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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
Oxid Med Cell Longev ; 2018: 9630175, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30116499

RESUMO

Thymosin beta 4 (Tß4), an actin-sequestering protein, is involved in tissue development and regeneration. It prevents inflammation and fibrosis in several tissues. We investigated the role of Tß4 in chronic ethanol- and acute lipopolysaccharide- (LPS-) induced mouse liver injury. C57BL/6 mice were fed 5% ethanol in liquid diet for 4 weeks plus binge ethanol (5 g/kg, gavage) with or without LPS (2 mg/kg, intraperitoneal) for 6 hours. Tß4 (1 mg/kg, intraperitoneal) was administered for 1 week. We demonstrated that Tß4 prevented ethanol- and LPS-mediated increase in liver injury markers as well as changes in liver pathology. It also prevented ethanol- and LPS-mediated increase in oxidative stress by decreasing ROS and lipid peroxidation and increasing the antioxidants, reduced glutathione and manganese-dependent superoxide dismutase. It also prevented the activation of nuclear factor kappa B by blocking the phosphorylation of the inhibitory protein, IκB, thereby prevented proinflammatory cytokine production. Moreover, Tß4 prevented fibrogenesis by suppressing the epigenetic repressor, methyl-CpG-binding protein 2, that coordinately reversed the expression of peroxisome proliferator-activated receptor-γ and downregulated fibrogenic genes, platelet-derived growth factor-ß receptor, α-smooth muscle actin, collagen 1, and fibronectin, resulting in reduced fibrosis. Our data suggest that Tß4 has antioxidant, anti-inflammatory, and antifibrotic potential during alcoholic liver injury.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Etanol/efeitos adversos , Fibrose/tratamento farmacológico , Inflamação/tratamento farmacológico , Lipopolissacarídeos/efeitos adversos , Proteínas dos Microfilamentos/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Timosina/uso terapêutico , Animais , Doença Hepática Induzida por Substâncias e Drogas/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/farmacologia , Timosina/farmacologia
11.
Expert Opin Biol Ther ; 18(sup1): 171-175, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30063848

RESUMO

INTRODUCTION: Thymosin ß4 (Tß4) is a thymic hormone with multiple and different intracellular and extracellular activities affecting wound healing, inflammation, fibrosis and tissue regeneration. As the failure to resolve inflammation leads to uncontrolled inflammatory pathology which underlies many chronic diseases, the endogenous pathway through which Tß4 may promote inflammation resolution becomes of great interest. In this review, we discuss data highlighting the efficacy of Tß4 in resolving inflammation by restoring autophagy. AREAS COVERED: The authors provide an overview of the Tß4's anti-inflammatory properties in several pathologies and provide preliminary evidence on the ability of Tß4 to resolve inflammation via the promotion of non-canonical autophagy associated with the activation of the DAP kinase anti-inflammatory function. EXPERT OPINION: Based on its multitasking activity in various animal studies, including tissue repair and prevention of chronic inflammation, Tß4 may represent a potential, novel treatment for inflammatory diseases associated with defective autophagy.


Assuntos
Autofagia/efeitos dos fármacos , Inflamação/prevenção & controle , Timosina/farmacologia , Animais , Autofagia/fisiologia , Regulação para Baixo/efeitos dos fármacos , Fibrose/prevenção & controle , Humanos , Inflamação/metabolismo , Inflamação/patologia , Cicatrização/efeitos dos fármacos
12.
Expert Opin Biol Ther ; 18(sup1): 177-184, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30063851

RESUMO

OBJECTIVES: Hepatic stellate cells (HSC) trans-differentiation is central to the development of liver fibrosis, marked by the expression of pro-fibrogenic genes and the proliferation and migration of activated HSC. Therefore, preventing and/or reverting the activation, proliferation, and migration of HSC may lead to new therapies for treating fibrosis/cirrhosis. Thymosin ß4 (Tß4) inhibits PDGF-BB-induced fibrogenesis, proliferation and migration of HSC by blocking Akt phosphorylation. Here, we utilized Tß4-derived peptides: amino-terminal-Ac-SDKPDMAEIEKFDKS (1-15aa) and actin-binding-LKKTETQ (17-23aa) to investigate the molecular mechanisms in the anti-fibrogenic actions of Tß4. METHODS: We used RT-PCR, Western blot, and proliferation and migration assays in early passages of human HSC cultures treated with PDGF-BB and/or Tß4 peptides. RESULTS: We showed that 17-23aa but not 1-15aa inhibited PDGF-BB-dependent up-regulation of PDGFß receptor, α-SMA, and collagen 1. It also blunted the phosphorylation of Akt at T 308 and S473, resulting in the inhibition of phosphorylation of PRAS40, and HSC proliferation and migration. Interestingly, 1-15aa blocked Akt phosphorylation at S473, but not T308 by inhibiting mTOR phosphorylation, thus, it did not have any effect on HSC proliferation and migration. CONCLUSION: These findings suggest that while 1-15aa has a minor effect on Akt phosphorylation, the anti-fibrogenic actions of Tß4 are exerted via 17-23aa.


Assuntos
Actinas/metabolismo , Becaplermina/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Timosina/farmacologia , Animais , Células Cultivadas , Células Estreladas do Fígado/fisiologia , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/prevenção & controle , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/fisiologia , Timosina/química , Timosina/metabolismo
13.
Expert Opin Biol Ther ; 18(sup1): 111-120, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30063852

RESUMO

INTRODUCTION: The establishment of induced pluripotent stem cells (iPSCs) and cardiomyocytes differentiated from them generated a new platform to study pathophysiological processes and to generate drug screening platforms and iPSC-derived tissues as therapeutic agents. Although major advances have been made in iPSC-reprogramming, cardiac differentiation and EHT production, reprogramming efficiency and the maturity of iPSC-CMs need to be further improved. AREAS COVERED: In this review, the authors summarize the current state of the field of iPSC research, the methodology of cardiac differentiation of iPSCs, the use of iPSC-CMs as disease models and toxicity screening platforms, and the potential of EHTs as therapeutic agents. The authors furthermore highlight the mechanisms by which Thymosin ß4 might enhance the production of iPSC-CMs and EHTs to improve their maturity and performance. EXPERT OPINION: iPSCs derived cardiomyocytes and EHTs represent a still young research field with many problems and pitfalls that need to be resolved to realize the full potential of iPSC-CMs and EHTs. Given that Thymosin ß4 directly enhances cardiac differentiation while also promoting angiogenic sprouting and vessel maturation, Tß4 might be of particular interest as a novel agent in tackling the difficulty of iPSC-CMs and engineered heart tissue grafts.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Timosina/farmacologia , Animais , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Engenharia Tecidual/métodos
14.
Expert Opin Biol Ther ; 18(sup1): 121-129, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30063857

RESUMO

INTRODUCTION: Despite recent advances in the treatment of coronary heart disease, a significant number of patients progressively develop heart failure. Reduction of infarct size after acute myocardial infarction and normalization of microvasculature in chronic myocardial ischemia could enhance cardiac survival. AREAS COVERED: Induction of neovascularization using vascular growth factors has emerged as a promising novel approach for cardiac regeneration. Thymosin ß4 (Tß4) might be a promising candidate for the treatment of ischemic heart disease. It has been characterized as a major G-actin-sequestering factor regulating cell motility, migration, and differentiation. During cardiac development, Thymosin ß4 seems essential for vascularization of the myocardium. In the adult organism, Thymosin ß4 has anti-inflammatory properties, increases myocyte and endothelial cell survival accompanied by differentiation of epicardial progenitor cells. In chronic myocardial ischemia, Tß4 overexpression enhances micro- and macrovasculature in the ischemic area and thereby improves myocardial function. A comparable effect is seen in diabetic and dyslipidemic pig ischemic hearts, suggesting an attractive therapeutic potential of adeno-associated virus encoding for Tß4 for patients with ischemic heart disease. EXPERT OPINION: Induction of mature micro-vessels is a prerequisite for chronic myocardial ischemia and might be achieved via a long-term overexpression of Thymosin ß4.


Assuntos
Cardiotônicos/farmacologia , Citoproteção/efeitos dos fármacos , Coração/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Timosina/farmacologia , Adulto , Animais , Sobrevivência Celular/efeitos dos fármacos , Doença das Coronárias/tratamento farmacológico , Doença das Coronárias/patologia , Humanos , Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Suínos , Timosina/fisiologia
15.
Biochimie ; 154: 99-106, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30096371

RESUMO

Thymosin α1 (Tα1), a hormone containing 28 amino acids, has been approved in several cancer therapies, but the lack of tumor-targeting hinders its full use in tumor treatment. We designed a new peptide by connecting Tα1 and RGDR, generating a product, Tα1-RGDR, where RGDR is located in the C-end with both tumor-homing and cell internalizing properties (C-end rule peptides, a consensus R/KXXR/K motif). This work aimed to study the antitumor and immunological activities of Tα1-RGDR, and its differences compared with the wild-type Tα1. The antitumor and immunological activities of Tα1-RGDR were measured using the B16F10 tumor and immunologic suppression models. Tα1-RGDR treatment led to significant inhibition of tumor growth at a dose at which Tα1 showed a slight effect in the B16F10 tumor growth model. In the immunologic suppression model, Tα1-RGDR shared almost equivalent immunomodulatory effect with Tα1. These results demonstrated the better therapeutic effects after treatment with Tα1-RGDR compared with Tα1. Moreover, both Tα1-RGDR and Tα1 shared a helical conformation in the presence of trifluoroethanol based on CD spectroscopy. Our dock information of Tα1-RGDR when combined with integrin αvß3 or neuropilin-1 further confirmed previous experimental results. All these findings suggest that Tα1-RGDR might be a useful therapy for tumors by overcoming its wild type limitation of tumor homing.


Assuntos
Antineoplásicos/química , Melanoma/metabolismo , Timosina/análogos & derivados , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Timalfasina , Timosina/química , Timosina/farmacologia
16.
Artif Cells Nanomed Biotechnol ; 46(sup3): S95-S104, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29989423

RESUMO

Thymosin ß4 (Tß4) is a multifunctional N-acetylated peptide with distinct activities important at various stages. Due to its potential multiple therapeutic uses in many fields, there is an increasing need of Tß4 at lower costs than with the use of chemical synthesis. In this research, we developed a method to produce rhTß4 with N-acetylation in E. coli. Firstly, the E. coli strain whose chromosome being integrated by the specific N-terminal acetyltransferase ssArd1 was constructed. Secondly, the rhTß4-Intein was constructed, in which rhTß4 was fused to the N-terminus of the smallest mini-intein Spl DnaX. The rhTß4 could be fully acetylated when the rhTß4-Intein was expressed in the engineering strain. After purification, the rhTß4-Intein fusion protein was induced with dithiothreitol (DTT) to release rhTß4 through intein-mediated N-terminal cleavage. Under the optimal conditions, the N-terminal serine residue was shown to be 100% acetylated and the yield of N-acetylated rhTß4 can reach 200 mg per litre. The N-acetylated rhTß4 could be stable at 2-8 °C for 24 months in PBS buffer without protein degradation and concentration change. The N-acetylated rhTß4 also showed the activity of binding with actins from different sources and excellent therapeutic effect on the rats with moderate to severe dry eye disease.


Assuntos
Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Timosina/biossíntese , Acetilação , Animais , Modelos Animais de Doenças , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Escherichia coli/genética , Feminino , Humanos , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Timosina/genética , Timosina/farmacologia
17.
J Gene Med ; 20(9): e3043, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29972714

RESUMO

BACKGROUND: The present study aimed to clarify the effects of thymosin ß4 (Tß4) on CCl4 -induced hepatic fibrosis in mice and to further explore the underlying mechanisms. METHODS: Expression of Tß4 in fibrotic liver tissues was assessed by a quantitative real time-reverse transcriptase polymerase chain reaction and immunohistochemistry. The effects of intraperitoneal adeno-associated virus-Tß4 (AAV-Tß4) on CCl4 -induced hepatic fibrosis were observed by the evaluation of collagen deposition, hepatic stellate cell (HSC) activation and pro-fibrotic cytokine expression. In vitro tests with HSCs and hepatocytes were performed to confirm the effects of Tß4. RESULTS: The expression of Tß4 was down-regulated in fibrotic mouse livers but was rapidly up-regulated by CCl4 -induced acute injury. AAV-Tß4 pre-treatment significantly attenuated liver injury, collagen deposition, HSC activation and pro-fibrotic cytokine over-expression, such as transforming growth factor ß1 (TGF-ß1), platelet-derived growth factor B (PDGF-B), connective tissue growth factor (CTGF) and plasminogen activator inhibitor-1 (PAI-1) in CCl4 -intoxicated mouse livers. In vitro experiments showed that Tß4 suppressed HSC proliferation, blunted TGF-ß1-induced HSC activation and reduced TGF-ß1-induced TGF-ß1, PDGF-B, CTGF and PAI-1 expression in both HSCs and hepatocytes. Ectopic Tß4 ameliorated the over-expression of TGF-ß receptor-II (TGF-ßRII) in the fibrotic mouse livers. Exogenous Tß4 down-regulated TGF-ßRII expression, whereas neutralizing endogenous extracellular Tß4 with a specific antibody up-regulated TGF-ßRII expression in cultured HSCs and hepatocytes. CONCLUSIONS: Tß4 possesses anti-fibrotic activity in the liver, which is attributable, at least partly, to down-regulating TGF-ßRII and thereby blunting TGF-ß1-mediated fibrogenetic signaling in both HSCs and hepatocytes.


Assuntos
Regulação da Expressão Gênica/genética , Cirrose Hepática/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Timosina/genética , Animais , Tetracloreto de Carbono , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Dependovirus/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Masculino , Camundongos , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Timosina/metabolismo , Timosina/farmacologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia
18.
Expert Opin Biol Ther ; 18(sup1): 159-164, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29873258

RESUMO

OBJECTIVES: Thymosin beta 4 (Tß4) has demonstrated neuroprotective potential in models of neurlogical injury. The neuroprotective potential of Tß4 has been associated with increased miR-200a and miR-200b within the brain following stroke. Here we tested the hypothesis that Tß4 treatment could also alter miRNA profiles within the plasma following severe traumatic brain injury (TBI). METHODS: We used the rat lateral fluid percusion injury model of severe TBI to test this hypothesis. Highly sensitive and quantitative droplet digital polymerase chain reaction (ddPCR) was used to measure the plasma concentrations of miR-200 family members. In addition, we conducted RNAseq analysis of plasma miRNA to further identify changes associated with TBI and treatment with Tß4. RESULTS: ddPCR demonstrated that miR-200a-3p andmiR-200b-3p were both significantly increased in plasma following treatment with Tß4 after severe TBI. RNAseq analysis suggested that miR-300-3p and miR-598-3p increased while miR-450-3p and miR-194-5p significantly decreased following TBI. In contrast, miR-194-5p significantly increased in Tß4 treated rats following TBI. In addition, we identified nine plasma miRNAs whose expression significantly changed following treatment with Tß4. CONCLUSIONS: Tß4 treatment significantly increased plasma levels of miR-200a-3p and miR-200b-3p, while RNAseq analysis identified miR-194-5p as a candidate miRNA that may be critical for neuroprotection.


Assuntos
Lesões Encefálicas Traumáticas/sangue , Lesões Encefálicas Traumáticas/tratamento farmacológico , MicroRNAs/sangue , Timosina/farmacologia , Timosina/uso terapêutico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Modelos Animais de Doenças , Masculino , MicroRNAs/efeitos dos fármacos , MicroRNAs/genética , Percussão , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Transcriptoma/efeitos dos fármacos
19.
Pharmacol Res Perspect ; 6(3): e00407, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29864245

RESUMO

The transcription factor Islet-1 marks a progenitor cell population of the second heart field during cardiogenesis. In the adult heart Islet-1 expression is limited to the sinoatrial node, the ventricular outflow tract, and parasympathetic ganglia. The regenerative effect in the injured mouse ventricle of thymosin beta-4 (TB4), a 43-aminoacid peptide, was associated with increased Islet-1 immunostaining, suggesting the induction of an Islet-1-positive progenitor state by TB4. Here we aimed to reassess this effect in a genetic model. Mice from the reporter mouse line Isl1-nLacZ were primed with TB4 and subsequently underwent myocardial infarction. Islet-1 expression was assessed 2, 7, and 14 days after infarction. We detected only a single Islet-1+ cell in 8 TB4 treated and infarcted hearts which located outside of the sinoatrial node, the outflow tract or cardiac ganglia (in ~2500 sections). Two cells were identified in 5 control infarcted hearts. TB4 did not induce LacZ positivity in ventricular explants cultures of Isl1-nLacZ mice nor did it affect the density of LacZ+ cells in explant cultures of nLacZ+ regions of the heart. In summary, we found no evidence that TB4 reactivates Islet-1 expression in adult mouse ventricle.


Assuntos
Ventrículos do Coração/efeitos dos fármacos , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Infarto do Miocárdio/genética , Timosina/administração & dosagem , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Camundongos , Camundongos Transgênicos , Nó Sinoatrial/citologia , Nó Sinoatrial/efeitos dos fármacos , Nó Sinoatrial/metabolismo , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Timosina/farmacologia
20.
Stem Cell Res Ther ; 9(1): 166, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29921287

RESUMO

BACKGROUND: Deer antlers are the only known mammalian organ with vascularized cartilage that can completely regenerate. Antlers are of real significance as a model of mammalian stem cell-based regeneration with particular relevance to the fields of chondrogenesis, angiogenesis, and regenerative medicine. Recent research found that thymosin beta 10 (TMSB10) is highly expressed in the growth centers of growing antlers. The present study reports here the expression, functions, and molecular interactions of deer TMSB10. METHODS: The TMSB10 expression level in both tissue and cells in the antler growth center was measured. The effects of both exogenous (synthetic protein) and endogenous deer TMSB10 (lentivirus-based overexpression) on antlerogenic periosteal cells (APCs; nonactivated antler stem cells with no basal expression of TMSB10) and human umbilical vein endothelial cells (HUVECs; endothelial cells with no basal expression of TMSB10) were evaluated to determine whether TMSB10 functions on chondrogenesis and angiogenesis. Differences in deer and human TMSB10 in angiogenesis and molecular structure were determined using animal models and molecular dynamics simulation, respectively. The molecular mechanisms underlying deer TMSB10 in promoting angiogenesis were also explored. RESULTS: Deer TMSB10 was identified as a novel proangiogenic factor both in vitro and in vivo. Immunohistochemistry revealed that TMSB10 was widely expressed in the antler growth center in situ, with the highest expression in the reserve mesenchyme, precartilage, and transitional zones. Western blot analysis using deer cell lines further supports this result. Both exogenous and endogenous deer TMSB10 significantly decreased proliferation of APCs (P < 0.05), while increasing the proliferation of HUVECs (P < 0.05). Moreover, deer TMSB10 enhanced chondrogenesis in micromass cultures and nerve growth as assessed using a dorsal root ganglion model (P < 0.05). Deer TMSB10 was proangiogenic using models of chicken chorioallantoic membrane, tube formation, and aortic arch assay. At the molecular level, endogenous deer TMSB10 elevated the expression of vascular endothelial growth factor (VEGF), VEGF-B, VEGF-C, and VEGF-D, and VEGFR2 and VEGFR3 in HUVECs (P < 0.05). CONCLUSIONS: Deer TMSB10, in contrast to its human counterpart, was identified as a novel stimulating factor for angiogenesis, cartilage formation, and nerve growth, which is understandable given that deer antlers (as the arguably fastest mammalian growing tissue) may require this extra boost during a period of rapid growth and regeneration.


Assuntos
Morfogênese/fisiologia , Regeneração/fisiologia , Timosina/uso terapêutico , Animais , Chifres de Veado , Cervos , Timosina/farmacologia
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