Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.262
Filtrar
1.
Medicine (Baltimore) ; 99(28): e20060, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32664054

RESUMO

OBJECTIVE: This study was designed to investigate the effects of leukocyte Rho kinase activity and serum Cystatin C (Cys C) on cardiovascular events in patients with acute coronary syndrome (ACS). METHODS: A total of 48 patients with ST-segment elevation myocardial infarction (STEMI), 23 patients with non-ST-segment elevation myocardial infarction (NSTEMI), 25 patients with unstable angina (UA) and 20 patients with no-acute coronary syndrome as control from January 2017 to June 2018 in Tianyou Hospital affiliated to Wuhan University of Science and Technology were selected in this study. Western blot was used to detect the leukocyte Rho kinase activity and Elisa kit was used to measure serum Cys C. Univariate and multivariate analysis were used to analyze the influencing factors of cardiovascular events in ACS patients. RESULTS: The activity of leukocyte Rho kinase and serum Cys C were gradually reduced in the STEMI, NSTEMI and UA patients, but all significantly higher than that in No-ASC patients, and there was a positive correlation between leukocyte Rho kinase activity and serum Cys C in ACS patients (r = 0.516, P < .001). The activity of leukocyte Rho kinase was positively correlated with the levels of serum TNF-α (r = 0.634, P < .001), IL-6 (r = 0.578, P < .001), IL-8 (r = 0.582, P < .001) in ACS patients, and the level of Cys C was positively correlated with the levels of serum TNF-α (r = 0.634, P < .001), IL-6 (r = 0.578, P < .001), IL-8 (r = 0.582, P < .001) in ACS patients. Univariate and multivariate analysis showed that the leukocyte Rho kinase activity (HR = 2.994, 95%CI = 1.328-6.054, P < .0001) and the levels of serum Cys C (HR = 1.692, 95%CI = 1.028-2.124, P < .0001) were independent influencing factors of cardiovascular events in ACS patients. CONCLUSION: The leukocyte Rho kinase activity and serum Cystatin C are high in acute coronary syndrome patients, and are the independent influencing factors of cardiovascular events in ACS patients.


Assuntos
Cistatina C/sangue , Isquemia Miocárdica/sangue , Quinases Associadas a rho/metabolismo , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Leucócitos/enzimologia , Masculino , Pessoa de Meia-Idade
2.
Life Sci ; 255: 117779, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32417374

RESUMO

OBJECTIVE: Kidney is the most common location of microangiopathy in diabetic patients, and we designed this study to investigate the effects of hirudin on renal microangiopathy in STZ-induced diabetes rats and in vitro. METHODS: We established a diabetes model by intraperitoneal injection of STZ and administered hirudin daily by subcutaneous injection. HE staining was used to assess kidney pathological changes. Western blot and immunochemistry was used to detect the protein expression. Glomerular endothelial cells (GEC) in normal rats were assessed by cell scratch test for migration ability and tubule formation experiment for angiogenesis ability. RESULTS: Compared with DN rats without any treatment, the serum creatinine, serum Cys C, 24-hour urine protein of DN rats with hirudin treatment were significantly decrease, the kidney/body weight and glomerular area of DN rats with hirudin treatment were all significantly decrease, and also significant improvement in renal pathology revealed by HE staining in DN rats after treating with hirudin. Moreover, we also found that hirudin coun not only significantly increase the prothrombin time and aivated partial thromboplastin time in DN rats, but also significantly decrease the expression of VEGF and TM-1 protein in kidney tissues of DN rats. In vitro, we found that high glucose could promote the migration and angiogensis of GEC, and significantly increased the expression of VEGF and Ang protein, but significantly decreased the expression of THBS1 and Arg1 protein. More importantly was that hirudin could inhibit the migration and angiogensis of GEC, and reversed HG-induced the expression of VEGF, Ang, THBS1 and Arg1 protein in GEC. In addition, we also found that hirudin could not only decrease HG-enhanced the activity of RhoA in GEC, but also decrease HG-enhanced the expression of p-MYPT1/MYPT1, p-p38/p38 protein in GEC. CONCLUSION: Hirudin reduces nephropathy microangiopathy in STZ-induced diabetes, and might be related to hirudin inhibiting glomerular endothelial cell migration and angiogenesis through Rho-kinase and subsequent p38MAPK/NF-kB signaling pathway.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Angiopatias Diabéticas/prevenção & controle , Nefropatias Diabéticas/prevenção & controle , Hirudinas/farmacologia , Neovascularização Patológica/prevenção & controle , Animais , Movimento Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Glucose/metabolismo , Glomérulos Renais/metabolismo , Masculino , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases Associadas a rho/metabolismo
3.
PLoS One ; 15(5): e0232356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357159

RESUMO

Lymphatic systems play important roles in the maintenance of fluid homeostasis and undergo anatomical and physiological changes during inflammation and aging. While lymphatic endothelial cells (LECs) undergo mesenchymal transition in response to transforming growth factor-ß (TGF-ß), the molecular mechanisms underlying endothelial-to-mesenchymal transition (EndMT) of LECs remain largely unknown. In this study, we examined the effect of TGF-ß2 and tumor necrosis factor-α (TNF-α), an inflammatory cytokine, on EndMT using human skin-derived lymphatic endothelial cells (HDLECs). TGF-ß2-treated HDLECs showed increased expression of SM22α, a mesenchymal cell marker accompanied by increased cell motility and vascular permeability, suggesting HDLECs to undergo EndMT. Our data also revealed that TNF-α could enhance TGF-ß2-induced EndMT of HDLECs. Furthermore, both cytokines induced the production of Activin A while decreasing the expression of its inhibitory molecule Follistatin, and thus enhancing EndMT. Finally, we demonstrated that human dermal lymphatic vessels underwent EndMT during aging, characterized by double immunostaining for LYVE1 and SM22α. These results suggest that both TGF-ß and TNF-α signals play a central role in EndMT of LECs and could be potential targets for senile edema.


Assuntos
Ativinas/metabolismo , Células Endoteliais/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/fisiologia , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Vasos Linfáticos/citologia , Proteína Smad2/fisiologia , Transativadores/fisiologia , Quinases Associadas a rho/metabolismo
5.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(3): 193-197, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32389165

RESUMO

Objective To investigate the role of Ras homolog gene (Rho) A/Rho-associated coiled-coil containing protein kinase (ROCK) signaling pathway in tumor necrosis factor α (TNF-α) promoting hyper-permeability of vascular endothelial cells infected by Listeria monocytogenes (Lm) . Methods The cultured human umbilical vein endothelial cells (HUVECs) were divided into a control group (uninfected cells), TNF-α treatment group (100 ng/mL TNF-α, for 2 hours), Lm infection group (infected with MOI=10 Lm for 2 hours, then added gentamicin for 0.5 hour), Lm infection and TNF-α treatment group (infected with Lm and then treated with 100 ng/mL TNF-α for 2 hours), and Y-27632 inhibitor group combined with Lm infection and TNF-α treatment (treated with 50 µmol/L ROCK inhibitor Y-27632 for 30 minutes, and then Lm infection and TNF-α treatment as above). The protein levels of RhoA, zonula occluden-1 (ZO-1), occludin and ROCK in HUVECs were detected by Western blot analysis; the permeability of HUVECs was analyzed by the horseradish peroxidase (HRP) leakage; and the distribution of F-actin in HUVECs was detected by fluorescein isothiocyanate (FITC)-labeled phalloidine staining. Results TNF-α reduced the expression of tight junction protein ZO-1 and occludin in Lm-infected HUVECs, promoted its hyper-permeability and cytoskeletal rearrangement, and up-regulated the expression of RhoA and ROCK. ROCK inhibitor Y-27632 obviously inhibited the cytoskeleton rearrangement and hyper-permeability of HUVECs induced by TNF-α. Conclusion TNF-α can enhance hyper-permeability of HUVECs infected by Lm, which may be regulated by RhoA/Rock signaling pathway.


Assuntos
Células Endoteliais da Veia Umbilical Humana/microbiologia , Listeria monocytogenes , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Permeabilidade
6.
Toxicol Appl Pharmacol ; 399: 115053, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32417439

RESUMO

Acute promyelocytic leukemia (APL) is a form of acute myeloid leukemia with a unique chromosome translocation t (15;17), commonly complicated by a complex coagulopathy. 4-Amino-2-trifuoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative, was synthesized by our group and known to possess obvious biological anti-tumor activities. It has previously been shown that ATPR could induce differentiation and inhibit proliferation of APL cells, although the mechanism responsible for this effect was not well understood. In this study, we demonstrated that ATPR remarkably inhibited the expression and activity of SHP2. Further experiments showed silencing SHP2 or using SHP2 inhibition (SHP099) enhanced the effect of ATPR on cell proliferation and maturation. In addition, we also demonstrated that Rho/ROCK1 might be regulated by SHP2. Using Y-27632, a ROCK inhibitor, further proved that ROCK1 played an important role in ATPR-induced differentiation and proliferation suppression. In conclusion, the results from this study revealed that ATPR induced APL cells terminal differentiation and growth arrest by blockade of SHP2/Rho/ ROCK1 pathway.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Leucemia Promielocítica Aguda/tratamento farmacológico , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Retinoides/farmacologia , Quinases Associadas a rho/metabolismo , Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
PLoS One ; 15(5): e0233057, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32396545

RESUMO

Poor survival of human pluripotent stem cells (hPSCs) following freezing, thawing, or passaging hinders the maintenance and differentiation of stem cells. Rho-associated kinases (ROCKs) play a crucial role in hPSC survival. To date, a typical ROCK inhibitor, Y-27632, has been the primary agent used in hPSC research. Here, we report that another ROCK inhibitor, fasudil, can be used as an alternative and is cheaper than Y-27632. It increased hPSC growth following thawing and passaging, like Y-27632, and did not affect pluripotency, differentiation ability, and chromosome integrity. Furthermore, fasudil promoted retinal pigment epithelium (RPE) differentiation and the survival of neural crest cells (NCCs) during differentiation. It was also useful for single-cell passaging of hPSCs and during aggregation. These findings suggest that fasudil can replace Y-27632 for use in stem research.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Pesquisa com Células-Tronco
9.
Anticancer Res ; 40(4): 1931-1942, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32234882

RESUMO

BACKGROUND: RhoA and its downstream effectors Rho-associated coiled-coil kinases (ROCK) 1 and 2 are central controllers of cytoskeleton dynamics, and therefore influence cell shape, adhesion and migration. Since modulation of these processes holds promise for an effective anticancer strategy, effects of ROCK inhibition have been evaluated in a number of malignancies. MATERIALS AND METHODS: Using immunohistochemistry, ROCK1 and ROCK2 expression was semi-quantitatively assessed in 129 patient-derived primary melanomas. RESULTS: There was a striking predilection for low melanocytic expression of both kinases in thick, ulcerated and mitogenic tumors, as well as in nodular histological type. ROCK1 and -2 expression in tumor-infiltrating lymphocytes (TILs) was preferentially down-regulated in advanced and aggressive tumors. Moreover, diminished ROCK2 reactivity in melanoma cells and TILs was associated with shorter melanoma-specific and recurrence-free survival. CONCLUSION: This is the first analysis of ROCK1 and -2 protein expression in clinical melanoma samples and the results indicated the suppression of ROCK signaling in melanocytes of aggressive and late-stage tumors. Functional models that more accurately represent the clinical setting are necessary to dissect the role of ROCK1 and -2 in melanoma. Additionally, our study indicates that ROCK activity in TILs may be involved in the pathogenesis of cancer, and thus merits further investigations.


Assuntos
Melanoma/genética , Neoplasias Cutâneas/genética , Quinases Associadas a rho/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Estimativa de Kaplan-Meier , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Masculino , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/epidemiologia , Melanoma/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Transdução de Sinais/genética , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/patologia , Adulto Jovem
10.
Life Sci ; 254: 117605, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32278692

RESUMO

AIMS: Although chloroquine and diclofenac are not cardiovascular drugs, their chronic administration may trigger cardiotoxicity. We, therefore, evaluated the cardiotoxic impact of diclofenac in chloroquine-treated adjuvant arthritic rats and the protective role of Rho-kinase inhibitors. METHODS: 90 male rats were equally distributed into 9 groups including control. Arthritis was induced by S.C injection of Complete Freund's adjuvant in hind paw plantar surface. Arthritic rats were subdivided into 8 groups, orally treated with: no drug, chloroquine (50 mg/kg), diclofenac sodium (1 mg/kg) and chloroquine + diclofenac. To study the role of Rho-kinase in chloroquine/diclofenac-triggered cardiotoxicity, four arthritic groups were also co-treated with Rho-kinase inhibitors (fasudil or atorvastatin) along with diclofenac and chloroquine + diclofenac. KEY FINDINGS: All treatments significantly elevated serum cardiac injury and dysfunction markers as well as left ventricular malondialdehyde but depleted antioxidants with the greatest effect in the combination group. Chloroquine and/or diclofenac; in particular, their combination shifted the balance between left ventricular pro- and anti-apoptotic proteins towards myocardial apoptosis. Surprisingly, treatment with diclofenac or chloroquine/diclofenac markedly up-regulated cardiac RhoA and Rho-kinase1. Such up-regulation was coupled with a greater increase in cardiac oxidative damage biomarkers in the combination group than in individually-treated ones. However, Rho-kinase inhibition protected against diclofenac-induced increase in myocardial oxidative damage markers. SIGNIFICANCE: Diclofenac greatly amplified cardiac oxidative damage in chloroquine-treated arthritic rats via up-regulation of Rho-kinase1. However, Rho-kinase inhibitors provided cardioprotection against diclofenac toxicity. Overall, they could be used as safer adjuvants to diclofenac during the treatment of rheumatoid arthritis with chloroquine.


Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Artrite Experimental/tratamento farmacológico , Cloroquina/uso terapêutico , Diclofenaco/toxicidade , Coração/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Masculino , Estresse Oxidativo , Ratos
11.
Curr Opin Ophthalmol ; 31(3): 192-198, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32235252

RESUMO

PURPOSE OF REVIEW: Retinal detachment initiates a series of events that lead to degenerative changes in retinal synaptic architecture as well as the well-known phenomena of gliosis and photoreceptor apoptosis. Retinal reattachment does not always result in complete visual recovery, even if the fovea is not directly involved in the detachment. Rho-kinase (ROCK) inhibitors may mitigate some of these deleterious changes including disruption of synaptic architecture, photoreceptor apoptosis, and initiation of the epithelial-mesenchymal transition that characterizes proliferative vitreoretinopathy (PVR). This review focuses on the use of ROCK inhibitors to modulate synaptic disjunction. RECENT FINDINGS: ROCK inhibition prevents retinal detachment-induced photoreceptor synaptic terminal retraction (i.e., synaptic disjunction), thereby diminishing the damage of the first synapse in the visual pathway. ROCK inhibition also reduces retinal detachment-induced photoreceptor apoptosis and suppresses PVR progression in preclinical models. SUMMARY: Inhibition of ROCK may help to optimize visual recovery after retinal detachment surgery or iatrogenic detachments during cell transplantation or viral subretinal injection and might play a role in reducing the risk of PVR after retinal detachment surgery.


Assuntos
Inibidores de Proteínas Quinases/uso terapêutico , Descolamento Retiniano/tratamento farmacológico , Quinases Associadas a rho/antagonistas & inibidores , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Resultado do Tratamento , Vitreorretinopatia Proliferativa/tratamento farmacológico , Vitreorretinopatia Proliferativa/metabolismo
13.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(2): 138-144, 2020 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-32314711

RESUMO

Objective To investigate the molecular mechanism of miR-153-3p regulating the proliferation, migration and invasion of human breast cancer cells. Methods Bioinformatics software was used to predict the candidate target gene of miR-153-3p. The double-luciferase reporter assay was used to validate the targeting relationship between miR-153-3p and candidate genes. Real-time fluorescence quantitative PCR and Western blotting were performed to detect the effect of miR-153-3p on the mRNA and protein expressions, respectively. The cell proliferation ability was examined by MTT assay. The migration ability of cells was determined in a scratch assay. The invasion ability was detected by TranswellTM assay. Results Software-predicted results showed a continuous binding region between miR-153-3p and Rho-associated coiled-coil containing protein kinase 1 (ROCK1). The miR-153-3p mimics co-transfected with ROCK1 wild-type reporter vector significantly decreased the relative activity of luciferase in MDA-MB-231 cells. The transfection of hsa-miR-153-3p mimics increased miR-153-3p expression and decreased ROCK1 protein expression. In comparison with the control cells, the proliferation, migration and invasion of MDA-MB-231 cells over-expressing miR-153-3p were dramatically inhibited. Conclusion The miR-153-3p can inhibit the proliferation, migration and invasion of breast cancer cells via down-regulation of ROCK1.


Assuntos
Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , MicroRNAs/genética , Quinases Associadas a rho/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica
14.
J Pharmacol Exp Ther ; 373(2): 214-219, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32144123

RESUMO

Sickle cell disease (SCD) is associated with overactive bladder (OAB). Detrusor overactivity, a component of OAB, is present in an SCD mouse, but the molecular mechanisms for this condition are not well-defined. We hypothesize that nitric oxide (NO)/ ras homolog gene family (Rho) A/Rho-associated kinase (ROCK) dysregulation is a mechanism for detrusor overactivity and that NO-releasing nanoparticles (NO-nps), a novel NO delivery system, may serve to treat this condition. Male adult SCD transgenic, combined endothelial NO synthases (eNOSs) and neuronal NOS (nNOS) gene-deficient (dNOS-/-), and wild-type (WT) mice were used. Empty nanoparticle or NO-np was injected into the bladder, followed by cystometric studies. The expression levels of phosphorylated eNOS (Ser-1177), protein kinase B (Akt) (Ser-473), nNOS (Ser-1412), and myosin phosphatase target subunit 1 (MYPT1) (Thr-696) were assessed in the bladder. SCD and dNOS-/- mice had a greater (P < 0.05) number of voiding and nonvoiding contractions compared with WT mice, and they were normalized by NO-np treatment. eNOS (Ser-1177) and AKT (Ser-473) phosphorylation were decreased (P < 0.05) in the bladder of SCD compared with WT mice and reversed by NO-np. Phosphorylated MYPT1, a marker of the RhoA/ROCK pathway, was increased (P < 0.05) in the bladder of SCD mice compared with WT and reversed by NO-np. nNOS phosphorylation on positive (Ser-1412) regulatory site was decreased (P < 0.05) in the bladder of SCD mice compared with WT and was not affected by NO-np. NO-nps did not affect any of the measured parameters in WT mice. In conclusion, dysregulation of NO and RhoA/ROCK pathways is associated with detrusor overactivity in SCD mice; NO-np reverses these molecular derangements in the bladder and decreases detrusor overactivity. SIGNIFICANCE STATEMENT: Voiding abnormalities commonly affect patients with sickle cell disease (SCD) but are problematic to treat. Clarification of the science for this condition in an animal model of SCD may lead to improved interventions for it. Our findings suggest that novel topical delivery of a vasorelaxant agent nitric oxide into the bladder of these mice corrects overactive bladder by improving deranged bladder physiology regulatory signaling.


Assuntos
Nanopartículas/uso terapêutico , Óxido Nítrico/fisiologia , Bexiga Urinária Hiperativa/tratamento farmacológico , Quinases Associadas a rho/fisiologia , Anemia Falciforme/complicações , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico Sintase/fisiologia , Fosforilação , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia
15.
PLoS Biol ; 18(3): e3000646, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203518

RESUMO

Interleukin 23 (IL-23) triggers pathogenic features in pro-inflammatory, IL-17-secreting T cells (Th17 and Tγδ17) that play a key role in the development of inflammatory diseases. However, the IL-23 signaling cascade remains largely undefined. Here, we used quantitative phosphoproteomics to characterize IL-23 signaling in primary murine Th17 cells. We quantified 6,888 phosphorylation sites in Th17 cells and found 168 phosphorylations regulated upon IL-23 stimulation. IL-23 increased the phosphorylation of the myosin regulatory light chain (RLC), an actomyosin contractibility marker, in Th17 and Tγδ17 cells. IL-23-induced RLC phosphorylation required Janus kinase 2 (JAK2) and Rho-associated protein kinase (ROCK) catalytic activity, and further study of the IL-23/ROCK connection revealed an unexpected role of IL-23 in the migration of Tγδ17 and Th17 cells through ROCK activation. In addition, pharmacological inhibition of ROCK reduced Tγδ17 recruitment to inflamed skin upon challenge with inflammatory agent Imiquimod. This work (i) provides new insights into phosphorylation networks that control Th17 cells, (ii) widely expands the current knowledge on IL-23 signaling, and (iii) contributes to the increasing list of immune cells subsets characterized by global phosphoproteomic approaches.


Assuntos
Inflamação/metabolismo , Subunidade p19 da Interleucina-23/metabolismo , Células Th17/metabolismo , Animais , Movimento Celular , Imiquimode/farmacologia , Inflamação/patologia , Subunidade p19 da Interleucina-23/genética , Janus Quinase 2 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Cadeias Leves de Miosina/metabolismo , Fosforilação , Proteômica/métodos , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Serina/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismo
16.
Phytomedicine ; 69: 153193, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32120245

RESUMO

BACKGROUND: Although mechanical barriers and modern surgical techniques have been developed to prevent postoperative adhesion formation, high incidence of adhesions still represents an important challenge in abdominal surgery. So far, there has been no available therapeutic drug in clinical practice. PURPOSE: In this study, we explored the efficacy of sodium aescinate (AESS) treatment against postoperative peritoneal adhesions, the potential molecular mechanism was also investigated. STUDY DESIGN AND METHODS: Sixty male Sprague-Dawley rats were randomly divided into 6 groups for the study: the blank, vehicle, positive control and three AESS administration groups (0.5, 1 and 2 mg/kg/d, intravenous administration for 7 days). Adhesions were induced by discretely ligating peritoneal sidewall. An IL-1ß-induced HMrSV5 cell model was also performed to explore possible functional mechanism. RESULTS: The results indicated that the incidence and severity of peritoneal adhesions were significantly lower in the AESS-treated groups than that in the vehicle and positive control group. AESS-treated groups showed that the secretion, activity, and expression of tPA in rat peritoneum were notably increased. The FIB levels in rat plasma were decreased. The immunohistochemical staining analysis demonstrated that collagen I and α-SMA deposition were significantly attenuated in AESS-treated peritoneal tissues. Besides, we found that AESS treatment reduced the protein levels of p-MYPT1. To further explore the mechanisms of AESS, both activator and inhibitors of RhoA/ROCK pathway were employed in this study. It was found that AESS-induced up-regulation of tPA was reversed by activator of ROCK, but the effects of ROCK inhibitors were consistent with AESS. CONCLUSION: Taken together, the findings of in vivo and in vitro experiments proved that AESS could significantly suppress postoperative peritoneal adhesion formation through inhibiting the RhoA/ROCK signaling pathway. Our researches provide important pharmacological basis for AESS development as a potential therapeutic agent on peritoneal adhesions.


Assuntos
Doenças Peritoneais/tratamento farmacológico , Complicações Pós-Operatórias/tratamento farmacológico , Saponinas/farmacologia , Triterpenos/farmacologia , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Linhagem Celular , Colágeno Tipo I/metabolismo , Fibrinogênio/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Masculino , Doenças Peritoneais/patologia , Doenças Peritoneais/prevenção & controle , Peritônio/citologia , Peritônio/cirurgia , Complicações Pós-Operatórias/patologia , Complicações Pós-Operatórias/prevenção & controle , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Aderências Teciduais
17.
J Neurosci ; 40(13): 2776-2788, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098904

RESUMO

Oligophrenin-1 (Ophn1) encodes a Rho GTPase activating protein whose mutations cause X-linked intellectual disability (XLID) in humans. Loss of function of Ophn1 leads to impairments in the maturation and function of excitatory and inhibitory synapses, causing deficits in synaptic structure, function and plasticity. Epilepsy is a frequent comorbidity in patients with Ophn1-dependent XLID, but the cellular bases of hyperexcitability are poorly understood. Here we report that male mice knock-out (KO) for Ophn1 display hippocampal epileptiform alterations, which are associated with changes in parvalbumin-, somatostatin- and neuropeptide Y-positive interneurons. Because loss of function of Ophn1 is related to enhanced activity of Rho-associated protein kinase (ROCK) and protein kinase A (PKA), we attempted to rescue Ophn1-dependent pathological phenotypes by treatment with the ROCK/PKA inhibitor fasudil. While acute administration of fasudil had no impact on seizure activity, seven weeks of treatment in adulthood were able to correct electrographic, neuroanatomical and synaptic alterations of Ophn1 deficient mice. These data demonstrate that hyperexcitability and the associated changes in GABAergic markers can be rescued at the adult stage in Ophn1-dependent XLID through ROCK/PKA inhibition.SIGNIFICANCE STATEMENT In this study we demonstrate enhanced seizure propensity and impairments in hippocampal GABAergic circuitry in Ophn1 mouse model of X-linked intellectual disability (XLID). Importantly, the enhanced susceptibility to seizures, accompanied by an alteration of GABAergic markers were rescued by Rho-associated protein kinase (ROCK)/protein kinase A (PKA) inhibitor fasudil, a drug already tested on humans. Because seizures can significantly impact the quality of life of XLID patients, the present data suggest a potential therapeutic pathway to correct alterations in GABAergic networks and dampen pathological hyperexcitability in adults with XLID.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Neurônios GABAérgicos/efeitos dos fármacos , Proteínas Ativadoras de GTPase/genética , Hipocampo/efeitos dos fármacos , Deficiência Intelectual/fisiopatologia , Inibidores de Proteínas Quinases/farmacologia , Convulsões/fisiopatologia , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Neurônios GABAérgicos/fisiologia , Hipocampo/fisiopatologia , Deficiência Intelectual/genética , Camundongos , Camundongos Knockout , Convulsões/genética
18.
PLoS One ; 15(2): e0228195, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053631

RESUMO

Tissue fibrosis is a pathological condition characterized by uncontrolled fibroblast activation that ultimately leads to organ failure. The TGFß1 pathway, one of the major players in establishment of the disease phenotype, is dependent on the transcriptional co-activators YAP/TAZ. We were interested whether fibroblasts can be sensitized to TGFß1 by activation of the GPCR/YAP/TAZ axis and whether this mechanism explains the profibrotic properties of diverse GPCR ligands. We found that LPA, S1P and thrombin cooperate in human dermal fibroblasts with TGFß1 to induce extracellular matrix synthesis, myofibroblast marker expression and cytokine secretion. Whole genome expression profiling identified a YAP/TAZ signature behind the synergistic profibrotic effects of LPA and TGFß1. LPA, S1P and thrombin stimulation led to activation of the Rho-YAP axis, an increase of nuclear YAP-Smad2 complexes and enhanced expression of profibrotic YAP/Smad2-target genes. More generally, dermal, cardiac and lung fibroblast responses to TGFß1 could be enhanced by increasing YAP nuclear levels (with GPCR ligands LPA, S1P, thrombin or Rho activator) and inhibited by decreasing nuclear YAP (with Rho inhibitor, forskolin, latrunculin B or 2-deoxy-glucose). Thus, we present here a conceptually interesting finding that fibroblast responses to TGFß1 can be predicted based on the nuclear levels of YAP and modulated by stimuli/treatments that change YAP nuclear levels. Our study contributes to better understanding of fibrosis as a complex interplay of signalling pathways and proposes YAP/TAZ as promising targets in the treatment of fibrosis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Fibroblastos/patologia , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Linhagem Celular , Ativação Enzimática , Fibroblastos/metabolismo , Fibrose , Humanos , Ligantes , Lisofosfolipídeos/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Trombina/metabolismo , Quinases Associadas a rho/metabolismo
19.
Adv Exp Med Biol ; 1223: 99-127, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32030687

RESUMO

The Rho-ROCK signaling network has a range of specialized functions of key biological importance, including control of essential developmental processes such as morphogenesis and physiological processes including homeostasis, immunity, and wound healing. Deregulation of Rho-ROCK signaling actively contributes to multiple pathological conditions, and plays a major role in cancer development and progression. This dynamic network is critical in modulating the intricate communication between tumor cells, surrounding diverse stromal cells and the matrix, shaping the ever-changing microenvironment of aggressive tumors. In this chapter, we overview the complex regulation of the Rho-ROCK signaling axis, its role in health and disease, and analyze progress made with key approaches targeting the Rho-ROCK pathway for therapeutic benefit. Finally, we conclude by outlining likely future trends and key questions in the field of Rho-ROCK research, in particular surrounding Rho-ROCK signaling within the tumor microenvironment.


Assuntos
Neoplasias/metabolismo , Transdução de Sinais , Microambiente Tumoral , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Humanos , Neoplasias/enzimologia
20.
Gene ; 737: 144474, 2020 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-32057928

RESUMO

Rho-associated protein kinases (ROCKs) have various cellular functions, which include actin cytoskeleton remodeling and vesicular trafficking, and there are two major mammalian ROCK isotypes, namely, ROCK1 (ROKß) and ROCK2 (ROKα). The ROCK2-specific inhibitor KD025 (SLx-2119) is currently undergoing phase II clinical trials, but its cellular functions have not been fully explored. In this study, we investigated the functions of KD025 at the genomics level by bioinformatics analysis using the GSE8686 microarray dataset from the NCBI GEO database, in three different primary human cell lines. An initial microarray analysis conducted by Boerma et al. focused on the effects of KD025 on cell adhesion and blood coagulation, but did not provide comprehensive information on the functions of KD025. Our analysis of differentially expressed genes (DEGs) showed ~70% coincidence with Boerma et al.'s findings, and newly identified that CCND1, CXCL2, NT5E, and SMOX were differentially expressed by KD025. However, due to low numbers of co-regulated DEGs, we were unable to extract the functions of KD025 with significance. To overcome this limitation, we used gene set enrichment analysis (GSEA) and the heatmap hierarchical clustering method. We confirmed KD025 regulated inflammation and adipogenesis pathways, as previously reported experimentally. In addition, we found KD025 has novel regulatory functions on various pathways, including oxidative phosphorylation, WNT signaling, angiogenesis, and KRAS signaling. Further studies are required to systematically characterize these newly identified functions of KD025.


Assuntos
Biologia Computacional , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA