RESUMO
ProBDNF is the precursor protein of brain-derived neurotrophic factor (BDNF) expressed in the central nervous system and peripheral tissues. Previous studies showed that the blood levels of both proBDNF and p75 neurotrophic receptors (p75NTR) in major depressive disorder (MDD) were increased, but which blood cell types express proBDNF and its receptors is not known. Furthermore, the relationship between proBDNF/p75NTR and inflammatory cytokines in peripheral blood of MDD is unclear. Peripheral blood mononuclear cells (PBMCs) and serum were obtained from depressive patients (n = 32) and normal donors (n = 20). We examined the expression of proBDNF and inflammatory markers and their correlative relationship in patients with major depression. Using flow cytometry analysis, we examined which blood cells express proBDNF and its receptors. Finally, the role of proBDNF/p75NTR signal in inflammatory immune activity of PBMCs was verified in vitro experiments. Inflammatory cytokines in PBMC from MDD patients were increased and correlated with the major depression scores. The levels of IL-1ß and IL-10 were also positively correlated with the major depression scores, while the levels of TNF-α and IL-6 were negatively correlated with the major depression scores. Intriguingly, the levels of sortilin were positively correlated with IL-1ß. Q-PCR and Western blots showed proBDNF, p75NTR, and sortilin levels were significantly increased in PBMCs from MDD patients compared with that from the normal donors. Flow cytometry studies showed that proBDNF and p75NTR were present mainly in CD4+ and CD8+ T cells. The number of proBDNF and p75NTR positive CD4+ and CD8+ T cells from MDD patients was increased and subsequently reversed after therapeutic management. Exogenous proBDNF protein or p75ECD-Fc treatment of cultured PBMC affected the release of inflammatory cytokines in vitro. ProBDNF promoted the expression of inflammatory cytokines, while p75ECD-Fc inhibited the expression of inflammatory cytokines. Given there was an inflammatory response of lymphocytes to proBDNF, it is suggested that proBDNF/p75NTR signaling may upstream inflammatory cytokines in MDD. Our data suggest that proBDNF/p75NTR signaling may not only serve as biomarkers but also may be a potential therapeutic target for MDD.
Assuntos
Transtorno Depressivo Maior , Humanos , Transtorno Depressivo Maior/metabolismo , Leucócitos Mononucleares/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Regulação para Cima , Linfócitos T CD8-Positivos/metabolismo , Depressão , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Citocinas/metabolismoRESUMO
P75 pan-neurotrophin receptor (p75NTR) is an important receptor for the role of neurotrophins in survival and death of neurons during development and after nerve injury. Our previous research found that the precursor of brain-derived neurotrophic factor (proBDNF) regulates pain as an inflammatory mediator. The current understanding of the role of proBDNF/p75NTR signaling pathway in inflammatory arthritis pain and rheumatoid arthritis (RA) is unclear. We recruited 20 RA patients, 20 healthy donors (HDs), and 10 osteoarthritis (OA) patients. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) of proBDNF and p75NTR in synovial membrane were performed and evaluated. We next examined the mRNA and protein expression of proBDNF/p75NTR signaling pathway in peripheral blood mononuclear cells (PBMCs) and synovial tissue. ELISA and flow cytometry were assessed between the blood of RA patients and HD. To induce RA, collagen-induced arthritis (CIA) were induced in mice. We found over-synovitis of RA synovial membrane compared to OA controls in histologic sections. P75NTR and sortilin mRNA, and proBDNF protein level were significantly increased in PBMCs of RA patients compared with the HD. Consistently, ELISA showed that p75NTR, sortilin, tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and interleukin-10 (IL-10) levels in the serum of RA patients were increased compared with HD and p75NTR, sortilin were positively correlated with Disease Activity Score in 28 joints (DAS28). In addition, using flow cytometry we showed that the increased levels of proBDNF and p75NTR characterized in CD4+ and CD8+ T cells of RA patients were subsequently reversed with methotrexate (MTX) treatment. Furthermore, we found pathological changes, inflammatory pain, upregulation of the mRNA and protein expression of proBDNF/p75NTR signaling pathway, and upregulation of inflammatory cytokines in spinal cord using a well-established CIA mouse model. We showed intravenous treatment of recombinant p75ECD-Fc that biologically blocked all inflammatory responses and relieved inflammatory pain of animals with CIA. Our findings showed the involvement of proBDNF/p75NTR pathway in the RA inflammatory response and how blocking it with p75ECD-Fc may be a promising therapeutic treatment for RA.
Assuntos
Artrite Reumatoide/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Interleucinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Animais , Feminino , Humanos , Interleucinas/sangue , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Precursores de Proteínas/metabolismo , Membrana Sinovial/metabolismo , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/sangueRESUMO
Three Orai (Orai1, Orai2, and Orai3) and two stromal interaction molecule (STIM1 and STIM2) mammalian protein homologues constitute major components of the store-operated Ca2+ entry mechanism. When co-expressed with STIM1, Orai1, Orai2 and Orai3 form highly selective Ca2+ channels with properties of Ca2+ release-activated Ca2+ (CRAC) channels. Despite the high level of homology between Orai proteins, CRAC channels formed by different Orai isoforms have distinctive properties, particularly with regards to Ca2+ -dependent inactivation, inhibition/potentiation by 2-aminoethyl diphenylborinate and sensitivity to reactive oxygen species. This study characterises and compares the regulation of Orai1, Orai2- and Orai3-mediated CRAC current (ICRAC ) by intracellular pH (pHi ). Using whole-cell patch clamping of HEK293T cells heterologously expressing Orai and STIM1, we show that ICRAC formed by each Orai homologue has a unique sensitivity to changes in pHi . Orai1-mediated ICRAC exhibits a strong dependence on pHi of both current amplitude and the kinetics of Ca2+ -dependent inactivation. In contrast, Orai2 amplitude, but not kinetics, depends on pHi , whereas Orai3 shows no dependence on pHi at all. Investigation of different Orai1-Orai3 chimeras suggests that pHi dependence of Orai1 resides in both the N-terminus and intracellular loop 2, and may also involve pH-dependent interactions with STIM1. KEY POINTS: It has been shown previously that Orai1/stromal interaction molecule 1 (STIM1)-mediated Ca2+ release-activated Ca2+ current (ICRAC ) is inhibited by intracellular acidification and potentiated by intracellular alkalinisation. The present study reveals that CRAC channels formed by each of the Orai homologues Orai1, Orai2 and Orai3 has a unique sensitivity to changes in intracellular pH (pHi ). The amplitude of Orai2 current is affected by the changes in pHi similarly to the amplitude of Orai1. However, unlike Orai1, fast Ca2+ -dependent inactivation of Orai2 is unaffected by acidic pHi . In contrast to both Orai1 and Orai2, Orai3 is not sensitive to pHi changes. Domain swapping between Orai1 and Orai3 identified the N-terminus and intracellular loop 2 as the molecular structures responsible for Orai1 regulation by pHi . Reduction of ICRAC dependence on pHi seen in a STIM1-independent Orai1 mutant suggested that some parts of STIM1 are also involved in ICRAC modulation by pHi .
Assuntos
Canais de Cálcio , Canais de Cálcio Ativados pela Liberação de Cálcio , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Proteína ORAI1/genética , Proteína ORAI2/metabolismo , Molécula 1 de Interação Estromal/metabolismoRESUMO
We investigated the potential of gelatin microspheres (GMs) loaded with platelet-rich plasma (PRP) to enhance their wound healing effect. Platelets from the PRP were immobilized onto GMs to form biomimetic bioreactor GM+PRP. The therapeutic effect of this agent was further investigated in vivo on a wound-healing model in rats. Wounds were locally injected with phosphate buffered saline (PBS), GM, PRP, and GM+PRP. Wound healing rate, vessel density, and inflammation level were measured histologically, by RT-PCR, and by Western blotting at days 3, 7, 14, and 21. Platelets on GM caused a continuous high release in both interleukin-10 and metalloproteinase-3 compared with PRP alone. Both GM+PRP and PRP successfully accelerated the wound healing process, while GM alone did not improve the wound healing process compared with the untreated control. Wounds treated with GM+PRP resulted in shorter healing period and improved dermal structure. GM+PRP improved angiogenesis in the wound by increasing expression of angiogenic factors. GM+PRP prolonged and enhanced the cytokine release profile compared with PRP. By promoting the inflammatory and angiogenic responses, GM+PRP has the potential to improve wound healing. Our findings demonstrate that GMs are an injectable carrier that enhanced the therapeutic effects of PRP.
Assuntos
Portadores de Fármacos/química , Microesferas , Plasma Rico em Plaquetas , Pele/lesões , Cicatrização/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Gelatina/química , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Injeções Subcutâneas , Interleucina-10/metabolismo , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Ratos , Pele/efeitos dos fármacos , Pele/imunologia , Pele/metabolismoRESUMO
Neural cell transplantation is an effective way for treatment of neurological diseases. However, the absence of transplantable human neurons remains a barrier for clinical therapies. Human urine-derived cells, namely renal cells and urine stem cells, have become a good source of cells for reprogramming or trans-differentiation research. Here, we show that human urine-derived cells can be partially converted into neuron-like cells by applying a cocktail of small molecules. Gene expression analysis has shown that these induced cells expressed some neuron-specific genes, and a proportion of the cells are GABAergic neurons. Moreover, whole-cell patch clamping recording has shown that some induced cells have neuron-specific voltage gated Na+ and K+ currents but have failed to generate Ca2+ currents and action potentials. Taken together, these results suggest that induced neuronal cells from human urine-derived cells may be useful for neurological disease modelling, drug screening and cell therapies.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Neurais/citologia , Neurônios/metabolismo , Urina/citologia , Potenciais de Ação/efeitos dos fármacos , Adulto , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Pessoa de Meia-Idade , Células-Tronco Neurais/metabolismo , Neurônios/patologia , Técnicas de Patch-ClampRESUMO
Transient Receptor Potential Melastatin (TRPM) 2 is a non-selective Ca2+ permeable cation channel and a member of the Transient Receptor Potential (TRP) channel family. TRPM2 has unique gating properties; it is activated by intracellular ADP-ribose (ADPR), whereas Ca2+ plays a role of an important co-factor in channel activation, increasing TRPM2 sensitivity to ADPR. TRPM2 is highly expressed in rat and mouse hepatocytes, where it has been shown to contribute to oxidative stress-induced cell death and liver damage due to paracetamol-overdose. The mechanisms regulating the activity of TRPM2 channels in hepatocytes, however, are not well understood. In this paper, we investigate the localisation of TRPM2 protein in hepatocytes. The presented results demonstrate that in rat hepatocytes under normal conditions, most of the TRPM2 protein is localised intracellularly. This was determined by confocal microscopy using TRPM2-and plasma membrane (PM)-specific antibodies and immunofluorescence, and biotinylation studies followed by western blotting. Interestingly, in hepatocytes treated with either H2O2 or paracetamol, the amount of TRPM2 co-localised with PM is significantly increased, compared to the untreated cells. It is concluded that trafficking of TRPM2 to the PM could potentially contribute to a positive feedback mechanism mediating Ca2+ overload in hepatocytes under conditions of oxidative stress.
Assuntos
Membrana Celular/metabolismo , Hepatócitos/metabolismo , Estresse Oxidativo , Canais de Cátion TRPM/metabolismo , Acetaminofen/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Hepatócitos/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Masculino , Ratos , Ratos WistarRESUMO
The release of Ca(2+) from the endoplasmic reticulum (ER) and subsequent replenishment of ER Ca(2+) by Ca(2+) entry through store-operated Ca(2+) channels (SOCE) play critical roles in the regulation of liver metabolism by adrenaline, glucagon and other hormones. Both ER Ca(2+) release and Ca(2+) entry are severely inhibited in steatotic hepatocytes. Exendin-4, a slowly-metabolised glucagon-like peptide-1 (GLP-1) analogue, is known to reduce liver glucose output and liver lipid, but the mechanisms involved are not well understood. The aim of this study was to determine whether exendin-4 alters intracellular Ca(2+) homeostasis in steatotic hepatocytes, and to evaluate the mechanisms involved. Exendin-4 completely reversed lipid-induced inhibition of SOCE in steatotic liver cells, but did not reverse lipid-induced inhibition of ER Ca(2+) release. The action of exendin-4 on Ca(2+) entry was rapid in onset and was mimicked by GLP-1 or dibutyryl cyclic AMP. In steatotic liver cells, exendin-4 caused a rapid decrease in lipid (half time 6.5min), inhibited the accumulation of lipid in liver cells incubated in the presence of palmitate plus the SOCE inhibitor BTP-2, and enhanced the formation of cyclic AMP. Hormone-stimulated accumulation of extracellular glucose in glycogen replete steatotic liver cells was inhibited compared to that in non-steatotic cells, and this effect of lipid was reversed by exendin-4. It is concluded that, in steatotic hepatocytes, exendin-4 reverses the lipid-induced inhibition of SOCE leading to restoration of hormone-regulated cytoplasmic Ca(2+) signalling. The mechanism may involve GLP-1 receptors, cyclic AMP, lipolysis, decreased diacylglycerol and decreased activity of protein kinase C.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Fígado Gorduroso/patologia , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Hepatócitos/metabolismo , Espaço Intracelular/metabolismo , Peptídeos/farmacologia , Peçonhas/farmacologia , Animais , Bucladesina/farmacologia , Cálcio/farmacologia , AMP Cíclico/metabolismo , Exenatida , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hormônios/farmacologia , Espaço Intracelular/efeitos dos fármacos , Ácido Palmítico/farmacologia , Ratos ZuckerRESUMO
Bone cancer pain is a common symptom in cancer patients with bone metastases and the underlying mechanisms are largely unknown. The aim of this study is to explore the endogenous analgesic mechanisms to develop new therapeutic strategies for bone-cancer induced pain (BCIP) as a result of metastases. MRMT-1 tumor cells were injected into bilateral tibia of rats and X-rays showed that the area suffered from bone destruction, accompanied by an increase in osteoclast numbers. In addition, rats with bone cancer showed apparent mechanical and thermal hyperalgesia at day 28 after intratibial MRMT-1 inoculation. However, intrathecal injection of morphine or lentivirus-mediated glial cell line-derived neurotrophic factor RNAi (Lvs-siGDNF) significantly attenuated mechanical and thermal hyperalgesia, as shown by increases in paw withdrawal thresholds and tail-flick latencies, respectively. Furthermore, Lvs-siGDNF interference not only substantially downregulated GDNF protein levels, but also reduced substance P immunoreactivity and downregulated the ratio of pERK/ERK, where its activation is crucial for pain signaling, in the spinal dorsal horn of this model of bone-cancer induced pain. In this study, Lvs-siGDNF gene therapy appeared to be a beneficial method for the treatment of bone cancer pain. As the effect of Lvs-siGDNF to relieve pain was similar to morphine, but it is not a narcotic, the use of GDNF RNA interference may be considered as a new therapeutic strategy for the treatment of bone cancer pain in the future.
Assuntos
Analgesia/métodos , Neoplasias Ósseas/complicações , Neoplasias Ósseas/secundário , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Manejo da Dor/métodos , Dor/etiologia , Interferência de RNA , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Hiperalgesia/terapia , Injeções Espinhais , Lentivirus , Morfina/uso terapêutico , Neuroglia/metabolismo , RNA Interferente Pequeno/genética , RatosRESUMO
Chronic stress causes a variety of psychiatric disorders such as anxiety and depression, but its mechanism is not well understood. Tripartite motif-containing protein 32 (TRIM32) was strongly associated with autism spectrum disorder, attention deficit hyperactivity disorder, anxiety and obsessive compulsive disorder based on a study of copy number variation, and deletion of TRIM32 increased neural proliferation and reduced apoptosis. Here, we propose that TRIM32 is involved in chronic stress-induced affective behaviors. Using a chronic unpredictable mild stress mouse depression model, we studied expression of TRIM32 in brain tissue samples and observed behavioral changes in Trim32 knockout mice. The results showed that TRIM32 protein but not its mRNA was significantly reduced in hippocampus in a time-dependent manner within 8 weeks of chronic stress. These stress-induced affective behaviors and reduction of TRIM32 protein expression were significantly reversed by antidepressant fluoxetine treatment. In addition, Trim32 knockout mice showed reduced anxiety and depressive behaviors and hyperactivities compared with Trim32 wild-type mice under normal and mild stress conditions. We conclude that TRIM32 plays important roles in regulation of hyperactivities and positively regulates the development of anxiety and depression disorders induced by chronic stress.
Assuntos
Ansiedade/metabolismo , Depressão/metabolismo , Hipocampo/metabolismo , Estresse Psicológico/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Ubiquitina-Proteína Ligases/genéticaRESUMO
Studies have shown that mutations in the matrilin-3 gene (MATN3) are associated with multiple epiphyseal dysplasia (MED) and spondyloepimetaphyseal dysplasia (SEMD). We tested whether MATN3 mutations affect the differentiation of chondroprogenitor and/or mesenchymal stem cells, which are precursors to chondrocytes. ATDC5 chondroprogenitors stably expressing wild-type (WT) MATN3 underwent spontaneous chondrogenesis. Expression of chondrogenic markers collagen II and aggrecan was inhibited in chondroprogenitors carrying the MED or SEMD MATN3 mutations. Hypertrophic marker collagen X remained attenuated in WT MATN3 chondroprogenitors, whereas its expression was elevated in chondroprogenitors expressing the MED or SEMD mutant MATN3 gene suggesting that these mutations inhibit chondrogenesis but promote hypertrophy. TGF-ß treatment failed to rescue chondrogenesis markers but dramatically increased collagen X mRNA expression in mutant MATN3 expressing chondroprogenitors. Synovium derived mesenchymal stem cells harboring the SEMD mutation exhibited lower glycosaminoglycan content than those of WT MATN3 in response to TGF-ß. Our results suggest that the properties of progenitor cells harboring MATN3 chondrodysplasia mutations were altered, as evidenced by attenuated chondrogenesis and premature hypertrophy. TGF-ß treatment failed to completely rescue chondrogenesis but instead induced hypertrophy in mutant MATN3 chondroprogenitors. Our data suggest that chondroprogenitor cells should be considered as a potential target of chondrodysplasia therapy.
Assuntos
Condrogênese/efeitos dos fármacos , Proteínas Matrilinas/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Western Blotting , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Proteínas Matrilinas/genética , Camundongos , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
CDK4/6 inhibitors have become game-changers in the treatment of estrogen receptor-positive (ER+) breast cancer, and in combination with endocrine therapy are the standard of care first-line treatment for ER+/HER2-negative advanced breast cancer. Although CDK4/6 inhibitors prolong survival for these patients, resistance is inevitable and there is currently no clear standard next-line treatment. There is an urgent unmet need to dissect the mechanisms which drive intrinsic and acquired resistance to CDK4/6 inhibitors and endocrine therapy to guide the subsequent therapeutic decisions. We will review the insights gained from preclinical studies and clinical cohorts into the diverse mechanisms of CDK4/6 inhibitor action and resistance, and highlight potential therapeutic strategies in the context of CDK4/6 inhibitor resistance.
RESUMO
Several endocrine therapies are currently available for the treatment of estrogen receptor (ER) positive breast cancer, but the clinical benefit of these agents is limited by endocrine therapy drug resistance. A common mechanism of endocrine therapy resistance is ESR1 mutations. The first-generation selective estrogen receptor degrader (SERD) fulvestrant has activity against ESR1 mutant tumors but requires intramuscular injection and has poor bioavailability that precludes optimal drug dosing. This led to the development of second-generation SERDs which are potent and have improved oral bioavailability and pharmacokinetics. Several of these oral SERDs are now in phase III trials in both the early and advanced ER positive breast cancer settings. This review summarizes the background of oral SERD development, the current status and future perspectives.
Assuntos
Neoplasias da Mama , Neoplasias Bucais , Mama , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Receptores de Estrogênio , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Moduladores Seletivos de Receptor Estrogênico/uso terapêuticoRESUMO
Polysaccharides have been used widely in many industries, from food technology and mining to cosmetics and biomedical applications. Over recent years there has been growing interest in the development of responsive polysaccharides with unique and switchable properties, particularly systems that display lower-critical solution temperatures (LCSTs). Therefore, in this study we aimed to investigate a novel strategy that would allow the conversion of non-responsive polysaccharides into thermoresponsive polysaccharides with tuneable LCSTs. Through the functionalisation of dextran with alkylamide groups (isopropyl amide, diethyl amide, piperidinyl and diisobutyl amide) using a carbodiimide coupling approach in conjunction with amic acid derivatives, we prepared a library of novel dextrans with various degrees of substitution (DS), which were characterised via nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC). The alkylamide-functionalised dextrans were found to have good solubility in aqueous solutions, with the exception of those having a high DS of large hydrophobic substituents. Determination of the thermoresponsive characteristics of the polymer solutions via UV-vis spectroscopy revealed that the LCST of the alkylamide-functionalised dextrans was highly dependent on the type of alkylamide group and the DS and could be tuned over a large range (5-35 °C). Above the LCST, all of the thermoresponsive alkylamide-functionalised dextrans formed colloidal dispersions with particles sizes ranging from 400 -600 nm, as determined by dynamic light scattering (DLS). In addition, the polymers were found to exhibit a fast and reversible phase transition in solution with narrow hysteresis (â¼ 1-5 °C). Finally, the injectability and biocompatibility of the novel thermoresponsive dextrans was confirmed in vivo via subcutaneous and intracranial ventricle injections, with no local or systemic toxicity noted over a 14 d period. Overall, the alkylamide-functionalised dextrans display interesting thermoresponsive properties and trends that may make them useful in biomedical applications, such as drug-delivery.
Assuntos
Polissacarídeos/química , Alquilação , Amidas/química , Animais , Materiais Biocompatíveis/administração & dosagem , Materiais Biocompatíveis/química , Materiais Biocompatíveis/toxicidade , Cromatografia em Gel , Dextranos/administração & dosagem , Dextranos/química , Dextranos/toxicidade , Difusão Dinâmica da Luz , Teste de Materiais , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Peso Molecular , Ressonância Magnética Nuclear Biomolecular , Tamanho da Partícula , Transição de Fase , Polissacarídeos/administração & dosagem , Polissacarídeos/toxicidade , Espectrofotometria , TemperaturaRESUMO
Alzheimer's disease (AD) is the most common cause of dementia among elderly people. Majority of AD cases are sporadic (SAD) with unknown cause. Transgenic animal models closely reflect the familial (genetic) aspect of the disease but not the sporadic type. However, most new drug candidates which are tested positive in transgenic animal models failed in clinical studies so far. Herein, we aim to develop an AD animal model that combines most of the neuropathological features seen in sporadic AD in humans with amyloid plaques observed in transgenic mice. Four-month-old wild-type and APP/PS1 AD mice were given a single intracerebroventricular (ICV) injection of 3 mg/kg streptozotocin (STZ), a diabetogenic agent. Three weeks later, their cognitive behavior was assessed, and their brain tissues were collected for biochemical and histological analysis. STZ produced cognitive deficits in both non-transgenic mice and AD mice. Biochemical analysis showed a severe decline in synaptic proteins, increase in tau phosphorylation, oxidative stress, disturbed brain insulin signaling with extensive neuroinflammation, and cell death. Significant increase was also observed in the level of the soluble beta amyloid precursor protein (APP) fragments and robust accumulation of amyloid plaques in AD mice compared to the control. These results suggest that STZ ICV treatment causes disturbance in multiple metabolic and cell signaling pathways in the brain that facilitated amyloid plaque accumulation and tau phosphorylation. Therefore, this animal model can be used to evaluate new AD therapeutic agents for clinical translation.
Assuntos
Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Presenilina-1/genética , Estreptozocina/toxicidade , Doença de Alzheimer/patologia , Animais , Feminino , Humanos , Injeções Intraventriculares , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estreptozocina/administração & dosagemRESUMO
PURPOSE: Excitatory amino acid transporters (EAATs) have an indispensable function in the reuptake of extracellular glutamate. To investigate the relationship and the expression of neuronal and astrocytic markers after brain ischemia, the temporal profile of glial EAATs in both peripheral and core regions of the cortex was examined. METHODS: Transient common carotid artery occlusion was used to induce unilateral transient forebrain ischemia of Mongolian gerbils, and post-ischemic brains (6 h to 2 w) were collected and prepared for immunohistochemical and Western blotting analysis of glutamine synthetase (GS), GLT-1, GLAST, S100ß, and NeuN, and for Alizarin red staining of calcium deposits. RESULTS: The expression of GLAST and GLT-1 were significantly escalated at 6 h both in the core and periphery regions, while reduced from 12 h to 2 w in the core region post-ischemia. GS-positive cells increased at 6 h both in the core and periphery regions, while the density of Alizarin red-positive cells increased and peaked at 12 h in the ischemic cortex. The density of S100ß-positive cells decreased in the ischemic core and increased in the periphery region. Immunofluorescence staining showed that S100ß and TUNEL double-positive cells increased at 12 h in the core region. CONCLUSION: The results of GLT-1 and GLAST expression in the cortex indicate that their up-regulation was time-dependent and occurred in the acute post-ischemia period, whereas their down-regulation was region-dependent and it is involved in the pathological progress of nerve cell and glial cell death, and has a series of cascade reactions.
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PURPOSE: Parkinson's disease (PD) is the second most common neurodegenerative disease. The α-Synuclein is a major component of Lewy bodies and Lewy neurites, the pathologic hallmark of PD. It is known that α-Synuclein is phosphorylated (p-α-Synuclein) in PD and tau-hyperphosphorylation (p-Tau) is also a pathologic feature of PD. However, the relationship between p-Synuclein and p-Tau in PD is not clear, in particular in the MPTP model of PD. The purpose of this study was to reveal their relationship in the mouse MPTP model. METHODS: Firstly, the p-α-Synuclein, α-Synuclein, p-Tau and Tau protein levels were analyzed. Then, GSK3ß activation was determined using immunoblot and immunohistochemical staining. Finally, the dopaminergic neurodegeneration was assessed using Tyrosine Hydroxylase (TH) staining and retrograde labeling and microglial marker were labeled. Microglial activation and nigrostriatal pathway degeneration were observed. RESULTS: The results showed that p-α-Synuclein, α-Synuclein, p-Tau and Tau were upregulated in both hippocampus and substantia nigra of the PD mouse model. Furthermore, p-α-Synuclein and p-Tau were localized in the same regions of substantial nigra (SN) and dentate gyrus (DG) of hippocampus (Hippo). The activated form of GSK3ß (phosphor GSK3ß Y216) was increased in multiple brain areas. The GSK3ß inhibitor AZD1080 injected in MPTP mice suppressed the expression of p-Tau and p-GSK3ß and improved motor functions. CONCLUSION: These findings revealed that p-α-Synuclein and p-Tau proteins are key pathological events leading to neurodegeneration and motor dysfunctions in the mouse MPTP model of PD. Our data suggest that the interference with the GSK3ß activity may be an effective approach for the treatment of PD.
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The cellular and molecular mechanisms via which MK2206, an AKT inhibitor, prevents the activation of AKT in toluene diisocyanate (TDI)induced asthma remain unclear. Thus, the present study aimed to evaluate the potential effects of MK2206 on airway AKT activation, inflammation and remodeling in a TDIinduced mouse model of asthma. A total of 24 BALB/c mice were selected and randomly divided into untreated (AOO), asthma (TDI), MK2206 (TDI + MK2206), and dexamethasone (TDI + DEX) groups. Phosphorylated AKT (pAKT), total AKT, airway remodeling indices, αsmooth muscle actin (αSMA) and collagen I levels in pulmonary tissue were measured using western blotting. Airway inflammation factors, including interleukin (IL)4, 5, 6, and 13 in bronchoalveolar lavage fluid (BALF) and IgE in serum, were determined using ELISA. Additionally, the airway hyperresponsiveness (AHR) and pulmonary pathology of all groups were evaluated. The results of the present study demonstrated that pAKT levels in lung protein lysate were upregulated, and neutrophil, eosinophil and lymphocyte counts were increased in the lungs obtained from the asthma group compared with the AOO group. Both MK2206 and DEX treatment in TDIinduced mice resulted not only in the attenuation of AKT phosphorylation, but also reductions in neutrophil, eosinophil and lymphocyte counts in the lungs of mice in the asthma group. Consistently, increases in the levels of the inflammatory cytokines IL4, 5, 6 and 13 analyzed in BALF, and serum IgE in the TDI group were demonstrated to be attenuated in the TDI + MK2206 and TDI + DEX groups. Furthermore, αSMA and AHR were significantly attenuated in the TDI + MK2206 group compared with the TDI group. These results revealed that MK2206 not only inhibited AKT activation, but also served a role in downregulating airway inflammation and airway remodeling in chemicalinduced asthma. Therefore, the findings of the present study may provide important insight into further combination therapy.
Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Anti-Inflamatórios/administração & dosagem , Asma/induzido quimicamente , Asma/tratamento farmacológico , Dexametasona/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Tolueno 2,4-Di-Isocianato/efeitos adversos , Animais , Asma/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Imunoglobulina E/sangue , Inflamação/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
Colorectal cancer (CRC) is one of the most common malignancies. The current treatments of metastatic colorectal cancer (mCRC) are ineffective and the bottleneck problem. It is of significance to explore effective new therapeutic strategies to eradicate mCRC. Photothermal therapy (PTT) is an emerging technology for tumor therapy, with the potential in the treatment of mCRC. In this review, the current treatment approaches to mCRC including surgery, radiotherapy, chemotherapy interventional therapy, biotherapy, and photothermal therapy are reviewed. In addition, we will focus on the various kinds of nanomaterials used in PTT for the treatment of CRC both in vitro and in vivo models. In conclusion, we will summarize the combined application of PTT with other theranostic methods, and propose future research directions of PTT in the treatment of CRC.
RESUMO
While it has been a great challenge to determine the positive status of metastasis lesions, intraoperative tumor imaging, which can show tumor localization and facilitate intraoperative staging of nodal metastases, have enabled surgeons to quickly and accurately perform radical resections. However, to date, there is no accurate method for evaluating nodal status intraoperatively. In this study, we synthesized activatable cell-penetrating peptides (ACPPs) that can specifically recognize colorectal cancer and their nodal status. ACPPs were labeled with Cy5 dye at the C-terminal, and named ACPP-Cy5. Laser scanning confocal microscopy and flow cytometry were used to measure the change in intracellular fluorescence intensity between cancer cells and normal cells. The results showed while the intracellular Cy5 fluorescent intensity can be visualized in both cancer and normal cells by 8 h after adding ACPP-Cy5, the relative fluorescence intensity of colorectal cancer cells was significantly higher than the normal cells. In addition, IVIS spectrum in vivo imaging system was used to observe the fluorescence intensity of ACPP-Cy5 after tail vein injection of mice with subcutaneous tumor or orthotopic colorectal cancer and liver metastasis. We found in mice with colorectal cancer and liver metastasis the Cy5 fluorescence intensity of cancer was significantly increased compared to the organs including liver, colorectum, lung, spleen, and heart. It is demonstrated here, this ACPPs can target colorectal cancer and liver metastasis, therefore ACPP-Cy5 may be a promising tool used for the diagnoses of colorectal cancer and to assist in tumor localization during surgery.
RESUMO
AIMS: After peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves. MAIN METHODS: In p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry. KEY FINDINGS: The results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice. SIGNIFICANCE: Our data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves.