration is recommended to increase the likelihood of vaginal delivery, but no particular method is currently recommended for cervical ripening of prolonged pregnancies. This trial evaluates whether the use of mechanical cervical ripening with a silicone double balloon catheter for induction of labour in prolonged pregnancies reduces the cesarean section rate for nonreassuring fetal status compared with pharmacological cervical ripening by a vaginal pessary for the slow release of dinoprostone (prostaglandin E2). METHODS AND FINDINGS: This is a multicentre, superiority, open-label, parallel-group, randomised controlled trial conducted in 15 French maternity units. Women with singleton pregnancies, a vertex presentation, ≥41+0 and ≤42+0 weeks' gestation, a Bishop score <6, intact membranes, and no history of cesarean delivery for whom induction of labour was decided were randomised to either mechanical cervical ripening with a Cook Cervical Ripening Balloon or pharmacological cervical ripening by a Propess vaginal pessary serving as a prostaglandin E2 slow-release system. The primary outcome was the rate of cesarean for nonreassuring fetal status, with an independent endpoint adjudication committee determining whether the fetal heart rate was nonreassuring. Secondary outcomes included delivery (time from cervical ripening to delivery, number of patients requiring analgesics), maternal and neonatal outcomes. Between January 2017 and December 2018, 1,220 women were randomised in a 1:1 ratio, 610 allocated to a silicone double balloon catheter, and 610 to the Propess vaginal pessary for the slow release of dinoprostone. The mean age of women was 31 years old, and 80% of them were of white ethnicity. The cesarean rates for nonreassuring fetal status were 5.8% (35/607) in the mechanical ripening group and 5.3% (32/609) in the pharmacological ripening group (proportion difference: 0.5%; 95% confidence interval (CI) -2.1% to 3.1%, p = 0.70). Time from cervical ripening to delivery was shorter in the pharmacological ripening group (23 hours versus 32 hours, median difference 6.5 95% CI 5.0 to 7.9, p < 0.001), and fewer women required analgesics in the mechanical ripening group (27.5% versus 35.4%, difference in proportion -7.9%, 95% CI -13.2% to -2.7%, p = 0.003). There were no statistically significant differences between the 2 groups for other delivery, maternal, and neonatal outcomes. A limitation was a low observed rate of cesarean section. CONCLUSIONS: In this study, we observed no difference in the rates of cesarean deliveries for nonreassuring fetal status between mechanical ripening with a silicone double balloon catheter and pharmacological cervical ripening with a pessary for the slow release of dinoprostone. TRIAL REGISTRATION: ClinicalTrials.gov NCT02907060.


Assuntos
Maturidade Cervical/efeitos dos fármacos , Dinoprostona/farmacologia , Ocitócicos/farmacologia , Silicones/farmacologia , Adulto , Maturidade Cervical/fisiologia , Cesárea/métodos , Parto Obstétrico/métodos , Dinoprostona/administração & dosagem , Feminino , Humanos , Trabalho de Parto Induzido/métodos , Ocitócicos/administração & dosagem , Pessários , Gravidez , Gravidez Prolongada/tratamento farmacológico
9.
Cells ; 10(2)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530465

RESUMO

Bone-muscle crosstalk plays an important role in skeletal biomechanical function, the progression of numerous pathological conditions, and the modulation of local and distant cellular environments. Previous work has revealed that the deletion of connexin (Cx) 43 in osteoblasts, and consequently, osteocytes, indirectly compromises skeletal muscle formation and function. However, the respective roles of Cx43-formed gap junction channels (GJs) and hemichannels (HCs) in the bone-muscle crosstalk are poorly understood. To this end, we used two Cx43 osteocyte-specific transgenic mouse models expressing dominant negative mutants, Δ130-136 (GJs and HCs functions are inhibited), and R76W (only GJs function is blocked), to determine the effect of these two types of Cx43 channels on neighboring skeletal muscle. Blockage of osteocyte Cx43 GJs and HCs in Δ130-136 mice decreased fast-twitch muscle mass with reduced muscle protein synthesis and increased muscle protein degradation. Both R76W and Δ130-136 mice exhibited decreased muscle contractile force accompanied by a fast-to-slow fiber transition in typically fast-twitch muscles. In vitro results further showed that myotube formation of C2C12 myoblasts was inhibited after treatment with the primary osteocyte conditioned media (PO CM) from R76W and Δ130-136 mice. Additionally, prostaglandin E2 (PGE2) level was significantly reduced in both the circulation and PO CM of the transgenic mice. Interestingly, the injection of PGE2 to the transgenic mice rescued fast-twitch muscle mass and function; however, this had little effect on protein synthesis and degradation. These findings indicate a channel-specific response: inhibition of osteocytic Cx43 HCs decreases fast-twitch skeletal muscle mass alongside reduced protein synthesis and increased protein degradation. In contrast, blockage of Cx43 GJs results in decreased fast-twitch skeletal muscle contractile force and myogenesis, with PGE2 partially accounting for the measured differences.


Assuntos
Osso e Ossos/metabolismo , Conexina 43/metabolismo , Músculos/metabolismo , Osteócitos/metabolismo , Animais , Osso e Ossos/efeitos dos fármacos , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Dinoprostona/farmacologia , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Camundongos Transgênicos , Contração Muscular/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculos/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo
10.
Mol Cell Endocrinol ; 526: 111219, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33610642

RESUMO

Prostaglandin E2 (PGE2) is a principal lipid mediator mediating various biological processes including immune responses and fluid secretion. As the first line of host defense against infection, vaginal epithelium plays orchestrated roles in vaginal innate immunity. However, the effect of PGE2 triggered by pro-inflammatory stimuli on vaginal epithelium remains elusive. This study aimed to investigate the regulatory role of PGE2 on vaginal epithelium after lipopolysaccharide (LPS) stimulation. RT-PCR and western blot analysis revealed that E-prostanoid (EP) receptors EP2 and EP4 were expressed in rat vagina. Basolateral application of PGE2 induced anion secretion mediated by cystic fibrosis transmembrane conductance regulator (CFTR) via EP-adenylate cyclase-cAMP signaling pathway in rat vaginal epithelial cells. The in vivo study showed that PGE2 promoted fluid secretion in rat vagina. Moreover, LPS stimulation facilitated cyclooxygenase-dependent PGE2 synthesis and vaginal fluid secretion in vivo. Conclusively, LPS stimulation triggered epithelium-derived PGE2 production in vaginal epithelium, leading to CFTR-mediated anion secretion and luminal flushing. This study provides valuable insights into the physiological role of PGE2 during vaginal bacterial infection.


Assuntos
Líquidos Corporais/metabolismo , Dinoprostona/farmacologia , Epitélio/metabolismo , Lipopolissacarídeos/farmacologia , Vagina/metabolismo , Animais , Ânions , Líquidos Corporais/efeitos dos fármacos , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Feminino , Modelos Biológicos , Ratos Sprague-Dawley , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Vagina/efeitos dos fármacos
11.
J Physiol Sci ; 71(1): 8, 2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33622238

RESUMO

Colonic motor activity is important for the formation and propulsion of feces. The production of prostaglandins (PGs) in colonic tissue is considered to play a critical role in the generation and regulation of colonic motility. In this study, we investigated the inhibitory effects of PGE2 and selective agonists of four EP receptors on the spontaneous phasic contractions, called 'giant contractions' (GCs), of mucosa-free circular smooth muscle strips from the rat middle colon. Neural blockade with tetrodotoxin (TTX) increased the frequency and amplitude of the GCs by about twofold. However, inhibiting PG production with piroxicam reduced the GC frequency in the presence of TTX, but did not affect the GC amplitude. In the presence of both TTX and piroxicam, exogenous PGE2 and each EP receptor agonist were cumulatively added to the tissue bath. In this setting, PGE2, the EP2 agonist ONO-AE1-259, and the EP4 agonist ONO-AE1-329, but not the EP1 agonist ONO-AE-DI-004 or the EP3 agonist ONO-AE-248, concentration-dependently reduced the GC frequency and amplitude. The PGE2-induced inhibition of GC frequency and amplitude was inhibited by the EP4 antagonist ONO-AE3-208, but not by the EP1/2 antagonist AH6809. Immunohistochemistry revealed the EP2 and EP4 receptors were localized in perinuclear sites in circular smooth muscle cells. EP2 immunoreactivity was also located in GFAP-immunoreactive enteroglia, whereas EP4 immunoreactivity was also located in HU (embryonic lethal, abnormal vision [ELAV] protein; a marker of all myenteric neurons)-immunoreactive myenteric nerve cell bodies. These results suggest that the PGs produced in the colonic tissue inhibit the GC frequency and amplitude of circular muscle in the rat middle colon, and is mediated by EP4 receptors expressed in the smooth muscle cells.


Assuntos
Colo/efeitos dos fármacos , Dinoprostona/farmacologia , Motilidade Gastrointestinal/fisiologia , Músculo Liso/efeitos dos fármacos , Piroxicam/farmacologia , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Animais , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Colo/fisiologia , Imuno-Histoquímica , Masculino , Ratos , Ratos Wistar , Receptores de Prostaglandina E Subtipo EP1 , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP3 , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Proteínas de Peixe-Zebra
12.
PLoS One ; 16(1): e0245400, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33444342

RESUMO

The Na+/K+ ATPase is a key regulator of the hepatocytes ionic homeostasis, which when altered may lead to many liver disorders. We demonstrated recently, a significant stimulation of the Na+/K+ ATPase in HepG2 cells treated with the S1P analogue FTY 720P, that was mediated through PGE2. The mechanism by which the prostaglandin exerts its effect was not investigated, and is the focus of this work. The type of receptors involved was determined using pharmacological inhibitors, while western blot analysis, fluorescence imaging of GFP-tagged Na+/K+ ATPase, and time-lapse imaging on live cells were used to detect changes in membrane abundance of the Na+/K+ ATPase. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. The enhanced activity of the ATPase was not observed when EP4 receptors were blocked but still appeared in presence inhibitors of EP1, EP2 and EP3 receptors. The involvement of EP4 was confirmed by the stimulation observed with EP4 agonist. The stimulatory effect of PGE2 did not appear in presence of Rp-cAMP, an inhibitor of PKA, and was imitated by db-cAMP, a PKA activator. Chelating intracellular calcium with BAPTA-AM abrogated the effect of db-cAMP as well as that of PGE2, but PGE2 treatment in a calcium-free PBS medium did not, suggesting an involvement of intracellular calcium, that was confirmed by the results obtained with 2-APB treatment. Live cell imaging showed movement of GFP-Na+/K+ ATPase-positive vesicles to the membrane and increased abundance of the ATPase at the membrane after PGE2 treatment. It was concluded that PGE2 acts via EP4, PKA, and intracellular calcium.


Assuntos
Cálcio/metabolismo , Carcinoma Hepatocelular/patologia , Dinoprostona/farmacologia , Neoplasias Hepáticas/patologia , Receptores de Prostaglandina E Subtipo EP4/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Ocitócicos/farmacologia , Proteína Quinase C/metabolismo , Receptores de Prostaglandina E Subtipo EP4/genética , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/genética
13.
Blood ; 137(4): 500-512, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33507291

RESUMO

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.


Assuntos
AMP Cíclico/fisiologia , Dexametasona/farmacologia , Dinoprostona/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Sistemas do Segundo Mensageiro/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Criança , Cromograninas/antagonistas & inibidores , Colforsina/farmacologia , AMP Cíclico/farmacologia , Dexametasona/administração & dosagem , Dinoprostona/administração & dosagem , Dinoprostona/antagonistas & inibidores , Dinoprostona/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gs de Proteínas de Ligação ao GTP/deficiência , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Modelos Animais , Terapia de Alvo Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Quimera por Radiação , Receptores de Glucocorticoides/biossíntese , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/fisiologia , Receptores de Prostaglandina E Subtipo EP4/biossíntese , Receptores de Prostaglandina E Subtipo EP4/genética , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Nat Chem Biol ; 17(1): 39-46, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32989297

RESUMO

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.


Assuntos
Técnicas Biossensoriais , Proteínas Quinases Dependentes de AMP Cíclico/genética , Miócitos Cardíacos/enzimologia , Neurônios/enzimologia , Imagem Óptica/métodos , Alprostadil/farmacologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Di-Hidroxifenilalanina/farmacologia , Dinoprostona/farmacologia , Corantes Fluorescentes/química , Expressão Gênica , Biblioteca Gênica , Genes Reporter , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Humanos , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Cultura Primária de Células , Transdução de Sinais
15.
Radiat Res ; 195(2): 115-127, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33302300

RESUMO

Identification of medical countermeasures (MCM) to mitigate radiation damage and/or protect first responders is a compelling unmet medical need. The prostaglandin E2 (PGE2) analog, 16,16 dimethyl-PGE2 (dmPGE2), has shown efficacy as a radioprotectant and radiomitigator that can enhance hematopoiesis and ameliorate intestinal mucosal cell damage. In this study, we optimized the time of administration of dmPGE2 for protection and mitigation against mortality from the hematopoietic acute radiation syndrome (H-ARS) in young adult mice, evaluated its activity in pediatric and geriatric populations, and investigated potential mechanisms of action. Windows of 30-day survival efficacy for single administration of dmPGE2 were defined as within 3 h prior to and 6-30 h after total-body γ irradiation (TBI). Radioprotective and radio-mitigating efficacy was also observed in 2-year-old geriatric mice and 6-week-old pediatric mice. PGE2 receptor agonist studies suggest that signaling through EP4 is primarily responsible for the radioprotective effects. DmPGE2 administration prior to TBI attenuated the drop in red blood cells and platelets, accelerated recovery of all peripheral blood cell types, and resulted in higher hematopoietic and mesenchymal stem cells in survivor bone marrow. Multiplex analysis of bone marrow cytokines together with RNA sequencing of hematopoietic stem cells indicated a pro-hematopoiesis cytokine milieu induced by dmPGE2, with IL-6 and G-CSF strongly implicated in dmPGE2-mediated radioprotective activity. In summary, we have identified windows of administration for significant radio-mitigation and radioprotection by dmPGE2 in H-ARS, demonstrated survival efficacy in special populations, and gained insight into radioprotective mechanisms, information useful towards development of dmPGE2 as a MCM for first responders, military personnel, and civilians facing radiation threats.


Assuntos
Síndrome Aguda da Radiação/tratamento farmacológico , Dinoprostona/farmacologia , Tolerância a Radiação/genética , Protetores contra Radiação/farmacologia , Síndrome Aguda da Radiação/genética , Síndrome Aguda da Radiação/patologia , Animais , Dinoprostona/análogos & derivados , Dinoprostona/genética , Relação Dose-Resposta à Radiação , Raios gama/efeitos adversos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Fator Estimulador de Colônias de Granulócitos/genética , Hematopoese/efeitos dos fármacos , Hematopoese/efeitos da radiação , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Interleucina-6/genética , Camundongos , Tolerância a Radiação/efeitos dos fármacos , Análise de Sequência de RNA , Irradiação Corporal Total
16.
Am J Physiol Renal Physiol ; 320(2): F212-F223, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33283648

RESUMO

Prostaglandin E2 (PGE2) instilled into the bladder generates symptoms of urinary urgency in healthy women and reduces bladder capacity and urethral pressure in both humans and female rats. Systemic capsaicin desensitization, which causes degeneration of C-fibers, prevented PGE2-mediated reductions in bladder capacity, suggesting that PGE2 acts as an irritant (Maggi CA, Giuliani S, Conte B, Furio M, Santicioli P, Meli P, Gragnani L, Meli A. Eur J Pharmacol 145: 105-112, 1988). In the present study, we instilled PGE2 in female rats after capsaicin desensitization but without the hypogastric nerve transection that was conducted in the Maggi et al. study. One week after capsaicin injection (125 mg/kg sc), rats underwent cystometric and urethral perfusion testing under urethane anesthesia with saline and 100 µM PGE2. Similar to naïve rats, capsaicin-desensitized rats exhibited a reduction in bladder capacity from 1.23 ± 0.08 mL to 0.70 ± 0.10 mL (P = 0.002, n = 9), a reduction in urethral perfusion pressure from 19.3 ± 2.1 cmH2O to 10.9 ± 1.2 cmH2O (P = 0.004, n = 9), and a reduction in bladder compliance from 0.13 ± 0.020 mL/cmH2O to 0.090 ± 0.014 mL/cmH2O (P = 0.011, n = 9). Thus, changes in bladder function following the instillation of PGE2 were not dependent on capsaicin-sensitive pathways. Further, these results suggest that urethral relaxation/weakness and/or increased detrusor pressure as a result of decreased compliance may contribute to urinary urgency and highlight potential targets for new therapies for overactive bladder.


Assuntos
Capsaicina/farmacologia , Dinoprostona/farmacologia , Bexiga Urinária/efeitos dos fármacos , Administração Intravesical , Animais , Dinoprostona/administração & dosagem , Feminino , Ocitócicos/farmacologia , Ratos , Ratos Wistar , Fármacos do Sistema Sensorial/farmacologia , Bexiga Urinária/fisiologia
17.
Biochem Biophys Res Commun ; 537: 29-35, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33383561

RESUMO

Articular chondrocytes are exposed to dynamic osmotic environments during normal joint loading, and thus, require effective volume regulatory mechanisms. A regulatory volume decrease (RVD) is one of the mechanisms for protecting chondrocytes from swelling and damage. Swelling-activated Cl- currents (ICl,swell) are responsible for the RVD, but the molecular identity in chondrocytes is largely unknown. In this study, we reveal that in human OUMS-27 chondrocytes, ICl,swell can be elicited by hypoosmotic stimulation (180 mOsm) and be inhibited by classical Cl- channel blockers, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid, and be attenuated by siRNA knockdown of ClC-3. Our molecular analyses revealed that ClC-3A is expressed as a major splice variant in both human articular chondrocytes and OUMS-27 cells. The onset and early phase of RVD following hypoosmotic stress in OUMS-27 cells were affected by DIDS and ClC-3 knockdown. Hypoosmotic stimulation caused Ca2+ influx and subsequent release of prostaglandin E2 (PGE2) in OUMS-27 cells, and both of these responses were reduced by DIDS and ClC-3 knockdown. These results strongly suggest that ClC-3 is responsible for ICl,swell and RVD under the hypoosmotic environments. It is likely that ClC-3 is associated with the pathogenesis of cartilage degenerative diseases including osteoarthritis via PGE2 release.


Assuntos
Canais de Cloreto/metabolismo , Condrócitos/metabolismo , Dinoprostona/farmacologia , Cartilagem Articular/citologia , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Soluções
18.
Mol Pain ; 16: 1744806920970368, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33307981

RESUMO

The embryonic rat dorsal root ganglion (DRG) neuron-derived 50B11 cell line is a promising sensory neuron model expressing markers characteristic of NGF and GDNF-dependent C-fibre nociceptors. Whether these cells have the capacity to develop into distinct nociceptive subtypes based on NGF- or GDNF-dependence has not been investigated. Here we show that by augmenting forskolin (FSK) and growth factor supplementation with NGF or GDNF, 50B11 cultures can be driven to acquire differential functional responses to common nociceptive agonists capsaicin and ATP respectively. In addition, to previous studies, we also demonstrate that a differentiated neuronal phenotype can be maintained for up to 7 days. Western blot analysis of nociceptive marker proteins further demonstrates that the 50B11 cells partially recapitulate the functional phenotypes of classical NGF-dependent (peptidergic) and GDNF-dependent (non-peptidergic) neuronal subtypes described in DRGs. Further, 50B11 cells differentiated with NGF/FSK, but not GDNF/FSK, show sensitization to acute prostaglandin E2 treatment. Finally, RNA-Seq analysis confirms that differentiation with NGF/FSK or GDNF/FSK produces two 50B11 cell subtypes with distinct transcriptome expression profiles. Gene ontology comparison of the two subtypes of differentiated 50B11 cells to rodent DRG neurons studies shows significant overlap in matching or partially matching categories. This transcriptomic analysis will aid future suitability assessment of the 50B11 cells as a high-throughput nociceptor model for a broad range of experimental applications. In conclusion, this study shows that the 50B11 cell line is capable of partially recapitulating features of two distinct types of embryonic NGF and GDNF-dependent nociceptor-like cells.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Gânglios Espinais/citologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Fator de Crescimento Neural/farmacologia , Nociceptores/citologia , Potenciais de Ação/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Animais , Biomarcadores/metabolismo , Capsaicina/farmacologia , Diferenciação Celular/genética , Linhagem Celular , Forma Celular/efeitos dos fármacos , Colforsina/farmacologia , Dinoprostona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Variação Genética , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nociceptores/efeitos dos fármacos , Fenótipo , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Canais de Sódio/metabolismo
19.
Bull Exp Biol Med ; 170(1): 49-52, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33222085

RESUMO

We studied the effects of IL-1ß, IL-8, TNFα, and prostaglandin E2α in concentrations typically observed in health and during inflammation on the growth of vaginal microbiota and its resistance to factors inhibiting the synthesis of proteins, nucleic acids, and peptidoglycans. An increase in the cytokine levels, characteristic of inflammation, inhibits the growth of Lactobacillus population and improves its resistance to adverse factors. The growth of the population of opportunistic microorganisms (S. aureus, E. coli) is stimulated under these conditions, while their resistance to adverse factors decreases. Hence, it seems that the cytokines regulate the behavior of the host cells and of its bacterial symbionts.


Assuntos
Dinoprostona/farmacologia , Mediadores da Inflamação/farmacologia , Interleucina-1beta/farmacologia , Interleucina-8/farmacologia , Microbiota/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Vaginose Bacteriana/microbiologia , Líquidos Corporais/microbiologia , Estudos de Casos e Controles , Dinoprostona/imunologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Feminino , Interações Hospedeiro-Patógeno , Humanos , Inflamação , Mediadores da Inflamação/imunologia , Interleucina-1beta/imunologia , Interleucina-8/imunologia , Lactobacillus/efeitos dos fármacos , Lactobacillus/crescimento & desenvolvimento , Microbiota/imunologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Fator de Necrose Tumoral alfa/imunologia , Vagina/imunologia , Vagina/microbiologia , Vaginose Bacteriana/imunologia
20.
Stem Cell Res Ther ; 11(1): 330, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33100224

RESUMO

BACKGROUND: We have previously verified the beneficial effects of exosomes from endothelial progenitor cells (EPC-EXs) in ischemic stroke. However, the effects of EPC-EXs in hemorrhagic stroke have not been investigated. Additionally, miR-137 is reported to regulate ferroptosis and to be involved in the neuroprotection against ischemic stroke. Hence, the present work explored the effects of miR-137-overexpressing EPC-EXs on apoptosis, mitochondrial dysfunction, and ferroptosis in oxyhemoglobin (oxyHb)-injured SH-SY5Y cells. METHODS: The lentiviral miR-137 was transfected into EPCs and then the EPC-EXs were collected. RT-PCR was used to detect the miR-137 level in EPCs, EXs, and neurons. The uptake mechanisms of EPC-EXs in SH-SY5Y cells were explored by the co-incubation of Dynasore, Pitstop 2, Ly294002, and Genistein. After the transfection of different types of EPC-EXs, flow cytometry and expression of cytochrome c and cleaved caspase-3 were used to detect the apoptosis of oxyHb-injured neurons. Neuronal mitochondrial function was assessed by reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP) depolarization, and cellular ATP content. Cell ferroptosis was measured by lipid peroxidation, iron overload, degradation of glutathione, and glutathione peroxidase 4. Additionally, recombinational PGE2 was used to detect if activation of COX2/PGE2 pathway could reverse the protection of miR-137 overexpression. RESULTS: The present work showed (1) EPC-EXs could be taken in by SH-SY5Y cells via caveolin-/clathrin-mediated pathways and macropinocytosis; (2) miR-137 was decreased in neurons after oxyHb treatment, and EXsmiR-137 could restore the miR-137 levels; (3) EXsmiR-137 worked better than EXs in reducing the number of apoptotic neurons and pro-apoptotic protein expression after oxyHb treatment; (4) EXsmiR-137 are more effective in improving the cellular MMP, ROS, and ATP level; (5) EXsmiR-137, but not EXs, protected oxyHb-treated SH-SY5Y cells against lipid peroxidation, iron overload, degradation of glutathione, and glutathione peroxidase 4; and (6) EXsmiR-137 suppressed the expression of the COX2/PGE2 pathway, and activation of the pathway could partially reverse the neuroprotective effects of EXsmiR-137. CONCLUSION: miR-137 overexpression boosts the neuroprotective effects of EPC-EXs against apoptosis and mitochondrial dysfunction in oxyHb-treated SH-SY5Y cells. Furthermore, EXsmiR-137 rather than EXs can restore the decrease in miR-137 levels and inhibit ferroptosis, and the protection mechanism might involve the miR-137-COX2/PGE2 signaling pathway.


Assuntos
Células Progenitoras Endoteliais , Exossomos , MicroRNAs , Fármacos Neuroprotetores , Apoptose , Linhagem Celular Tumoral , Ciclo-Oxigenase 2 , Dinoprostona/farmacologia , MicroRNAs/genética , Fármacos Neuroprotetores/farmacologia , Oxiemoglobinas/farmacologia
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