RESUMO
Peripheral nerve defect are common clinical problem caused by trauma or other diseases, often leading to the loss of sensory and motor function in patients. Autologous nerve transplantation has been the gold standard for repairing peripheral nerve defects, but its clinical application is limited due to insufficient donor tissue. In recent years, the application of tissue engineering methods to synthesize nerve conduits for treating peripheral nerve defect has become a current research focus. This study introduces a novel approach for treating peripheral nerve defects using a tissue-engineered PLCL/SF/NGF@TA-PPy-RGD conduit. The conduit was fabricated by combining electrospun PLCL/SF with an NGF-loaded conductive TA-PPy-RGD gel. The gel, synthesized from RGD-modified tannic acid (TA) and polypyrrole (PPy), provides growth anchor points for nerve cells. In vitro results showed that this hybrid conduit could enhance PC12 cell proliferation, migration, and reduce apoptosis under oxidative stress. Furthermore, the conduit activated the PI3K/AKT signalling pathway in PC12 cells. In a rat model of sciatic nerve defect, the PLCL/SF/NGF@TA-PPy-RGD conduit significantly improved motor function, gastrocnemius muscle function, and myelin sheath axon thickness, comparable to autologous nerve transplantation. It also promoted angiogenesis around the nerve defect. This study suggests that PLCL/SF/NGF@TA-PPy-RGD conduits provide a conducive environment for nerve regeneration, offering a new strategy for peripheral nerve defect treatment, this study provided theoretical basis and new strategies for the research and treatment of peripheral nerve defect.
Assuntos
Hidrogéis , Fator de Crescimento Neural , Regeneração Nervosa , Oligopeptídeos , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Nervo Isquiático , Transdução de Sinais , Animais , Regeneração Nervosa/efeitos dos fármacos , Ratos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células PC12 , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/lesões , Oligopeptídeos/farmacologia , Oligopeptídeos/química , Hidrogéis/química , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/metabolismo , Ratos Sprague-Dawley , Masculino , Proliferação de Células/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Alicerces Teciduais/química , Engenharia Tecidual/métodos , Polímeros/químicaRESUMO
Nerve growth factor (NGF) plays a crucial role in cellular growth and neurodifferentiation. To achieve significant neuronal regeneration and repair using in vitro NGF delivery, spatiotemporal control that follows the natural neuronal processes must be developed. Notably, a challenge hindering this is the uncontrolled burst release from the growth factor delivery systems. The rapid depletion of NGF reduces treatment efficacy, leading to poor cellular response. To address this, we developed a highly controllable system using graphene oxygen (GO) and GelMA hydrogels modulated by electrical stimulation. Our system showed superior control over the release kinetics, reducing the burst up 30-fold. We demonstrate that the system is also able to sequester and retain NGF up to 10-times more efficiently than GelMA hydrogels alone. Our controlled release system enabled neurodifferentiation, as revealed by gene expression and immunostaining analysis. The increased retention and reduced burst release from our system show a promising pathway for nerve tissue engineering research toward effective regeneration.
Assuntos
Materiais Biocompatíveis , Estimulação Elétrica , Grafite , Hidrogéis , Fator de Crescimento Neural , Regeneração Nervosa , Hidrogéis/química , Hidrogéis/farmacologia , Grafite/química , Grafite/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Animais , Tamanho da Partícula , Teste de Materiais , Ratos , Células PC12 , Engenharia TecidualRESUMO
The nerve growth factor (NGF) participates in cell survival and glucose-stimulated insulin secretion (GSIS) processes in rat adult beta cells. GSIS is a complex process in which metabolic events and ionic channel activity are finely coupled. GLUT2 and glucokinase (GK) play central roles in GSIS by regulating the rate of the glycolytic pathway. The biphasic release of insulin upon glucose stimulation characterizes mature adult beta cells. On the other hand, beta cells obtained from neonatal, suckling, and weaning rats are considered immature because they secrete low levels of insulin and do not increase insulin secretion in response to high glucose. The weaning of rats (at postnatal day 20 in laboratory conditions) involves a dietary transition from maternal milk to standard chow. It is characterized by increased basal plasma glucose levels and insulin levels, which we consider physiological insulin resistance. On the other hand, we have observed that incubating rat beta cells with NGF increases GSIS by increasing calcium currents in neonatal cells. In this work, we studied the effects of NGF on the regulation of cellular distribution and activity of GLUT2 and GK to explore its potential role in the maturation of GSIS in beta cells from P20 rats. Pancreatic islet cells from both adult and P20 rats were isolated and incubated with 5.6 mM or 15.6 mM glucose with and without NGF for 4 hours. Specific immunofluorescence assays were conducted following the incubation period to detect insulin and GLUT2. Additionally, we measured glucose uptake, glucokinase activity, and insulin secretion assays at 5.6 mM or 15.6 mM glucose concentrations. We observed an age-dependent variation in the distribution of GLUT2 in pancreatic beta cells and found that glucose plays a regulatory role in GLUT2 distribution independently of age. Moreover, NGF increases GLUT2 abundance, glucose uptake, and GSIS in P20 beta cells and GK activity in adult beta cells. Our results suggest that besides increasing calcium currents, NGF regulates metabolic components of the GSIS, thereby contributing to the maturation process of pancreatic beta cells.
Assuntos
Glucoquinase , Transportador de Glucose Tipo 2 , Glucose , Células Secretoras de Insulina , Fator de Crescimento Neural , Animais , Masculino , Ratos , Células Cultivadas , Glucoquinase/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Ratos WistarRESUMO
We examined the mechanism by which 24(R)-ethyllophenol (MAB28) isolated from the branches of Morus alba caused neurite outgrowth in rat pheochromocytoma cells (PC12). MAB28 significantly promoted neurite outgrowth to a similar degree as the positive control, nerve growth factor (NGF). After incubation with MAB28 in PC12 cells, phosphorylation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and cyclic AMP response element-binding protein was detected, but the time course of phosphorylation was different from that induced by NGF. The expression of chloride intracellular channel protein 3 (CLIC3) was significantly decreased by MAB28. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), an outward rectifying chloride channel inhibitor, significantly promoted neurite outgrowth in PC12 cells. These data suggested that MAB28 could induce neurite outgrowth by downregulating CLIC3 expression.
Assuntos
Morus , Neuritos , Animais , Células PC12 , Ratos , Morus/química , Neuritos/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Nitrobenzoatos/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fenóis/farmacologia , Western Blotting , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Canais de CloretoRESUMO
Purpose: Recombinant human nerve growth factor (rhNGF; cenegermin-bkbj, OXERVATE) is the first and only U.S. Food and Drug Administration-approved treatment for moderate to severe neurotrophic keratopathy. The aim of this study was to determine the feasibility of incorporating a version of rhNGF in a mucoadhesive hydrogel capable of sustained drug release to the ocular surface. Methods: Hydrogels loaded with rhNGF were synthesized by conjugating chitosan with azidobenzoic acid (Az-Ch), adding rhNGF, and exposing the solution to ultraviolet (UV) radiation to induce photocrosslinking. Az-Ch hydrogels were evaluated for physical properties and rhNGF release profiles. Cytocompatbility of Az-Ch was assessed using immortalized human corneal limbal epithelial (HCLE) cells. TF1 erythroleukemic cell proliferation and HCLE cell proliferation and migration were used to assess the bioactivity of rhNGF released from Az-Ch hydrogels. Results: Az-Ch formed hydrogels in <10 seconds of UV exposure and demonstrated high optical transparency (75-85 T%). Az-Ch hydrogels exhibited good cytocompatibility with no demonstratable effect on HCLE cell morphology or viability. rhNGF was released gradually over 24 hours from Az-Ch hydrogels and retained its ability to induce TF1 cell proliferation. No significant difference was observed between rhNGF released from Az-Ch and freshly prepared rhNGF solutions on HCLE cell proliferation or percent wound closure after 12 hours; however, both were significantly better than control (P < 0.01). Conclusions: rhNGF-loaded Az-Ch hydrogels exhibited favorable physical, optical, and drug-release properties, as well as retained drug bioactivity. This drug delivery system has the potential to be further developed for in vivo and translational clinical applications. Translational Relevance: Az-Ch hydrogels may be used to enhance rhNGF therapy in patients with NK.
Assuntos
Proliferação de Células , Quitosana , Hidrogéis , Fator de Crescimento Neural , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/química , Fator de Crescimento Neural/administração & dosagem , Humanos , Quitosana/química , Quitosana/farmacologia , Hidrogéis/química , Hidrogéis/farmacologia , Hidrogéis/síntese química , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Raios Ultravioleta , Reagentes de Ligações Cruzadas/química , Limbo da Córnea/efeitos dos fármacos , Limbo da Córnea/citologia , Proteínas Recombinantes/química , Sistemas de Liberação de Medicamentos/métodosRESUMO
Spinal pain affects individuals of all ages and is the most common musculoskeletal problem globally. Its clinical management remains a challenge as the underlying mechanisms leading to it are still unclear. Here, we report that significantly increased numbers of senescent osteoclasts (SnOCs) are observed in mouse models of spinal hypersensitivity, like lumbar spine instability (LSI) or aging, compared to controls. The larger population of SnOCs is associated with induced sensory nerve innervation, as well as the growth of H-type vessels, in the porous endplate. We show that deletion of senescent cells by administration of the senolytic drug Navitoclax (ABT263) results in significantly less spinal hypersensitivity, spinal degeneration, porosity of the endplate, sensory nerve innervation, and H-type vessel growth in the endplate. We also show that there is significantly increased SnOC-mediated secretion of Netrin-1 and NGF, two well-established sensory nerve growth factors, compared to non-senescent OCs. These findings suggest that pharmacological elimination of SnOCs may be a potent therapy to treat spinal pain.
Assuntos
Senescência Celular , Osteoclastos , Animais , Camundongos , Osteoclastos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Senescência Celular/efeitos dos fármacos , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Células Receptoras Sensoriais/metabolismo , Modelos Animais de Doenças , Masculino , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Netrina-1/metabolismo , Netrina-1/genética , Camundongos Endogâmicos C57BLRESUMO
Nerve guidance conduits (NGCs) are considered as promising treatment strategy and frontier trend for peripheral nerve regeneration, while their therapeutic outcomes are limited by the lack of controllable drug delivery and available physicochemical cues. Herein, novel aligned piezoelectric nanofibers derived hydrogel NGCs with ultrasound (US)-triggered electrical stimulation (ES) and controllable drug release for repairing peripheral nerve injury are proposed. The inner layer of the NGCs is the barium titanate piezoelectric nanoparticles (BTNPs)-doped polyvinylidene fluoride-trifluoroethylene [BTNPs/P(VDF-TrFE)] electrospinning nanofibers with improved piezoelectricity and aligned orientation. The outer side of the NGCs is the thermoresponsive poly(N-isopropylacrylamide) hybrid hydrogel with bioactive drug encapsulation. Such NGCs can not only induce neuronal-oriented extension and promote neurite outgrowth with US-triggered wireless ES, but also realize the controllable nerve growth factor release with the hydrogel shrinkage under US-triggered heating. Thus, the NGC can positively accelerate the functional recovery and nerve axonal regeneration of rat models with long sciatic nerve defects. It is believed that the proposed US-responsive aligned piezoelectric nanofibers derived hydrogel NGCs will find important applications in clinic neural tissue engineering.
Assuntos
Hidrogéis , Nanofibras , Regeneração Nervosa , Animais , Regeneração Nervosa/efeitos dos fármacos , Hidrogéis/química , Nanofibras/química , Ratos , Ondas Ultrassônicas , Compostos de Bário/química , Nervo Isquiático/fisiologia , Nervo Isquiático/efeitos dos fármacos , Titânio/química , Polivinil/química , Alicerces Teciduais/química , Resinas Acrílicas/química , Traumatismos dos Nervos Periféricos/terapia , Ratos Sprague-Dawley , Fator de Crescimento Neural/química , Fator de Crescimento Neural/farmacologia , Liberação Controlada de Fármacos , Estimulação Elétrica , Nanopartículas/química , Engenharia Tecidual/métodosRESUMO
The re-bridging of the deficient nerve is the main problem to be solved after the functional impairment of the peripheral nerve. In this study, a directionally aligned polycaprolactone/triiron tetraoxide (PCL/Fe3O4) fiber scaffolds were firstly prepared by electrospinning technique, and further then grafted with IKVAV peptide for regulating DRG growth and axon extension in peripheral nerve regeneration. The results showed that oriented aligned magnetic PCL/Fe3O4 composite scaffolds were successfully prepared by electrospinning technique and possessed good mechanical properties and magnetic responsiveness. The PCL/Fe3O4 scaffolds containing different Fe3O4 concentrations were free of cytotoxicity, indicating the good biocompatibility and low cytotoxicity of the scaffolds. The IKVAV-functionalized PCL/Fe3O4 scaffolds were able to guide and promote the directional extension of axons, the application of external magnetic field and the grafting of IKVAV peptides significantly further promoted the growth of DRGs and axons. The ELISA test results showed that the AP-10â¯F group scaffolds promoted the secretion of nerve growth factor (NGF) from DRG under a static magnetic field (SMF), thus promoting the growth and extension of axons. Importantly, the IKVAV-functionalized PCL/Fe3O4 scaffolds could significantly up-regulate the expression of Cntn2, PCNA, Sox10 and Isca1 genes related to adhesion, proliferation and magnetic receptor function under the stimulation of SMF. Therefore, IKVAV-functionalized PCL/Fe3O4 composite oriented scaffolds have potential applications in neural tissue engineering.
Assuntos
Poliésteres , Alicerces Teciduais , Animais , Poliésteres/química , Ratos , Alicerces Teciduais/química , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Gânglios Espinais/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/química , Regeneração Nervosa/efeitos dos fármacos , Campos Magnéticos , Compostos Férricos/química , Compostos Férricos/farmacologia , Ratos Sprague-Dawley , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Células PC12RESUMO
Self-assembly of biological elements into biomimetic cargo carriers for targeting and delivery is a promising approach. However, it still holds practical challenges. We developed a functionalization approach of DNA origami (DO) nanostructures with neuronal growth factor (NGF) for manipulating neuronal systems. NGF bioactivity and its interactions with the neuronal system were demonstrated in vitro and in vivo models. The DO elements fabricated by molecular self-assembly have manipulated the surrounding environment through static spatially and temporally controlled presentation of ligands to the cell surface receptors. Our data showed effective bioactivity in differentiating PC12 cells in vitro. Furthermore, the DNA origami NGF (DON) affected the growth directionality and spatial capabilities of dorsal root ganglion neurons in culture by introducing a chemotaxis effect along a gradient of functionalized DO structures. Finally, we showed that these elements provide enhanced axonal regeneration in a rat sciatic nerve injury model in vivo. This study is a proof of principle for the functionality of DO in neuronal manipulation and regeneration. The approach proposed here, of an engineered platform formed out of programmable nanoscale elements constructed of DO, could be extended beyond the nervous system and revolutionize the fields of regenerative medicine, tissue engineering, and cell biology.
Assuntos
DNA , Gânglios Espinais , Fator de Crescimento Neural , Regeneração Nervosa , Animais , Ratos , Células PC12 , DNA/química , Gânglios Espinais/citologia , Fator de Crescimento Neural/química , Fator de Crescimento Neural/farmacologia , Nanoestruturas/química , Neurônios , Nervo Isquiático , Alicerces Teciduais/química , Ratos Sprague-DawleyRESUMO
Neurotrophins are a family of growth factors that play a key role in the development and regulation of the functioning of the central nervous system. Their use as drugs is made difficult by their poor stability, cellular permeability, and side effects. Continuing our effort to use peptides that mimic the neurotrophic growth factor (NGF), the family model protein, and specifically the N-terminus of the protein, here we report on the spectroscopic characterization and resistance to hydrolysis of the 14-membered cyclic peptide reproducing the N-terminus sequence (SSSHPIFHRGEFSV (c-NGF(1-14)). Far-UV CD spectra and a computational study show that this peptide has a rigid conformation and left-handed chirality typical of polyproline II that favors its interaction with the D5 domain of the NGF receptor TrkA. c-NGF(1-14) is able to bind Cu2+ with good affinity; the resulting complexes have been characterized by potentiometric and spectroscopic measurements. Experiments on PC12 cells show that c-NGF(1-14) acts as an ionophore, influencing the degree and the localization of both the membrane transporter (Ctr1) and the copper intracellular transporter (CCS). c-NGF(1-14) induces PC12 differentiation, mimics the protein in TrkA phosphorylation, and activates the kinase cascade, inducing Erk1/2 phosphorylation. c-NGF(1-14) biological activities are enhanced when the peptide interacts with Cu2+ even with the submicromolar quantities present in the culture media as demonstrated by ICP-OES measurements. Finally, c-NGF(1-14) and Cu2+ concur to activate the cAMP response element-binding protein CREB that, in turn, induces the brain-derived neurotrophic factor (BDNF) and the vascular endothelial growth factor (VEGF) release.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Cobre , Fator de Crescimento Neural , Peptídeos Cíclicos , Fator A de Crescimento do Endotélio Vascular , Células PC12 , Animais , Ratos , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cobre/metabolismo , Cobre/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ionóforos/farmacologia , Proteínas de Transporte de Cátions/metabolismo , Receptor trkA/metabolismoRESUMO
The authors' previous research has shown the pivotal roles of cyclin-dependent kinase 5 (CDK5) and its regulatory protein p35 in nerve growth factor (NGF)-induced differentiation of sympathetic neurons in PC12 cells. During the process of differentiation, neurons are susceptible to environmental influences, including the effects of drugs. Metformin is commonly used in the treatment of diabetes and its associated symptoms, particularly in diabetic neuropathy, which is characterized by dysregulation of the sympathetic neurons. However, the impacts of metformin on sympathetic neuronal differentiation remain unknown. In this study, we investigated the impact of metformin on NGF-induced sympathetic neuronal differentiation using rat pheochromocytoma PC12 cells as a model. We examined the regulation of TrkA-p35/CDK5 signaling in NGF-induced PC12 differentiation. Our results demonstrate that metformin reduces NGF-induced PC12 differentiation by inactivating the TrkA receptor, subsequently inhibiting ERK and EGR1. Inhibition of this cascade ultimately leads to the downregulation of p35/CDK5 in PC12 cells. Furthermore, metformin inhibits the activation of the presynaptic protein Synapsin-I, a substrate of CDK5, in PC12 differentiation. In addition, metformin alters axonal and synaptic bouton formation by inhibiting p35 at both the axons and axon terminals in fully differentiated PC12 cells. In summary, our study elucidates that metformin inhibits sympathetic neuronal differentiation in PC12 cells by disrupting TrkA/ERK/EGR1 and p35/CDK5 signaling. This research contributes to uncovering a novel signaling mechanism in drug response during sympathetic neuronal differentiation, enhancing our understanding of the intricate molecular processes governing this critical aspect of neurodevelopment.NEW & NOTEWORTHY This study unveils a novel mechanism influenced by metformin during sympathetic neuronal differentiation. By elucidating its inhibitory effects from the nerve growth factor (NGF) receptor, TrkA, to the p35/CDK5 signaling pathways, we advance our understanding of metformin's mechanisms of action and emphasize its potential significance in the context of drug responses during sympathetic neuronal differentiation.
Assuntos
Diferenciação Celular , Quinase 5 Dependente de Ciclina , Metformina , Fator de Crescimento Neural , Neurônios , Receptor trkA , Animais , Metformina/farmacologia , Ratos , Células PC12 , Quinase 5 Dependente de Ciclina/metabolismo , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Receptor trkA/metabolismo , Receptor trkA/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Diferenciação Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , FosfotransferasesRESUMO
Neuronal differentiation is regulated by nerve growth factor (NGF) and other neurotrophins. We explored the impact of NGF on mitochondrial dynamics and metabolism through time-lapse imaging, metabolomics profiling, and computer modeling studies. We show that NGF may direct differentiation by stimulating fission, thereby causing selective mitochondrial network fragmentation and mitophagy, ultimately leading to increased mitochondrial quality and respiration. Then, we reconstructed the dynamic fusion-fission-mitophagy cycling of mitochondria in a computer model, integrating these processes into a single network mechanism. Both the computational model and the simulations are able to reproduce the proposed mechanism in terms of mitochondrial dynamics, levels of reactive oxygen species (ROS), mitophagy, and mitochondrial quality, thus providing a computational tool for the interpretation of the experimental data and for future studies aiming to detail further the action of NGF on mitochondrial processes. We also show that changes in these mitochondrial processes are intertwined with a metabolic function of NGF in differentiation: NGF directs a profound metabolic rearrangement involving glycolysis, TCA cycle, and the pentose phosphate pathway, altering the redox balance. This metabolic rewiring may ensure: (a) supply of both energy and building blocks for the anabolic processes needed for morphological reorganization, as well as (b) redox homeostasis.
Assuntos
Diferenciação Celular , Mitocôndrias , Dinâmica Mitocondrial , Mitofagia , Fator de Crescimento Neural , Neurônios , Espécies Reativas de Oxigênio , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/genética , Dinâmica Mitocondrial/efeitos dos fármacos , Animais , Neurônios/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células PC12 , Ratos , Mitofagia/efeitos dos fármacos , Ciclo do Ácido Cítrico/efeitos dos fármacos , Glicólise , Simulação por Computador , Reprogramação MetabólicaRESUMO
Nerve growth factor (NGF) and its receptor, tropomyosin receptor kinase A (TrkA), are known to play important roles in the immune and nervous system. However, the effects of NGF on the osteogenic differentiation of dental pulp stem cells (DPSCs) remain unclear. This study aimed to investigate the role of NGF on the osteogenic differentiation of DPSCs in vitro and the underlying mechanisms. DPSCs were cultured in osteogenic differentiation medium containing NGF (50 ng/mL) for 7 days. Then osteogenic-related genes and protein markers were analysed using qRT-PCR and Western blot, respectively. Furthermore, addition of NGF inhibitor and small interfering RNA (siRNA) transfection experiments were used to elucidate the molecular signalling pathway responsible for the process. NGF increased osteogenic differentiation of DPSCs significantly compared with DPSCs cultured in an osteogenic-inducing medium. The NGF inhibitor Ro 08-2750 (10 µM) and siRNA-mediated gene silencing of NGF receptor, TrkA and ERK signalling pathways inhibitor U0126 (10 µM) suppressed osteogenic-related genes and protein markers on DPSCs. Furthermore, our data revealed that NGF-upregulated osteogenic differentiation of DPSCs may be associated with the activation of MEK/ERK signalling pathways via TrkA. Collectively, NGF was capable of promoting osteogenic differentiation of DPSCs through MEK/ERK signalling pathways, which may enhance the DPSCs-mediated bone tissue regeneration.
Assuntos
Fator de Crescimento Neural , Osteogênese , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/metabolismo , Polpa Dentária , Células-Tronco/metabolismo , Diferenciação Celular , Células Cultivadas , RNA Interferente Pequeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proliferação de CélulasRESUMO
The essential role of the neural network in enhancing bone regeneration has often been overlooked in biomaterial design, leading to delayed or compromised bone healing. Engineered mesenchymal stem cells (MSCs)-derived exosomes are becoming increasingly recognized as potent cell-free agents for manipulating cellular behavior and improving therapeutic effectiveness. Herein, MSCs are stimulated with nerve growth factor (NGF) to regulate exosomal cargoes to improve neuro-promotive potential and facilitate innervated bone regeneration. In vitro cell experiments showed that the NGF-stimulated MSCs-derived exosomes (N-Exos) obviously improved the cellular function and neurotrophic effects of the neural cells, and consequently, the osteogenic potential of the osteo-reparative cells. Bioinformatic analysis by miRNA sequencing and pathway enrichment revealed that the beneficial effects of N-Exos may partly be ascribed to the NGF-elicited multicomponent exosomal miRNAs and the subsequent regulation and activation of the MAPK and PI3K-Akt signaling pathways. On this basis, N-Exos were delivered on the micropores of the 3D-printed hierarchical porous scaffold to accomplish the sustained release profile and extended bioavailability. In a rat model with a distal femoral defect, the N-Exos-functionalized hierarchical porous scaffold significantly induced neurovascular structure formation and innervated bone regeneration. This study provided a feasible strategy to modulate the functional cargoes of MSCs-derived exosomes to acquire desirable neuro-promotive and osteogenic potential. Furthermore, the developed N-Exos-functionalized hierarchical porous scaffold may represent a promising neurovascular-promotive bone reparative scaffold for clinical translation.
Assuntos
Exossomos , Células-Tronco Mesenquimais , Ratos , Animais , Exossomos/metabolismo , Diferenciação Celular/genética , Porosidade , Fosfatidilinositol 3-Quinases , Fator de Crescimento Neural/análise , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Regeneração Óssea/fisiologia , Osteogênese , Impressão TridimensionalRESUMO
Traumatic brain injuries(TBI) pose significant challenges to human health, specifically neurological disorders and related motor activities. After TBI, the injured neuronal tissue is known for hardly regenerated and recovered to their normal neuron physiology and tissue compositions. For this reason, tissue engineering strategies that promote neuronal regeneration have gained increasing attention. This study explored the development of a novel neural tissue regeneration cryogel by combining brain-derived decellularized extracellular matrix (ECM) with heparin sulfate crosslinking that can perform nerve growth factor (NGF) release ability. Morphological and mechanical characterizations of the cryogels were performed to assess their suitability as a neural regeneration platform. After that, the heparin concnentration dependent effects of varying NGF concentrations on cryogel were investigated for their controlled release and impact on neuronal cell differentiation. The results revealed a direct correlation between the concentration of released NGF and the heparin sulfate ratio in cryogel, indicating that the cryogel can be tailored to carry higher loads of NGF with heparin concentration in cryogel that induced higher neuronal cell differentiation ratio. Furthermore, the study evaluated the NGF loaded cryogels on neuronal cell proliferation and brain tissue regeneration in vivo. The in vivo results suggested that the NGF loaded brain ECM derived cryogel significantly affects the regeneration of brain tissue. Overall, this research contributes to the development of advanced neural tissue engineering strategies and provides valuable insights into the design of regenerative cryogels that can be customized for specific therapeutic applications.
Assuntos
Lesões Encefálicas Traumáticas , Engenharia Tecidual , Humanos , Encéfalo , Lesões Encefálicas Traumáticas/terapia , Criogéis , Matriz Extracelular , Heparina , Fator de Crescimento Neural/farmacologia , Regeneração Nervosa , Sulfatos , Engenharia Tecidual/métodosRESUMO
Neural differentiation is triggered by the activation of multiple signaling pathways initiated by various neurotrophic factors. An elucidation of these mechanisms is anticipated to facilitate the prevention of diseases and the development of novel therapeutic approaches. Alternative small-molecule inducers for neuroscience studies are required instead of protein-based reagents for more efficient and convenient experiments. We demonstrated that small molecules of thieno[2,3-b]pyridine derivatives that induce neural differentiation, compounds 3a and 9a in particular, exhibited significant neuritogenic activity in rat pheochromocytoma (PC12) cells. Moreover, 3a displayed pronounced fluorescence and a discernible Stokes shift. Furthermore, the outcome of the experiment conducted on the NGF-insensitive clones of rat PC12 cells, and the results of the intercellular uptake analyses suggested that the 3a-mediated activation of neural differentiation occurred independently of the TrkA receptor. Therefore, 3a portrays potential applicability both as a small molecule reagent to replace novel neurotrophic factors and as a potent fluorescent reagent for various techniques, including bioimaging.
Assuntos
Fatores de Crescimento Neural , Quinolinas , Animais , Ratos , Diferenciação Celular/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Células PC12/efeitos dos fármacos , FosforilaçãoRESUMO
BACKGROUND: Asthma is the most common allergic disease characterized by an inflammatory response in the airways. Mechanismly, urban particulate matter (PM) is the most widely air pollutant associated with increased asthma morbidity and airway inflammation. Current research found that vitamin D is an essential vitamin with anti-inflammatory, antioxidant and other medical efficacy. Inadequate or deficient vitamin D often leads to the pathogenesis and stability of asthma. NGF exacerbates airway inflammation in asthma by promoting smooth muscle cell proliferation and inducing the Th2 immune response. Activation of the Nrf2/HO-1 signaling pathway can exert a protective effect on the inflammatory response in bronchial asthma. However, the specific mechanism of this pathway in PM-involved asthmatic airway smooth muscle cells remains unclear. METHODS: Mice were sensitized and challenged with Ovalbumin (OVA) to establish an asthma model. They were then exposed to either PM, vitamin D or a combination of both, and inflammatory responses were observed. Including, acetylcholine stimulation at different concentrations measured airway hyperresponsiveness in mice. Bronchoalveolar lavage fluid (BALF) and serum were collected for TNF-α, IL-1ß, IL-6, and Nerve growth factor (NGF) analysis. Additionally, lung tissues underwent histopathological examination to observe alveolar structure and inflammatory cell infiltration. Specific ELISA kits were utilized to determine the levels of the inflammatory factors TNF-α, IL-1ß, IL-6, and Nerve growth factor (NGF). Nrf2/HO-1 signaling pathways were examined by western blot analysis. Meanwhile, we constructed a cell system with low HO-1 expression by lentiviral transfection of airway smooth muscle cells. The changes of Nrf2, HO-1, and NGF were observed after the treatment of OVA, PM, and Vit D were given. RESULTS: The in vivo results showed that vitamin D significantly alleviated pathological changes in lung tissue of PM-exposed mice models. Mechanismly, vitamin D decreased substantial inflammatory cell infiltration in lung tissue, as well as the number of inflammatory cells in BALF. Furthermore, vitamin D reduced the heightened inflammatory factors including of TNF-α, IL-1ß, IL-6, and NGF caused by PM exposure, and triggered the activity of nucleus Nrf2 and HO-1 in PM-exposed asthmatic mice. Notably, knockdown HO-1 weakens the Vitamin D- mediated inhibition to pollution toxicity in asthma. Importantly, in vitro experiments on OVA-stimulated mice airway smooth muscle cells, the results showed that OVA and PM, respectively, reduced Nrf2/HO-1 and increased NGF's expression, while vitamin D reversed the process. And in the HO-1 knockdown cell line of Lenti-si-HO-1 ASMCs, OVA and PM reduced Nrf2's expression, while HO-1 and NGF's expression were unchanged. CONCLUSIONS: The above results demastrate that vitamin D downregulated the inflammatory response and the expression of NGF by regulating the Nrf2/HO-1 signaling pathways in airway smooth muscle cells, thereby showing potent anti-inflammatory activity in asthma.
Assuntos
Asma , Material Particulado , Camundongos , Animais , Material Particulado/toxicidade , Fator 2 Relacionado a NF-E2/metabolismo , Vitamina D/farmacologia , Vitamina D/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Fator de Crescimento Neural/uso terapêutico , Asma/induzido quimicamente , Asma/tratamento farmacológico , Pulmão/patologia , Inflamação , Transdução de Sinais , Líquido da Lavagem Broncoalveolar , Anti-Inflamatórios/farmacologia , Vitaminas/uso terapêutico , Ovalbumina , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Citocinas/metabolismoRESUMO
Despite recent advances in the field of microphysiological systems (MPSs), availability of models capable of mimicking the interactions between the nervous system and innervated tissues is still limited. This represents a significant challenge in identifying the underlying processes of various pathological conditions, including neuropathic, cardiovascular and metabolic disorders. In this novel study, we introduce a compartmentalized three-dimensional (3D) coculture system that enables physiologically relevant tissue innervation while recording neuronal excitability. By integrating custom microelectrode arrays into tailored glass chips microfabricated via selective laser-etching, we developed an entirely novel class of innervation MPSs (INV-MPS). This INV-MPS allows for manipulation, visualization, and electrophysiological analysis of individual axons innervating complex 3D tissues. Here, we focused on sensory innervation of 3D tumor tissue as a model case study since cancer-induced pain represents a major unmet medical need. The system was compared with existing nociception models and successfully replicated axonal chemoattraction mediated by nerve growth factor (NGF). Remarkably, in the absence of NGF, 3D cancer spheroids cocultured in the adjacent compartment induced sensory neurons to consistently cross the separating barrier and establish fine innervation. Moreover, we observed that crossing sensory fibers could be chemically excited by distal application of known pain-inducing agonists only when cocultured with cancer cells. To our knowledge, this is the first system showcasing morphological and electrophysiological analysis of 3D-innervated tumor tissuein vitro, paving the way for a plethora of studies into innervation-related diseases and improving our understanding of underlying pathophysiology.
Assuntos
Neoplasias , Fator de Crescimento Neural , Humanos , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Microeletrodos , Células Receptoras Sensoriais/metabolismo , Dor/metabolismo , Gânglios Espinais/fisiologiaRESUMO
Spinal cord injuries are very common worldwide, leading to permanent nerve function loss with devastating effects in the affected patients. The challenges and inadequate results in the current clinical treatments are leading scientists to innovative neural regenerative research. Advances in nanoscience and neural tissue engineering have opened new avenues for spinal cord injury (SCI) treatment. In order for designed nerve guidance conduit (NGC) to be functionally useful, it must have ideal scaffold properties and topographic features that promote the linear orientation of damaged axons. In this study, it is aimed to develop channeled polycaprolactone (PCL)/Poly-D,L-lactic-co-glycolic acid (PLGA) hybrid film scaffolds, modify their surfaces by IKVAV pentapeptide/gold nanoparticles (AuNPs) or polypyrrole (PPy) and investigate the behavior of motor neurons on the designed scaffold surfaces in vitro under static/bioreactor conditions. Their potential to promote neural regeneration after implantation into the rat SCI by shaping the film scaffolds modified with neural factors into a tubular form is also examined. It is shown that channeled groups decorated with AuNPs highly promote neurite orientation under bioreactor conditions and also the developed optimal NGC (PCL/PLGA G1-IKVAV/BDNF/NGF-AuNP50) highly regenerates SCI. The results indicate that the designed scaffold can be an ideal candidate for spinal cord regeneration.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Ouro , Nanopartículas Metálicas , Fator de Crescimento Neural , Traumatismos da Medula Espinal , Alicerces Teciduais , Animais , Ratos , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Ouro/química , Nanopartículas Metálicas/química , Fator de Crescimento Neural/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Oligopeptídeos/farmacologia , Poliésteres/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/terapia , Traumatismos da Medula Espinal/patologia , Alicerces Teciduais/químicaRESUMO
MSCs (Mesenchymal Stem Cells) can differentiate into various lineages, including neurons and glial cells. In the past few decades, MSCs have been well explored in the context of neuronal differentiation and have been reported to have the immense potential to form distinct kinds of neurons. The distinguishing features of MSCs make them among the most desired cell sources for stem cell therapy. This study involved the trans-differentiation of Adipose-derived human Mesenchymal Stem Cells (ADMSCs) into neurons. The protocol employs a cocktail of chemical inducers in different combinations, including Brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), and Nerve growth factor (NGF) Fibroblastic growth factor (FGF), in induction media. Both types have been successfully differentiated into neurons, confirmed by morphological aspects and the presence of neural-specific markers through RT-PCR (Reverse transcription polymerase chain reaction) studies and immunocytochemistry assay. They have shown excellent morphology with long neurites, synaptic connections, and essential neural markers to validate their identity. The results may significantly contribute to cell replacement therapy for neurological disorders.