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1.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064584

RESUMO

Liver fibrosis results from many chronic injuries and may often progress to cirrhosis and hepatocellular carcinoma (HCC). In fact, up to 90% of HCC arise in a cirrhotic liver. Conversely, stress is implicated in liver damage, worsening disease outcome. Hence, stress could play a role in disrupting liver homeostasis, a concept that has not been fully explored. Here, in a murine model of TAA-induced liver fibrosis we identified nerve growth factor (NGF) to be a crucial regulator of the stress-induced fibrogenesis signaling pathway as it activates its receptor p75 neurotrophin receptor (p75NTR), increasing liver damage. Additionally, blocking the NGF decreased liver fibrosis whereas treatment with recombinant NGF accelerated the fibrotic process to a similar extent than stress challenge. We further show that the fibrogenesis induced by stress is characterized by specific changes in the hepatoglycocode (increased ß1,6GlcNAc-branched complex N-glycans and decreased core 1 O-glycans expression) which are also observed in patients with advanced fibrosis compared to patients with a low level of fibrosis. Our study facilitates an understanding of stress-induced liver injury and identify NGF signaling pathway in early stages of the disease, which contributes to the established fibrogenesis.


Assuntos
Regulação da Expressão Gênica , Cirrose Hepática/patologia , Fator de Crescimento Neural/metabolismo , Polissacarídeos/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Estresse Fisiológico , Tioacetamida/toxicidade , Animais , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/genética
2.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064906

RESUMO

Nerve growth factor (NGF) is a protein essential to neurons survival, which interacts with its receptor as a non-covalent dimer. Peptides belonging to NGF N-terminal domain are able to mimic the activity of the whole protein. Such activity is affected by the presence of copper ions. The metal is released in the synaptic cleft where proteins, not yet identified, may bind and transfer to human copper transporter 1 (hCtr1), for copper uptake in neurons. The measurements of the stability constants of copper complexes formed by amyloid beta and hCtr1 peptide fragments suggest that beta-amyloid (Aß) can perform this task. In this work, the stability constant values of copper complex species formed with the dimeric form of N-terminal domain, sequence 1-15 of the protein, were determined by means of potentiometric measurements. At physiological pH, NGF peptides bind one equivalent of copper ion with higher affinity of Aß and lower than hCtr1 peptide fragments. Therefore, in the synaptic cleft, NGF may act as a potential copper chelating molecule, ionophore or chaperone for hCtr1 for metal uptake. Copper dyshomeostasis and mild acidic environment may modify the balance between metal, NGF, and Aß, with consequences on the metal cellular uptake and therefore be among causes of the Alzheimer's disease onset.


Assuntos
Transportador de Cobre 1/metabolismo , Cobre/metabolismo , Fator de Crescimento Neural/metabolismo , Fragmentos de Peptídeos/metabolismo , Sítios de Ligação , Humanos , Ligação Proteica
3.
Chem Biol Interact ; 341: 109454, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33798505

RESUMO

Doxycycline has been used as antibiotic since the 1960s. Recently, studies have shown that doxycycline is neuroprotective in models of neurodegenerative diseases and brain injuries, mainly due to anti-inflammatory and anti-apoptotic effects. However, it is not known if doxycycline has neurotrophic potential, which is relevant, considering the role of axonal degeneration at the early stages of neurodegeneration in Alzheimer's disease, Amyotrophic Lateral Sclerosis and Parkinson's disease as well as in normal aging. Axons are preceded by the formation of neurites, the hallmark of the neuronal differentiation induced by neurotrophins like NGF. Therefore, the modulation of neurotrophin receptors aimed at formation and regeneration of axons has been proposed as a strategy to delay the progression of neurodegeneration and has gained relevance as new techniques for early diagnosis arise. Based on these premises, we investigated the potential of doxycycline to mimic the effects of Nerve Growth Factor (NGF) with focus on the signaling pathways and neuronal modulators of neurite initiation, growth and branching. We used PC12 cells, a neuronal model widely employed to study the neurotrophic pathways and mechanisms induced by NGF. Results showed that doxycycline induced neurite outgrowth via activation of the trkA receptor and the downstream signaling pathways, PI3K/Akt and MAPK/ERK, without inducing the expression of NGF. Doxycycline also increased the expression of GAP-43, synapsin I and NF200, proteins involved in axonal and synaptic plasticity. Altogether, these data demonstrate, for the first time, the neurotrophic potential of doxycycline, which might be useful to restore the neuronal connectivity lost at the initial phase of neurodegeneration.


Assuntos
Antibacterianos/farmacologia , Doxiciclina/farmacologia , Fator de Crescimento Neural/metabolismo , Animais , Carbazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Proteína GAP-43/metabolismo , Alcaloides Indólicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Proteínas de Neurofilamentos/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Células PC12 , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptor trkA/antagonistas & inibidores , Receptor trkA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinapsinas/metabolismo
4.
Int J Mol Sci ; 22(8)2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917718

RESUMO

BACKGROUND: Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) play a critical role in neurodevelopment, where breast milk is a significant dietary source. The impact of previous COVID-19 infection and mastitis on the concentration of BDNF and NGF in human milk was investigated. METHODS: Concentrations of BDNF and NGF were measured via ELISA in human milk samples collected from 12 mothers with a confirmed COVID-19 PCR, 13 mothers with viral symptoms suggestive of COVID-19, and 22 unexposed mothers (pre-pandemic Ctl-2018). These neurotrophins were also determined in 12 mothers with previous mastitis and 18 mothers without mastitis. RESULTS: The NGF concentration in human milk was lower in the COVID-19 PCR and viral symptoms groups than in the unexposed group, but BDNF did not differ significantly. Within the COVID-19 group, BDNF was higher in mothers who reported headaches or loss of smell/taste when compared with mothers without the respective symptom. BDNF was lower in mothers with mastitis than in mothers without mastitis. CONCLUSIONS: Previous COVID-19 and mastitis infections changed differently the secretion of NGF and BDNF in human milk. Whether the changes in NGF and BDNF levels in milk from mothers with infection influence their infant's development remains to be investigated.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , COVID-19/metabolismo , Mastite/metabolismo , Leite Humano/química , Fator de Crescimento Neural/metabolismo , Adulto , Secreções Corporais/química , Fator Neurotrófico Derivado do Encéfalo/análise , COVID-19/complicações , Feminino , Humanos , Mastite/complicações , Mães , Fator de Crescimento Neural/análise
5.
Mol Med Rep ; 23(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33649819

RESUMO

Nerve growth factor (NGF), a prototypical neurotrophic factor essential for neuronal cell proliferation and survival, has been implicated as a marker of tumor progression, as well as a potential target for novel therapeutic approaches in cancer. To investigate the functional potential of NGF in liver cancer in the present study, a stable NGF­overexpressing HepG2 cell line was generated. The scratch­wound assay was used to investigate cell motility and polarity. Western blotting was performed to evaluate the expression levels of epithelial­mesenchymal transition (EMT)­related proteins, including E­cadherin, N­cadherin and vimentin. Moreover, immunofluorescence was performed to investigate the arrangement of the actin cytoskeleton. Cell anoikis resistance was examined using a suspension culture model and cell apoptosis was examined via flow cytometry. The present results indicated that NGF overexpression in HepG2 cells disrupted HepG2 cell polarity and promoted cell motility. Furthermore, NGF overexpression induced EMT and actin cytoskeleton rearrangement in HepG2 cells, as well as enhanced anoikis resistance and prevented cellular apoptosis. Notably, a tropomyosin receptor kinase A receptor inhibitor blocked NGF­induced cell motility and apoptosis. Therefore, it was suggested that NGF serves a critical role in the invasion and metastasis of liver cancer. The use of NGF as a biomarker or potential new target could lead to the development of novel factors for diagnosis or for improving therapeutic strategies in liver cancer.


Assuntos
Movimento Celular/genética , Polaridade Celular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Fator de Crescimento Neural/genética , Anoikis/genética , Apoptose/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Fator de Crescimento Neural/metabolismo
6.
Carbohydr Polym ; 258: 117684, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33593557

RESUMO

To control the release of nerve growth factor (NGF) in the injured peripheral nerve, NGF-loaded chitosan/PLGA composite microspheres ionically cross-linked by tripolyphosphate (TPP/Chitosan/PLGA-NGF) were prepared. The encapsulation efficiency of NGF ranged from 83.4 ± 1.5 % to 72.1 ± 1.6 % with TPP concentrations from 1 % to 10 %. Zeta potential and FT-IR analyses together with confocal microscopy demonstrated that multiple NGF-loaded PLGA microspheres were embedded in chitosan matrix, the mean size of TPP/Chitosan/PLGA-NGF microspheres ranged from 40.2 ± 3.4 to 49.3 ± 3.1 µm. The increase of TPP concentration improved the network stability and decreased the swelling ratio, resulting in the decreased NGF release from 67.7 ± 1.2 % to 45.7 ± 0.8 % in 49 days. The sustained release of NGF could promote PC12 cells differentiation and neurite growth in vitro. Moreover, in comparison with NGF solution without microencapsulation, TPP/Chitosan/PLGA-NGF microspheres enhanced sciatic nerve regeneration and prevented gastrocnemius muscle atrophy in rats. These results demonstrate the feasibility of using TPP/Chitosan/PLGA-NGF microspheres for neural tissue repair.


Assuntos
Quitosana/análogos & derivados , Microesferas , Fator de Crescimento Neural/metabolismo , Sistema Nervoso Periférico/patologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Animais , Diferenciação Celular , Quitosana/química , Reagentes para Ligações Cruzadas/farmacologia , Sistemas de Liberação de Medicamentos , Íons , Masculino , Microscopia Confocal , Músculo Esquelético/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/metabolismo , Células PC12 , Tamanho da Partícula , Polímeros/química , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/lesões , Nervo Isquiático/cirurgia , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
7.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467640

RESUMO

Mesenchymal stem cells (MSCs) are a promising therapy to improve vascular repair, yet their role in ischemic retinopathy is not fully understood. The aim of this study is to investigate the impact of modulating the neurotrophin receptor; p75NTR on the vascular protection of MSCs in an acute model of retinal ischemia/reperfusion (I/R). Wild type (WT) and p75NTR-/- mice were subjected to I/R injury by increasing intra-ocular pressure to 120 mmHg for 45 min, followed by perfusion. Murine GFP-labeled MSCs (100,000 cells/eye) were injected intravitreally 2 days post-I/R and vascular homing was assessed 1 week later. Acellular capillaries were counted using trypsin digest 10-days post-I/R. In vitro, MSC-p75NTR was modulated either genetically using siRNA or pharmacologically using the p75NTR modulator; LM11A-31, and conditioned media were co-cultured with human retinal endothelial cells (HREs) to examine the angiogenic response. Finally, visual function in mice undergoing retinal I/R and receiving LM11A-31 was assessed by visual-clue water-maze test. I/R significantly increased the number of acellular capillaries (3.2-Fold) in WT retinas, which was partially ameliorated in p75NTR-/- retinas. GFP-MSCs were successfully incorporated and engrafted into retinal vasculature 1 week post injection and normalized the number of acellular capillaries in p75NTR-/- retinas, yet ischemic WT retinas maintained a 2-Fold increase. Silencing p75NTR on GFP-MSCs coincided with a higher number of cells homing to the ischemic WT retinal vasculature and normalized the number of acellular capillaries when compared to ischemic WT retinas receiving scrambled-GFP-MSCs. In vitro, silencing p75NTR-MSCs enhanced their secretome, as evidenced by significant increases in SDF-1, VEGF and NGF release in MSCs conditioned medium; improved paracrine angiogenic response in HREs, where HREs showed enhanced migration (1.4-Fold) and tube formation (2-Fold) compared to controls. In parallel, modulating MSCs-p75NTR using LM11A-31 resulted in a similar improvement in MSCs secretome and the enhanced paracrine angiogenic potential of HREs. Further, intervention with LM11A-31 significantly mitigated the decline in visual acuity post retinal I/R injury. In conclusion, p75NTR modulation can potentiate the therapeutic potential of MSCs to harness vascular repair in ischemic retinopathy diseases.


Assuntos
Células-Tronco Mesenquimais/citologia , Receptores de Fator de Crescimento Neural/genética , Traumatismo por Reperfusão/metabolismo , Vasos Retinianos/metabolismo , Animais , Capilares/metabolismo , Movimento Celular , Proliferação de Células , Técnicas de Cocultura , Meios de Cultivo Condicionados/química , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Endotélio/metabolismo , Deleção de Genes , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Injeções Intravítreas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica , Fator de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Receptor de Fator de Crescimento Neural/metabolismo , Traumatismo por Reperfusão/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Eur J Pharmacol ; 895: 173882, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33482180

RESUMO

Fabry disease (FD) is an X-linked metabolic storage disorder arising from the deficiency of lysosomal α-galactosidase A, which leads to the gradual accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), throughout the body. Pain in the extremities is an early symptom of FD; however, the underlying pathophysiological mechanisms remain unknown. α-Galactosidase A knockout animals exhibit nociceptive behaviors, with enhanced expression levels of several ion channels. These characteristics are observed in animals treated with nerve growth factor (NGF). Here, we aimed to elucidate the potential of NGF signaling as a cause of FD-associated pain, using intraplantar Gb3-treated mice displaying mechanical allodynia. Treatment with a neutralizing antibody against a precursor of NGF (proNGF) or its receptor, p75 neurotrophin receptor (p75NTR), resulted in the recovery from Gb3-induced pain. Conversely, anti-NGF and anti-tropomyosin receptor kinase A antibodies failed to exert analgesic effects. Gb3 injection had no effects on the expression levels of proNGF and p75NTR in the plantar skin and dorsal root ganglia, suggesting that Gb3 activates the pain pathway, possibly mediated through functional up-regulation of proNGF-p75NTR signaling. Furthermore, by pharmacological approaches using a protein kinase A (PKA) inhibitor and a cholesterol-removing agent, we found that p75NTR-phosphorylating PKA and lipid rafts for phosphorylated p75NTR translocation were required for Gb3-induced pain. These results suggest that acute exposure to Gb3 induces mechanical allodynia via activation of the proNGF-p75NTR pathway, which involves lipid rafts and PKA. Our findings provide new pathological insights into FD-associated pain, and suggest the need to develop therapeutic interventions targeting proNGF-p75NTR signaling.


Assuntos
Gânglios Espinais/metabolismo , Hiperalgesia/metabolismo , Fator de Crescimento Neural/metabolismo , Limiar da Dor , Precursores de Proteínas/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Pele/metabolismo , Triexosilceramidas , Analgésicos/farmacologia , Animais , Anticorpos Neutralizantes/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Gânglios Espinais/fisiopatologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/fisiopatologia , Hiperalgesia/prevenção & controle , Masculino , Microdomínios da Membrana/metabolismo , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/antagonistas & inibidores , Limiar da Dor/efeitos dos fármacos , Precursores de Proteínas/antagonistas & inibidores , Receptor trkA/metabolismo , Receptores de Fator de Crescimento Neural/antagonistas & inibidores , Transdução de Sinais
9.
Mol Med Rep ; 23(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33179111

RESUMO

Astragaloside (AST) is derived from the Chinese herb Astragalus membranaceus, and studies have demonstrated that it promotes differentiation of bone marrow­derived mesenchymal stem cells (BMSCs). To the best of our knowledge, however, the functions of the component AST­IV in osteogenesis have not previously been elucidated. The present study aimed to verify the effects of AST­IV in osteogenesis. First, the proliferation and differentiation status of human BMSCs incubated with AST­IV were analysed and compared with a control (no AST­IV treatment). In order to determine the involvement of the glycogen synthase kinase (GSK)3ß signalling pathway in AST­IV, overexpression and inhibition of GSK3ß was induced during incubation of BMSCs with AST­IV. In order to investigate how neuronal growth factor (NGF) contributes to BMSCs differentiation, BMSCs were co­incubated with an anti­NGF antibody and AST IV, and then levels of osteogenesis markers were assessed. The results demonstrated for the first time that AST­IV contributed to BMSCs differentiation. Furthermore, the GSK3ß/ß­catenin signalling pathway was revealed to be involved in AST­IV­induced osteogenesis; moreover, AST­IV accelerated differentiation by enhancing the expression levels of NGF. In summary, the present study demonstrated that AST­IV promotes BMSCs differentiation, thus providing a potential target for the treatment of osteoporosis.


Assuntos
Células-Tronco Mesenquimais/citologia , Osteogênese , Osteoporose/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Fator de Crescimento Neural/metabolismo , Osteoporose/tratamento farmacológico
10.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(11): 1438-1445, 2020 Nov 15.
Artigo em Chinês | MEDLINE | ID: mdl-33191703

RESUMO

Objective: To investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis. Methods: BMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model ( n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR. Results: At 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B ( P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days ( P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A ( P<0.05). Conclusion: Silencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration.


Assuntos
Células-Tronco Mesenquimais , Proteínas do Tecido Nervoso , Receptores de Fatores de Crescimento , Animais , Células da Medula Óssea , Matriz Óssea , Diferenciação Celular , Células Cultivadas , Inativação Gênica , Lentivirus , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Osteogênese , Ratos , Ratos Sprague-Dawley , Receptor de Fator de Crescimento Neural , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Transfecção
11.
J Headache Pain ; 21(1): 105, 2020 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-32842964

RESUMO

BACKGROUND: Different pain syndromes may be characterized by different profiles of mediators reflecting pathophysiological differences, and these alterations may be measured in a simple saliva sample. The aims of the current study were to compare concentration of glutamate, serotonin (5-HT), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and substance P (SP) in saliva and plasma from a well-defined group of patients with chronic temporomandibular disorders myalgia (TMD-myalgia) with a group of pain-free controls, and further investigate the relationship between these markers and clinical characteristics. METHODS: Patients diagnosed according to the diagnostic criteria for TMD (n = 39), and matched healthy pain-free controls (n = 39) were included. Stimulated whole saliva and plasma samples were collected in the morning. Glutamate was analysed using a colorimetric assay, and 5-HT and SP were analysed by commercially available ELISA. Levels of NGF and BDNF were determined using multiplex electrochemiluminescence assay panel. RESULTS: Patients expressed higher salivary and plasma levels of glutamate (saliva: 40.22 ± 13.23 µmol/L; plasma: 30.31 ± 18.73 µmol/L) than controls (saliva: 33.24 ± 11.27 µmol/L; plasma: 20.41 ± 15.96 µmol/L) (p < 0.05). Salivary NGF (0.319 ± 0.261 pg/ml) and BDNF (3.57 ± 1.47 pg/ml) were lower in patients compared to controls (NGF: 0.528 ± 0.477 pg/ml; BDNF 4.62 ± 2.51 pg/ml)(p's < 0.05). Contrary, plasma BDNF, was higher in patients (263.33 ± 245.13 pg/ml) than controls (151.81 ± 125.90 pg/ml) (p < 0.05). 5-HT was undetectable in saliva. Neither plasma 5-HT, nor SP levels differed between groups. BDNF and NGF concentrations correlated to levels of psychological distress (p < 0.0005). CONCLUSION: The higher levels of salivary and plasma glutamate in patients with TMD-myalgia compared to controls strengthens its importance in the pathophysiology of TMD-myalgia. However, the lack of correlation to pain levels question its role as a putative biomarker. Patients with TMD-myalgia further had lower levels of salivary NGF and BDNF, but higher plasma BDNF. These results and their correlations to psychological distress warrant further investigations.


Assuntos
Saliva/metabolismo , Transtornos da Articulação Temporomandibular/sangue , Transtornos da Articulação Temporomandibular/metabolismo , Adulto , Biomarcadores/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mialgia/metabolismo , Fator de Crescimento Neural/metabolismo , Substância P/metabolismo , Adulto Jovem
12.
Anticancer Res ; 40(7): 3685-3696, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32620607

RESUMO

BACKGROUND/AIM: Although chemotherapy agents, such as oxaliplatin, cisplatin, paclitaxel and bortezomib frequently cause severe peripheral neuropathy, very few studies have reported the effective strategy to prevent this side effect. In this study, we first investigated whether these drugs show higher neuropathy compared to a set of 15 other anticancer drugs, and then whether antioxidants, such as sodium ascorbate, N-acetyl-L-cysteine, and vitamin B12 have any protective effect against them. MATERIALS AND METHODS: Rat PC12 cells were induced to differentiate into neuronal cells by repeated overlay of serum-free medium supplemented with nerve growth factor. The cytotoxic levels of anticancer drugs against four human oral squamous cell carcinoma cell lines, three normal oral cells, and undifferentiated and differentiated PC12 cells were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Cells were sorted for apoptotic cells (distributed into subG1 phase) and cells at different stages of cell cycle (G1, S and G2/M). RESULTS: All 19 anticancer drugs showed higher cytotoxicity against PC12 compared to oral normal cells. Among them, bortezomib showed the highest cytotoxicity against both undifferentiated and differentiated PC12 cell and, committed them to undergo apoptosis. Sodium ascorbate and N-acetyl-L-cysteine, but not vitamin B12, completely reversed the cytotoxicity of bortezomib. CONCLUSION: Bortezomib-induced neuropathy might be ameliorated by antioxidants.


Assuntos
Antioxidantes/farmacologia , Bortezomib/efeitos adversos , Síndromes Neurotóxicas/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Fator de Crescimento Neural/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Síndromes Neurotóxicas/metabolismo , Células PC12 , Doenças do Sistema Nervoso Periférico/metabolismo , Ratos
13.
Am J Pathol ; 190(9): 1921-1930, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32479822

RESUMO

Nerves are emerging promoters of cancer progression, but the innervation of esophageal cancer and its clinicopathologic significance remain unclear. In this study, nerves were analyzed by immunohistochemistry in a cohort of 260 esophageal cancers, including 40 matched lymph node metastases and 137 normal adjacent esophageal tissues. Nerves were detected in 38% of esophageal cancers and were more associated with squamous cell carcinomas (P = 0.04). The surrounding or invasion of nerves by cancer cells (perineural invasion) was detected in 12% of esophageal cancers and was associated with reduced survival (P = 0.04). Nerves were found to express the following receptors for nerve growth factor (NGF): neurotrophic receptor tyrosine kinase 1 and nerve growth factor receptor. An association was suggested between high production of NGF by cancer cells and the presence of nerves (P = 0.02). In vitro, NGF production in esophageal cancer cells was shown by Western blot, and esophageal cancer cells were able to induce neurite outgrowth in the PC12 neuronal cells. The neurotrophic activity of esophageal cancer cells was inhibited by anti-NGF blocking antibodies. Together, these data suggest that innervation is a feature in esophageal cancers that may be driven by cancer cell-released NGF.


Assuntos
Neoplasias Esofágicas/patologia , Invasividade Neoplásica/patologia , Fator de Crescimento Neural/metabolismo , Nervos Periféricos/patologia , Adulto , Idoso , Neoplasias Esofágicas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Microambiente Tumoral
14.
PLoS One ; 15(6): e0231542, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497034

RESUMO

Many receptors elicit signal transduction by activating multiple intracellular pathways. This transduction can be triggered by a non-specific ligand, which simultaneously activates all the signaling pathways of the receptors. However, the binding of one biased ligand preferentially trigger one pathway over another, in a process called biased signaling. The identification the functional motions related to each of these distinct pathways has a direct impact on the development of new effective and specific drugs. We show here how to detect specific functional motions by considering the case of the NGF/TrkA-Ig2 complex. NGF-mediated TrkA receptor activation is dependent on specific structural motions that trigger the neuronal growth, development, and survival of neurons in nervous system. The R221W mutation in the ngf gene impairs nociceptive signaling. We discuss how the large-scale structural effects of this mutation lead to the suppression of collective motions necessary to induce TrkA activation of nociceptive signaling. Our results suggest that subtle changes in the NGF interaction network due to the point mutation are sufficient to inhibit the motions of TrkA receptors putatively linked to nociception. The methodological approach presented in this article, based jointly on the normal mode analysis and the experimentally observed functional alterations due to point mutations provides an essential tool to reveal the structural changes and motions linked to the disease, which in turn could be necessary for a drug design study.


Assuntos
Modelos Moleculares , Fator de Crescimento Neural/metabolismo , Mutação Puntual , Receptor trkA/genética , Receptor trkA/metabolismo , Transdução de Sinais , Movimento , Fator de Crescimento Neural/química , Ligação Proteica , Conformação Proteica , Receptor trkA/química
15.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118714, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32246947

RESUMO

Ceramide kinase (CerK) phosphorylates ceramide to ceramide-1-phosphate (C1P). CerK is highly expressed in the brain, and its association with the neuronal function has been reported. Previous reports showed that the activity of CerK is regulated by post-translational modifications including phosphorylation, whereas the cellular fate of CerK protein and its role in neuronal functions have not been clearly elucidated. Therefore, we investigated these issues in PC12 cells. Treatment with nerve growth factor (NGF) for 6 h increased the formation of C1P but not CerK mRNA. Knockdown of CerK and overexpression of HA-tagged CerK down- and up-regulated the formation of C1P, respectively. In PC12-CerK-HA cells, serum withdrawal caused ubiquitination of CerK-HA protein and down-regulated both CerK-HA protein and C1P formation within 6 h, and these down-regulations were abolished by co-treatments with NGF or proteasome inhibitors such as MG132 and clasto-lactacystin. Microscopic analysis showed that treatment with the proteasome inhibitors increased CerK-HA in puncture structures, possibly endosomes and/or vesicles, in cells. Treatment with the lysosome inhibitors reduced serum withdrawal-induced down-regulation of CerK-HA protein but not C1P formation. When knockdown or overexpression of CerK was performed, Ca2+-induced release of [3H] noradrenaline was reduced or enhanced, respectively, but neurite extension was not modified. There was a positive correlation between noradrenaline release and formation of C1P and/or CerK-HA levels in NGF- and clasto-lactacystin-treated cells. These results suggest that levels of CerK were down-regulated by the ubiquitin/proteasome and lysosome pathways and the former pathway-sensitive pool of CerK was suggested to be linked with exocytosis in PC12 cells.


Assuntos
Exocitose/genética , Fator de Crescimento Neural/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Ciclo Celular , Proliferação de Células , Ceramidas , Lisossomos/genética , Lisossomos/metabolismo , Redes e Vias Metabólicas/genética , Fator de Crescimento Neural/metabolismo , Células PC12 , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos
16.
J Nanobiotechnology ; 18(1): 46, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32169062

RESUMO

BACKGROUND: Peripheral nerve injury is one common clinical disease worldwide, in which sciatic nerve is anatomically the most challenging to regenerate given its length and large cross-sectional area. For the present, autologous nerve grafting remains to be the most ideal strategy when treating with sciatic nerve injury. However, this method sacrifices healthy nerves and requires highly intensive surgery, still calling for other advanced alternatives for nerve grafting. RESULTS: In this study, we utilized previously well-established gene delivery system to dually deliver plasmid DNA (pDNA) encoding vascular endothelial growth factor (VEGF) and nerve growth factor (NGF), exploring therapeutics for sciatic nerve injury. Low-molecular-weight branched polyethylenimine (bPEI) was constructed as the backbone structure of gene vectors, and it was further crosslinked to synthesize degradable polycations via the conjugation of dialdehydes. Potential synergistic effect between VEGF and NGF proteins were observed on rat sciatic nerve crush injury model in this study. CONCLUSIONS: We concluded that dual delivery of plasmid VEGF and NGF as gene therapy could enhance sciatic nerve regeneration.


Assuntos
Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Regeneração Nervosa/fisiologia , Nervo Isquiático/crescimento & desenvolvimento , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Anoplura/química , Autoenxertos , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Nanopartículas/química , Tamanho da Partícula , Polietilenoimina , Piridinas , Ratos , Nervo Isquiático/lesões , Nervo Isquiático/patologia , Neuropatia Ciática
17.
Sci Rep ; 10(1): 3375, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32099056

RESUMO

Optic neuropathy is a major cause of irreversible blindness worldwide, and no effective treatment is currently available. Secondary degeneration is believed to be the major contributor to retinal ganglion cell (RGC) death, the endpoint of optic neuropathy. Partial optic nerve transection (pONT) is an established model of optic neuropathy. Although the mechanisms of primary and secondary degeneration have been delineated in this model, until now how this is influenced by therapy is not well-understood. In this article, we describe a clinically translatable topical, neuroprotective treatment (recombinant human nerve growth factor, rh-NGF) predominantly targeting secondary degeneration in a pONT rat model. Topical application of rh-NGF twice daily for 3 weeks significantly improves RGC survival as shown by reduced RGC apoptosis in vivo and increased RGC population in the inferior retina, which is predominantly affected in this model by secondary degeneration. Topical rh-NGF also promotes greater axonal survival and inhibits astrocyte activity in the optic nerve. Collectively, these results suggest that topical rh-NGF exhibits neuroprotective effects on retinal neurons via influencing secondary degeneration process. As topical rh-NGF is already involved in early clinical trials, this highlights its potential in multiple indications in patients, including those affected by glaucomatous optic neuropathy.


Assuntos
Apoptose/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Fármacos Neuroprotetores/farmacologia , Administração Tópica , Animais , Axônios/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Esquema de Medicação , Humanos , Masculino , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Traumatismos do Nervo Óptico/tratamento farmacológico , Traumatismos do Nervo Óptico/patologia , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Retina/metabolismo , Retina/patologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo
18.
Muscle Nerve ; 61(5): 662-670, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32083755

RESUMO

INTRODUCTION: We investigated the mechanisms underlying immobilization-induced muscle pain in rats. METHODS: In rat skeletal muscle, pressure pain threshold (PPT) of the gastrocnemius muscle was measured, and nerve growth factor (NGF) level, peripheral nerve fiber density, macrophage number, and interleukin-1ß (IL-1ß) mRNA expression were examined. An NGF receptor inhibitor was injected intramuscularly to assess the relationship between PPT and NGF levels. RESULTS: Immobilization resulted in a decrease in PPT and increases in NGF level, C-fiber density, M1 macrophage number, and IL-1ß mRNA expression. Injection of NGF receptor inhibitor reversed the decrease in PPT. DISCUSSION: NGF upregulation may be a major contributor to immobilization-induced muscle pain. The increases in C-fiber density, M1 macrophage number, and IL-1ß mRNA expression may be related to immobilization-induced muscle pain.


Assuntos
Hiperalgesia/metabolismo , Imobilização , Interleucina-1beta/genética , Macrófagos/patologia , Músculo Esquelético/metabolismo , Fator de Crescimento Neural/metabolismo , Limiar da Dor/fisiologia , RNA Mensageiro/metabolismo , Animais , Carbazóis/farmacologia , Moldes Cirúrgicos , Inibidores Enzimáticos/farmacologia , Membro Posterior , Hiperalgesia/patologia , Hiperalgesia/fisiopatologia , Alcaloides Indólicos/farmacologia , Masculino , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Fibras Nervosas/patologia , Fibras Nervosas Amielínicas/patologia , Limiar da Dor/efeitos dos fármacos , Pressão , Distribuição Aleatória , Ratos , Ratos Wistar , Receptor trkA/antagonistas & inibidores
19.
Sci Rep ; 10(1): 2768, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066806

RESUMO

Postoperative cognitive dysfunction (POCD) is a major complication affecting patients of any age undergoing surgery. This syndrome impacts everyday life up to months after hospital discharge, and its pathophysiology still remains unclear. Translational research focusing on POCD is based on a wide variety of rodent models, such as the murine tibial fracture, whose severity can limit mouse locomotion and proper behavioral assessment. Besides, influence of skeletal muscle injury, a lesion encountered in a wide range of surgeries, has not been explored in POCD occurrence. We propose a physical model of muscle injury in CX3CR1GFP/+ mice (displaying green fluorescent microglial cells) to study POCD, with morphological, behavioral and molecular approaches. We highlighted: alteration of short- and long-term memory after muscle regeneration, wide microglial reactivity in the brain, including hippocampus area, 24 hours after muscle injury, and an alteration of central brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) balance, 28 days after muscle injury. Our results suggest for the first time that muscle injury can have early as well as late impacts on the brain. Our CX3CR1GFP/+ model can also facilitate microglial investigation, more specifically their pivotal role in neuroinflammation and synaptic plasticity, in the pathophysiology of POCD.


Assuntos
Encéfalo/cirurgia , Músculo Esquelético/cirurgia , Complicações Cognitivas Pós-Operatórias/patologia , Complicações Pós-Operatórias/metabolismo , Envelhecimento/patologia , Animais , Encéfalo/patologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Hipocampo/lesões , Hipocampo/patologia , Hipocampo/cirurgia , Humanos , Masculino , Camundongos , Microglia/patologia , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Fator de Crescimento Neural/metabolismo , Complicações Cognitivas Pós-Operatórias/etiologia , Complicações Cognitivas Pós-Operatórias/metabolismo , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/patologia
20.
Sci Rep ; 10(1): 1718, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015355

RESUMO

To determine the association of opening the paravesical space in relation to its occurrence of de novo SUI in an animal model. Thirty five female Sprague Dawley rats were divided into 5 groups of 7 rats each: Control group, Sham groups(F, H), and Study groups(MF, MH). Groups labeled with "F" had the paravesical space opened, "H" had tissue dissection with no opening of the space, and "M" had mesh implanted inside the vaginal wall. Urodynamic studies, immunohistochemical analysis, and western blot were done at day 40. The mean weight and age of 35 rats were 302.1 ± 25.1 grams and 12.8 ± 1.2 weeks old. No significant differences were noted among the control, Sham F, Sham H, Study MF, and Study MH on the voiding pressure and voided volume. The Sham F and Study MF (opened paravesical space) groups had significantly lower values on leak point pressures (LPP) (p = 0.026; p < 0.001) and shorter voiding intervals (p = 0.032; p = 0.005) when compared to other groups. Immunohistochemical analysis showed IL-1 and TNF-α to be intensely increased for the Study MF group (p = 0.003; p = <0.001). MMP-2 and CD 31 markers were also significantly higher in the Study MH and MF group. NGF expression was significantly increased in the Study MF and Sham F groups. Thus, opening of the paravesical space causes an increased inflammatory reaction, which leads to tissue destruction and lower urinary tract dysfunction, exemplified in the study with low leak point pressure and shortened voiding intervals.


Assuntos
Inflamação/imunologia , Sintomas do Trato Urinário Inferior/imunologia , Prolapso de Órgão Pélvico/cirurgia , Pelve/anatomia & histologia , Uretra/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Interleucina-1/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Fator de Crescimento Neural/metabolismo , Pelve/cirurgia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo , Uretra/patologia , Urodinâmica , Procedimentos Cirúrgicos Urogenitais
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