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1.
Eur Rev Med Pharmacol Sci ; 25(1): 431-437, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33506933

RESUMO

OBJECTIVE: Thymosin beta 4 (TB4) is the most abundant member of the beta-thymosin family in humans. The main physiological role of TB4 is the regulation of actin polymerization. TB4 is also involved in angiogenesis, cell survival, cell migration and fetal development. The aim of this study was to evaluate the activity of TB4 as a fetal growth promoter when administered during pregnancy. MATERIALS AND METHODS: Our protocols have been carried out in full conformity with the rules and guidelines expected for this kind of trial. 10 pregnant mice received the same injection regimen. Only 6 of these 10 are part of this experiment because they were pregnant. At 10:00 a.m. on day E14 and E17 of gestation mice were weighed and treated with an intraperitoneal injection of TB4 (Regene RX, Rockville, MD, USA; 6 mg/kg in PBS). RESULTS: The mothers treated with TB4 for two days precisely E14 and E17, showed a higher cranio-caudal length when compared to control newborns. At histology, maternal TB4 treatment was associated with more advanced development of lungs, heart, kidney, cerebral cortex and notochord. CONCLUSIONS: Our study shows that TB4 administration during gestation may act as a powerful fetal growth promoter, by accelerating the development of newborn organs and tissues.


Assuntos
Desenvolvimento Fetal/efeitos dos fármacos , Nascimento Prematuro , Timosina/farmacologia , Animais , Feminino , Humanos , Recém-Nascido , Injeções Intraperitoneais , Camundongos , Gravidez , Timosina/administração & dosagem
2.
Int J Mol Sci ; 21(18)2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32961846

RESUMO

Prior work has indicated that thymosin beta 4 (Tß4) administered with ciprofloxacin markedly improves disease outcome for Pseudomonas aeruginosa (PA)-induced keratitis. As a result, the goal of the current study was to elucidate mechanisms by which Tß4 mitigates the corneal response; specifically, regarding its bactericidal influence and potential synergy with ciprofloxacin. An in vitro approach was carried out using minimum inhibitory concentration (MIC) assays to assess bactericidal activity against PA. In addition, antimicrobial peptide (AMP) production was evaluated at the mRNA levels using human corneal epithelial cells in response to lipopolysaccharide (LPS) challenge. The results of the MIC assays did not show direct bactericidal activity with Tß4 alone, although ciprofloxacin exhibited significant killing at concentrations far lower than clinically dosed. Tß4, however, displayed an indirect effect on bacterial killing, as shown by an upregulation of AMPs and related molecules. The cumulative data from this study indicate an indirect bactericidal role of Tß4, as well as a synergistic relationship with ciprofloxacin. Furthermore, ciprofloxacin alone was found to influence cellular functions that otherwise have yet to be reported. These results highlight a mechanism of intracellular communication for Tß4 and further strengthen its development as an adjunct therapy with antibiotics for corneal infections.


Assuntos
Ciprofloxacina/farmacologia , Córnea/efeitos dos fármacos , Ceratite/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Timosina/farmacologia , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , Córnea/patologia , Sinergismo Farmacológico , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Humanos , Ceratite/enzimologia , Ceratite/microbiologia , Lipopolissacarídeos/farmacologia , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/enzimologia
3.
Gene ; 758: 144946, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32649978

RESUMO

Hepatic injury is one of the most challenging diseases in clinical medicine. Hepatic injury is accompanied by hepatocyte apoptosis and leads to hepatic fibrosis and cirrhosis, which may cause liver cancer and increased mortality. Therefore, it is essential to investigate the regulation mechanism and therapeutic strategies for hepatic injury. In the study, the effects of Thymosin ß4 (Tß4) on Long intergenic noncoding RNA-p21 (lincRNA-p21)-mediated liver injury were investigated. Results showed that lincRNA-p21 overexpression promoted hepatocytes apoptosis, which was blocked by Tß4. Besides, Tß4 reversed the levels of cleaved caspase-3 and caspase-9 induced by lincRNA-p21. LincRNA-p21 overexpression also caused the pathological injury and fibrosis in hepatic tissues and increased the levels of fibrosis-related proteins (Collagen I, α-SMA and TIMP-1), and induced hydroxyproline and ALT production. However, Tß4 reversed the effects of overexpression of lincRNA-p21 on hepatic injury and fibrosis. In vitro experiments, after lincRNA-p21 was overexpressed in hepatic stellate cells (HSCs), the proliferation ability and the levels of HSCs markers α-SMA and Desmin were increased. However, Tß4 reversed the effects of lincRNA-p21 on HSCs. Furthermore, the PI3K-AKT-NF-κB pathway was activated by lincRNA-p21, which was then reversed by the Tß4 administration. After the mice treated by insulin-like growth factor-1 (IGF-1) (the activator of PI3K-AKT), the inhibitory effect of Tß4 on activated the PI3K-AKT-NF-κB pathway was abrogated. Besides, IGF-1 abolished the protective effects of Tß4 on hepatic apoptosis and fibrosis induced by lincRNA-p21. Therefore, Tß4 reversed. lincRNA-p21-mediated liver injury through inhibiting PI3K-AKT-NF-κB pathway. Tß4 may be a promising drug for fibrosis therapy.


Assuntos
Apoptose/efeitos dos fármacos , Cirrose Hepática/prevenção & controle , Fígado/lesões , RNA Longo não Codificante/genética , Timosina/farmacologia , Actinas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Hepatócitos/patologia , Proteínas I-kappa B/antagonistas & inibidores , Fígado/metabolismo , Cirrose Hepática/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Inibidor Tecidual de Metaloproteinase-1/metabolismo
4.
Mol Cell Biochem ; 469(1-2): 133-142, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32304006

RESUMO

Pro-inflammatory cytokines prevent bone regeneration in vivo and activation of nuclear factor-κB (NF-κB) signaling has been proposed to lead to suppression of bone morphogenetic protein (BMP)-induced osteogenesis via direct binding of p65 to Smad4 in vitro. Application of a small nuclear acidic protein (MTI-II) and its delivered peptide, MPAID (MTI-II peptide anti-inflammatory drug) has been described to elicit therapeutic potential via strong anti-inflammatory action following the physical association of MTI-II and MPAID with p65. However, it is unclear whether MTI-II attenuates tumor necrosis factor (TNF)-α inhibition of BMP-induced osteogenesis. Herein, we found that TNF-α-mediated suppression of responses associated with BMP4-induced osteogenesis, including expression of the osteocalcin encoding gene Ocn, Smad binding element (SBE)-dependent luciferase activity, alkaline phosphatase activity, and alizarin red S staining were largely restored by MTI-II and MPAID in MC3T3-E1 cells. Mechanistically, MTI-II and MPAID did not inhibit nuclear translocation of p65 or disassociate Smad4 from p65. Further, results from chromatin immunoprecipitation (ChIP) analyses revealed that Smad4 enrichment in cells over-expressing MTI-II and treated with TNF-α was equivalent to that in cells without TNF-α treatment. Alternatively, Smad4 enrichment was considerably decreased following TNF-α treatment in control cells. Moreover, p65 enrichment in the Id-1 promoter SBE was detected only when cells over-expressing MTI-II were stimulated with TNF-α. Overall, our study concludes that MTI-II restored TNF-α-inhibited suppression of BMP-Smad-induced osteogenic differentiation by enhancing accessibility of the Smad4-p65 complex to the SBE rather than by liberating Smad4 from p65.


Assuntos
Anti-Inflamatórios/farmacologia , Proteínas Morfogenéticas Ósseas/farmacologia , Osteogênese/efeitos dos fármacos , Proteína Smad4/metabolismo , Timosina/análogos & derivados , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Imunoprecipitação da Cromatina , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Timosina/farmacologia
5.
Int J Mol Sci ; 21(6)2020 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-32245208

RESUMO

Thymosin ß4 (Tß4) is a G-actin sequestering protein that contributes to diverse cellular activities, such as migration and angiogenesis. In this study, the beneficial effects of combined cell therapy with Tß4 and human adipose-derived stem cells (hASCs) in a mouse ischemic hindlimb model were investigated. We observed that exogenous treatment with Tß4 enhanced endogenous TMSB4X mRNA expression and promoted morphological changes (increased cell length) in hASCs. Interestingly, Tß4 induced the active state of hASCs by up-regulating intracellular signaling pathways including the PI3K/AKT/mTOR and MAPK/ERK pathways. Treatment with Tß4 significantly increased cell migration and sprouting from microbeads. Moreover, additional treatment with Tß4 promoted the endothelial differentiation potential of hASCs by up-regulating various angiogenic genes. To evaluate the in vivo effects of the Tß4-hASCs combination on vessel recruitment, dorsal window chambers were transplanted, and the co-treated mice were found to have a significantly increased number of microvessel branches. Transplantation of hASCs in combination with Tß4 was found to improve blood flow and attenuate limb or foot loss post-ischemia compared to transplantation with hASCs alone. Taken together, the therapeutic application of hASCs combined with Tß4 could be effective in enhancing endothelial differentiation and vascularization for treating hindlimb ischemia.


Assuntos
Membro Posterior/metabolismo , Isquemia/metabolismo , Células-Tronco Mesenquimais/metabolismo , Timosina/metabolismo , Timosina/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Transplante de Células , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Membro Posterior/irrigação sanguínea , Humanos , Isquemia/genética , Isquemia/terapia , Sistema de Sinalização das MAP Quinases/genética , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Nus , Neovascularização Fisiológica/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Timosina/genética , Timosina/uso terapêutico , Cicatrização/genética
6.
Cells ; 9(4)2020 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-32290541

RESUMO

Endothelial progenitor cells (EPCs) are bone-marrow derived cells that are critical in the maintenance of endothelial wall integrity and protection of ischemic myocardium through the formation of new blood vessels (vasculogenesis) or proliferation of pre-existing vasculature (angiogenesis). Diabetes mellitus (DM) and the metabolic syndrome are commonly associated with ischemic heart disease through its pathological effects on the endothelium and consequent endothelial dysfunction. Thymosin-ß4 (Tß4) which expressed in the embryonic heart is critical in epicardial and coronary artery formation. In this study, we explored the effects of Tß4 treatment on diabetic EPCs in vitro and intramyocardial injection of Tß4-treated and non-Tß4 treated EPCs following acute myocardial infarction (MI) of diabetic rats in vivo. It was found that 10 ng/mL Tß4 increased migration, tubule formation, and angiogenic factor secretion of diabetic EPCs in vitro. In vivo, although implantation of Tß4 treated diabetic EPCs significantly increased capillary density and attracted more c-Kit positive progenitor cells into the infarcted hearts as compared with implantation of non-Tß4 treated diabetic EPCs, the significantly improved left ventricular ejection fraction was only found in the rats which received non-Tß4 treated EPCs. The data suggests that a low dose Tß4 increases diabetic EPC migration, tubule formation, and angiogenic factor secretion. However, it did not improve the effects of EPCs on left ventricular pump function in diabetic rats with MI.


Assuntos
Diabetes Mellitus Experimental/terapia , Ecocardiografia/métodos , Células Progenitoras Endoteliais/transplante , Proteínas dos Microfilamentos/uso terapêutico , Infarto do Miocárdio/terapia , Timosina/uso terapêutico , Animais , Modelos Animais de Doenças , Humanos , Masculino , Proteínas dos Microfilamentos/farmacologia , Obesidade , Ratos , Ratos Zucker , Timosina/farmacologia
7.
J Pharmacol Sci ; 143(2): 127-131, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32156464

RESUMO

The inhibition of retinal ischemia-induced damage by post-ischemic prothymosin alpha (ProTα) was not affected in toll-like receptor 4 knockout (TLR4-/-) mice but blocked by the pretreatment with antibody against F0/F1 ATPase α- or ß-subunit, novel candidate for ProTα-receptor. In addition to the previous observation of ProTα-induced ATP release from cells, the present study showed a ProTα-induced enhancement of ATP hydrolysis activity of recombinant ATP5A1/5B complex. As the protection of retinal function by post-ischemic ProTα was abolished by anti-P2Y12 antibody, the activation of F0/F1 ATPase and subsequent P2Y12 receptor system may play roles in beneficial actions by post-ischemic ProTα.


Assuntos
Isquemia/metabolismo , Isquemia/prevenção & controle , Precursores de Proteínas/administração & dosagem , Precursores de Proteínas/farmacologia , ATPases Translocadoras de Prótons/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Retina , Timosina/análogos & derivados , Animais , Hidrólise/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas Recombinantes/metabolismo , Timosina/administração & dosagem , Timosina/farmacologia
8.
Aesthet Surg J ; 40(9): NP519-NP529, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32144415

RESUMO

BACKGROUND: Autologous fat grafting is a common procedure to improve tissue deficiencies. However, the survival rate of fat grafting is unpredictable. Thymosin beta 4 (Tß4), a multifunctional peptide containing 43 amino acids, is effective in angiogenesis, inhibiting apoptosis and inflammation. OBJECTIVES: The authors initially investigated the potential effect of Tß4 in fat grafting. METHODS: Adipose tissue premixed exogenous Tß4 were transplanted into rabbit ears. Rabbits were randomly assigned to 3 groups: group A, 5 µg/mL Tß4; group B, 10 µg/mL Tß4; and group C, phosphate-buffered saline buffer as a blank control. The fat grafts were subjected to magnetic resonance imaging at 2, 4, and 12 weeks in vivo. Each harvested graft was analyzed at 3 time points after transplantation. RESULTS: The fat grafts in the Tß4-treated groups showed better volume and weight retention, greater adipose tissue integrity, adipocyte viability, and angiogenesis. The results of dynamic contrast-enhanced magnetic resonance imaging also showed that the experimental groups increased microcirculation perfusion of the grafts. CONCLUSIONS: The study proved that Tß4 could improve adipose tissue survival and neovascularization. It may be useful for fat grafting as a potential protective reagent.


Assuntos
Sobrevivência de Enxerto , Timosina , Tecido Adiposo , Animais , Coelhos , Timosina/farmacologia , Transplante Autólogo
9.
Peptides ; 126: 170265, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31982448

RESUMO

Prothymosin alpha (ProTα)-mimetic hexapeptide (amino acid: NEVDQE, P6Q) inhibits cerebral or retinal ischemia-induced behavioral, electrophysiological and histological damage. P6Q also abolishes cerebral hemorrhage induced by ischemia with tissue plasminogen activator (tPA). In the present study we examined the beneficial effects of P6Q on other post-stroke prognostic psychology-related symptoms, which obstruct the motivation toward physical therapy. Intravenous (i.v.) administration with tPA (10 mg/kg) at 6 h after photochemically induced thrombosis (PIT) in mice resulted in bilateral central post-stroke pain in thermal and mechanical nociception tests and loss of social activity in the nest building test, both of which were significantly blocked by P6Q (30 mg/kg, i.v.) given at 5 h after PIT. P6Q (30 mg/kg, i.v.) also improved the memory-learning deficit in the step-through test and depression-like behavior in the tail suspension test when it was given 1 day after bilateral common carotid arteries occlusion (BCCAO) in mice. Thus, these studies suggest that P6Q could be a promising candidate to prevent negative prognostic psychological symptoms following focal and global ischemia.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Depressão/tratamento farmacológico , Transtornos da Memória/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Dor/tratamento farmacológico , Precursores de Proteínas/farmacologia , Acidente Vascular Cerebral/tratamento farmacológico , Timosina/análogos & derivados , Animais , Isquemia Encefálica/induzido quimicamente , Isquemia Encefálica/patologia , Isquemia Encefálica/psicologia , Depressão/etiologia , Depressão/patologia , Aprendizagem , Masculino , Transtornos da Memória/etiologia , Transtornos da Memória/patologia , Camundongos , Camundongos Endogâmicos C57BL , Dor/etiologia , Dor/patologia , Fragmentos de Peptídeos/farmacologia , Acidente Vascular Cerebral/induzido quimicamente , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/psicologia , Timosina/farmacologia , Ativador de Plasminogênio Tecidual/toxicidade
10.
Dig Liver Dis ; 52(3): 324-330, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31542221

RESUMO

Liver fibrosis is an important health problem without adequate and effective therapeutics. In this study, effects of thymosinß4 (Tß4) on hepatic fibrogenesis and the underlying molecular mechanisms were explored in bile duct ligation (BDL)-induced mice cholestatic liver fibrosis model. Results showed exogenous Tß4 significantly reduced the mortality and liver/body weight ratio in BDL mice. Histological examinations and biochemical analyses demonstrated that BDL induced evident portal fibrosis and a significant increase in hepatic collagen contents. However, these changes were significantly attenuated by exogenous Tß4. Quantitative real-time PCR assays showed that Tß4 suppressed BDL-induced increases in many fibrotic genes expression including α-smooth muscle actin (α-SMA), collagen I, III and fibronectin, TGFß1, TGFßR II, Smad2, Smad3, and PDGFRß. Results from immunohistochemistry and Western blots also showed that Tß4 reduced TGFß1 and PDGFRß protein levels in the liver tissues of BDL mice. In vitro studies using LX-2 cells demonstrated that Tß4 could decrease PDGFRß and TGFßR II levels in hepatic stellate cells. Taken together, findings in our present studies suggested that exogenous Tß4 alleviated BDL-induced cholestatic liver fibrosis through downregulating PDGF/PDGFR and TGFß/Smad pathways.


Assuntos
Cirrose Hepática/prevenção & controle , Fígado/patologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Timosina/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Colágeno Tipo I/metabolismo , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo
11.
Mater Sci Eng C Mater Biol Appl ; 106: 110268, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753373

RESUMO

Microfiber yarns (MY) have been widely employed to construct tendon tissue grafts. However, suboptimal ultrastructure and inappropriate environments for cell interactions limit their clinical application. Herein, we designed a modified electrospinning device to coat poly(lactic-co-glycolic acid) PLGA nanofibers onto polylactic acid (PLA) MY to generate PLGA/PLA hybrid yarns (HY), which had a well-aligned nanofibrous structure, resembling the ultrastructure of native tendon tissues and showed enhanced failure load compared to PLA MY. PLGA/PLA HY significantly improved the growth, proliferation, and tendon-specific gene expressions of human adipose derived mesenchymal stem cells (HADMSC) compared to PLA MY. Moreover, thymosin beta-4 (Tß4) loaded PLGA/PLA HY presented a sustained drug release manner for 28 days and showed an additive effect on promoting HADMSC migration, proliferation, and tenogenic differentiation. Collectively, the combination of Tß4 with the nano-topography of PLGA/PLA HY might be an efficient strategy to promote tenogenesis of adult stem cells for tendon tissue engineering.


Assuntos
Nanofibras/química , Poliésteres/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Timosina/química , Engenharia Tecidual , Tecido Adiposo/citologia , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Tendões/citologia , Tendões/metabolismo , Timosina/metabolismo , Timosina/farmacologia , Tecidos Suporte/química
12.
Life Sci Alliance ; 2(6)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31719116

RESUMO

Chronic granulomatous disease (CGD) is a genetic disorder of the NADPH oxidase characterized by increased susceptibility to infections and hyperinflammation associated with defective autophagy and increased inflammasome activation. Herein, we demonstrate that thymosin ß4 (Tß4), a g-actin sequestering peptide with multiple and diverse intracellular and extracellular activities affecting inflammation, wound healing, fibrosis, and tissue regeneration, promoted in human and murine cells noncanonical autophagy, a form of autophagy associated with phagocytosis and limited inflammation via the death-associated protein kinase 1. We further show that the hypoxia inducible factor-1 (HIF-1)α was underexpressed in CGD but normalized by Tß4 to promote autophagy and up-regulate genes involved in mucosal barrier protection. Accordingly, inflammation and granuloma formation were impaired and survival increased in CGD mice with colitis or aspergillosis upon Tß4 treatment or HIF-1α stabilization. Thus, the promotion of endogenous pathways of inflammation resolution through HIF-1α stabilization is druggable in CGD by Tß4.


Assuntos
Doença Granulomatosa Crônica/tratamento farmacológico , Doença Granulomatosa Crônica/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Timosina/farmacologia , Actinas/metabolismo , Animais , Autofagia/fisiologia , Reparo do DNA , Feminino , Doença Granulomatosa Crônica/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Células RAW 264.7
13.
Int J Mol Sci ; 20(19)2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31590392

RESUMO

The American cockroach (Periplaneta americana) is a medicinal insect. Its extract is used clinically to promote wound healing and tissue regeneration, but the effective medicinal components and mechanisms are not yet clear. It has been reported that human thymosin beta 4 (Tß4) may accelerate skin wound healing, however, the role of P. americana thymosin (Pa-THYs) is still poorly understood. In the present study, we identify and analyze the DNA sequences of Pa-THYs by bioinformatics analysis. Then we clone, express, and purify the Pa-THYs proteins and evaluate the activity of recombinant Pa-THYs proteins by cell migration and proliferation assays in NIH/3T3 cells. To elucidate the role of Pa-THYs in wound healing, a mouse model is established, and we evaluate wound contraction, histopathological parameters, and the expressions of several key growth factors after Pa-THYs treatment. Our results showed that three THY variants were formed by skipping splicing of exons. Pa-THYs could promote fibroblast migration, but have no effect on fibroblast proliferation. In wound repair, Pa-THYs proteins could effectively promote wound healing through stimulating dermal tissue regeneration, angiogenesis, and collagen deposition. On the molecular mechanism, Pa-THYs also stimulated the expression of several key growth factors to promote wound healing. The data suggest that Pa-THYs could be a potential drug for promoting wound repair.


Assuntos
Baratas/genética , Proteínas de Insetos/farmacologia , Timosina/farmacologia , Cicatrização/efeitos dos fármacos , Células 3T3 , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Baratas/metabolismo , Proteínas de Insetos/genética , Masculino , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Timosina/genética
14.
Mol Med Rep ; 20(5): 4186-4192, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31545437

RESUMO

The aim of the present study was to investigate the protective effects of thymosin ß4 (Tß4) on neuronal apoptosis in rat middle cerebral artery occlusion ischemia/reperfusion (MCAO I/R) injury, and determine the mechanisms involved in this process. Forty­eight adult male Sprague­Dawley rats were randomly divided into three groups (n=16 per group): A sham control group, an ischemia/reperfusion group (I/R group), and a Tß4 group. The focal cerebral I/R model was established by blocking the right MCA for 2 h, followed by reperfusion for 24 h. The Zea­Longa method was used to assess neurological deficits. Cerebral infarct volume was assessed using 2,3,5­triphenyltetrazolium chloride staining, and pathological changes were observed via hematoxylin and eosin staining. The terminal dexynucleotidyl transferase (TdT)­mediated dUTP nick end labeling (TUNEL) assay was used to detect apoptosis. The expression of glucose­regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and caspase­12 (CASP12) protein was assessed using immunohistochemistry and western blotting 24 h after reperfusion. Infarct volume and neuronal damage in the I/R and Tß4 groups were significantly greater than those observed in the sham group. The Zea­Longa score, neuronal apoptosis, and expression of GRP78, CHOP, and CASP12 in the I/R and Tß4 groups were significantly higher than those reported in the sham group. However, the Longa score and neuronal apoptosis were lower in the Tß4 group compared to the I/R group. The expression of GRP78 was significantly increased, whereas that of CHOP and CASP12 was significantly decreased in the Tß4 group compared to the I/R group. The present data revealed that Tß4 can inhibit neuronal apoptosis by upregulating GRP78 and downregulating CHOP and CASP12, thereby reducing cerebral I/R injury.


Assuntos
Apoptose/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Timosina/farmacologia , Animais , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Ratos
15.
Can J Physiol Pharmacol ; 97(10): 945-951, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31397599

RESUMO

The aim of our work was to test whether thymosin beta 4 protected endothelial progenitor cells against apoptosis induced by advanced glycation endproducts and investigate the underlying mechanism. Treatment with thymosin beta 4 or transfection with microRNA-34a inhibitor enhanced cell viability, reduced apoptosis, abated oxidative stress, and attenuated mitochondrial dysfunction in endothelial progenitor cells exposed to advanced glycation endproducts. Incubation with advanced glycation endproducts led to increased levels of microRNA-34a, which was attenuated by treatment with thymosin beta 4. Transfection with microRNA-34a reversed the beneficial effect of thymosin beta 4 against injuries induced by advanced glycation endproducts. The microRNA-34a could directly bind to the 3'UTRs of the mRNA of B-cell lymphoma 2, and thymosin beta 4 treatment upregulated B-cell lymphoma 2 expression in endothelial progenitor cells exposed to advanced glycation endproducts. More importantly, knockdown of B-cell lymphoma 2 abolished the protection of thymosin beta 4 and microRNA-34a inhibitor against advanced glycation endproducts. In conclusion, inhibition of microRNA-34a mediated protection of thymosin beta 4 in endothelial progenitor cells against advanced glycation endproducts by targeting B-cell lymphoma 2, which was helpful for understanding the therapeutic potential of thymosin beta 4 for diabetic patients.


Assuntos
Células Progenitoras Endoteliais/patologia , Produtos Finais de Glicação Avançada/metabolismo , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Timosina/farmacologia , Apoptose/genética , Células Cultivadas , Células Progenitoras Endoteliais/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Voluntários Saudáveis , Humanos , Leucócitos Mononucleares , MicroRNAs/antagonistas & inibidores , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Regulação para Cima
16.
Protein J ; 38(6): 675-682, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31332687

RESUMO

Thymosin beta 10 (TB10) is one of the common members among beta-thymosins. Human TB10 is reported to play a role in anti-angiogenesis and inhibition of cell migration during the tumorigenesis or metastasis of some certain cancers. Thus, it would be a potent clinical agent. In the present study, the coding sequence of TB10 was optimized based on the codon preference of Escherichia coli and cloned to pET28a (+) by chemical synthesis and molecular cloning methods. The recombinant protein was highly expressed employing E. coli expressing system and purified by a simple step of Ni2+ affinity chromatography. The TEV proteinase recognition site was inserted in the His6-tag and the target protein for easy removal of the His6-tag. To improve the biological activity of TB10, the transactivator of transcription (TAT) short peptide, a transduction domain, was added to the N-terminus of TB10. About 14.3 mg of the recombinant TB10 proteins was obtained from 1 L bacterial culture. The functional analyses demonstrated that the recombinant TB10 proteins displayed the distinct inhibition on angiogenesis by chick embryo chorioallantoic membrane assay and endothelial cell migration by wound healing assay. The TAT-fused TB10 even had stronger effects, probably due to the better transduction into the cells.


Assuntos
Proteínas Recombinantes de Fusão , Timosina , Inibidores da Angiogênese/farmacologia , Clonagem Molecular , Escherichia coli/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Timosina/isolamento & purificação , Timosina/farmacologia
17.
Mar Drugs ; 17(2)2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30795639

RESUMO

ß-thymosin is known for having 43 amino acids, being water-soluble, having a light molecular weight and ubiquitous polypeptide. The biological activities of ß-thymosin are diverse and include the promotion of wound healing, reduction of inflammation, differentiation of T cells and inhibition of apoptosis. Our previous studies showed that oyster ß-thymosin originated from the mantle of the Pacific oyster, Crassostrea gigas and had antimicrobial activity. In this study, we investigated the anti-inflammatory effects of oyster ß-thymosin in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells using human ß-thymosin as a control. Oyster ß-thymosin inhibited the nitric oxide (NO) production as much as human ß-thymosin in LPS-induced RAW264.7 cells. It also showed that oyster ß-thymosin suppressed the expression of prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, oyster ß-thymosin reduced inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). Oyster ß-thymosin also suppressed the nuclear translocation of phosphorylated nuclear factor-κB (NF-κB) and degradation of inhibitory κB (IκB) in LPS-induced RAW264.7 cells. These results suggest that oyster ß-thymosin, which is derived from the mantle of the Pacific oyster, has as much anti-inflammatory effects as human ß-thymosin. Additionally, oyster ß-thymosin suppressed NO production, PGE2 production and inflammatory cytokines expression via NF-κB in LPS-induced RAW264.7 cells.


Assuntos
Anti-Inflamatórios/farmacologia , Crassostrea/química , Dinoprostona/biossíntese , Macrófagos/efeitos dos fármacos , Óxido Nítrico/biossíntese , Timosina/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Queratinócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Alinhamento de Sequência , Transdução de Sinais/efeitos dos fármacos , Timosina/isolamento & purificação
18.
Eur Rev Med Pharmacol Sci ; 23(3): 1279-1290, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30779097

RESUMO

OBJECTIVE: To investigate the inhibitory effect of thymosin-ß4 (Tß4) on the activation of the human hepatic stellate cell line (HSC-LX2) induced by interleukin (IL)-1ß. MATERIALS AND METHODS: There were 5 groups in this study, i.e., blank control group, negative control group (SI-NC, empty plasmid), model group (20 ng/ml of IL-1ß), siRNA-Tß4 knockdown group (IL-1ß and si-Tß4) and Tß4 treatment group (IL-1ß and 1000 ng/ml of Tß4). Cell proliferation rate was measured using the Cell Counting Kit-8 (CCK-8) method. The cell cycle change and percentage of apoptotic cells were determined by Propidium Iodide (PI) DNA staining and Annexin V-fluorescein isothiocyanate (FITC) double staining. Cellular nucleic acid levels of p-IKB and nuclear factor-kappa B (NF-κB)/p65 proteins were measured by fluorescent quantitative Real Time-Polymerase Chain Reaction (RT-PCR). Double immunofluorescence staining and Western blot were used to detect nuclear translocation of NF-κB and p65 and levels of cytoplasmic p-IKB protein and nuclear p65 protein. RESULTS: Due to the G0/G1 phase arrest, the number of cells in the Tß4 treatment group increased, compared with the model group and the siRNA-Tß4 knockdown group (p<0.01). In the same between-group comparison, apoptotic rate in the Tß4 treatment group increased significantly (p<0.05). The cellular nucleic acid levels of p-IKB and NF-κB/p65 were markedly higher in the model group and the siRNA-Tß4 knockdown group than in the blank control group (p<0.01). The cellular nucleic acid levels of p-IKB and NF-κB/p65 were remarkably lower in the Tß4 treatment group than in the siRNA-Tß4 knockdown group (p<0.01). The expression levels of NF-κB/p65 and NF-κB/p50 were significantly lower in the Tß4 treatment group. The expression levels of cytoplasmic p-IKB and nuclear NF-κB/p65 were lower in the Tß4 treatment group than in the model group (p<0.01). CONCLUSIONS: Tß4 significantly inhibited IL-1ß-induced HSC-LX2 cell proliferation. The mechanism may involve decreased activation of the NF-κB pathway, decreased expression of p-IKB and nuclear translocation of p65. Therefore, Tß4 had the effect of reversing liver fibrosis.


Assuntos
Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Timosina/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Técnicas de Silenciamento de Genes , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Interleucina-1beta/farmacologia , Cirrose Hepática/patologia , RNA Interferente Pequeno/genética , Ratos , Transdução de Sinais , Timosina/genética , Timosina/farmacologia
19.
Neurosci Res ; 147: 1-8, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30326249

RESUMO

Angiopoietin-1 (Ang1) and its receptor Tie2 regulate vascular function. Our previous study demonstrated that thymosin beta 4 (Tß4) ameliorates neurological function of diabetic peripheral neuropathy. Mechanisms underlying the therapeutic effect of Tß4 on diabetic peripheral neuropathy have not been fully investigated. The present in vivo study investigated whether the Ang1/Tie2 signaling pathway is involved in Tß4-improved neurovascular remodeling in diabetic peripheral neuropathy. Diabetic BKS. Cg-m+/+Leprdb/J (db/db) mice at age 20 weeks were treated with Tß4 and neutralizing antibody against mouse Tie2 for 4 consecutive weeks. Neurological functional and neurovascular remodeling were measured. Administration of the neutralizing antibody against Tie2 attenuated the therapeutic effect of Tß4 on improved diabetic peripheral neuropathy as measured by motor and sensory nerve conduction velocity and thermal hypoesthesia compared to diabetic db/db mice treated with Tß4 only. Histopathological analysis revealed that the neutralizing antibody against Tie2 abolished Tß4-increased microvascular density in sciatic nerve and intraepidermal nerve fiber density, which were associated with suppression of Tß4-upregulated occludin expression and Tß4-reduced protein levels of nuclear factor-κB (NF-κB) and vascular cell adhesion molecule-1 (VCAM1). Our data provide in vivo evidence that the Ang1/Tie2 pathway contributes to the therapeutic effect of Tß4 on diabetic peripheral neuropathy.


Assuntos
Angiopoietina-1/metabolismo , Neuropatias Diabéticas/metabolismo , Receptor TIE-2/antagonistas & inibidores , Receptor TIE-2/metabolismo , Timosina/farmacologia , Animais , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Neuropatias Diabéticas/tratamento farmacológico , Camundongos , Camundongos Transgênicos , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/patologia , Nervo Isquiático/irrigação sanguínea , Nervo Isquiático/patologia , Transdução de Sinais/efeitos dos fármacos
20.
Brain Res ; 1707: 198-207, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30500399

RESUMO

Diabetes induces neurovascular dysfunction leading to peripheral neuropathy. MicroRNAs (miRNAs) affect many biological processes and the development of diabetic peripheral neuropathy. In the present study, we investigated whether thymosin-ß4 (Tß4) ameliorates diabetic peripheral neuropathy and whether miR-146a mediates the effect of Tß4 on improved neurovascular function. Male Type II diabetic BKS. Cg-m+/+Leprdb/J (db/db) mice at age 20 weeks were treated with Tß4 for 8 consecutive weeks, and db/db mice treated with saline were used as a control group. Compared to non-diabetic mice, diabetic mice exhibited substantially reduced miR-146a expression, and increased IL-1R-associated kinase-1 (IRAK1), tumor necrosis factor (TNFR)-associated factor 6 (TRAF6) levels and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) activity in sciatic nerve tissues. Treatment of diabetic mice with Tß4 significantly elevated miR-146a levels and overcame the effect of diabetes on these proteins. Tß4 treatment substantially improved motor and sensory conduction velocity of the sciatic nerve, which was associated with improvements in sensory function. Tß4 treatment significantly increased intraepidermal nerve fiber density and augmented local blood flow and the density of fluorescein isothiocyanate (FITC)-dextran perfused vessels in the sciatic nerve tissue. In vitro, treatment of dorsal root ganglion (DRG) neurons and mouse dermal endothelial cells (MDEs) with Tß4 significantly increased axonal outgrowth and capillary-like tube formation, whereas blocking miR-146a attenuated Tß4-induced axonal outgrowth and capillary tube formation, respectively. Our data indicate that miR-146a may mediate Tß4-induced neurovascular remodeling in diabetic mice, by suppressing pro-inflammatory signals.


Assuntos
Neuropatias Diabéticas/terapia , MicroRNAs/genética , Timosina/farmacologia , Animais , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/fisiopatologia , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Gânglios Espinais/metabolismo , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Nervo Isquiático/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF/metabolismo , Timosina/metabolismo
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