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BACKGROUND: To explore whether nobiletin has a protective effect on high-fat diet (HFD)-induced enteric nerve injury and its underlying mechanism. METHODS: An obesity model was induced by a HFD. Nobiletin (100 mg/kg and 200 mg/kg) and vehicle were administered by gastric gavage for 4 weeks. Lee's index, body weight, OGTT and intestinal propulsion assays were performed before sacrifice. After sampling, lipids were detected using Bodipy 493/503; lipid peroxidation was detected using MDA and SOD kits and the expression of PGP 9.5, Trem2, GFAP, ß-tubulin 3, Bax, Bcl2, Nestin, P75 NTR, SOX10 and EDU was detected using immunofluorescence. The GDNF, p-AKT, AKT, p-FOXO3a, FOXO3a and P21 proteins were detected using western blotting. The relative mRNA expression levels of NOS2 were detected via qPCR. Primary enteric neural stem cells (ENSCs) were cultured. After ENSCs were treated with palmitic acid (PA) and nobiletin, CCK-8 and caspase-3/7 activity assays were performed to evaluate proliferation and apoptosis. RESULTS: HFD consumption caused colon lipid accumulation and peroxidation, induced enteric nerve damage and caused intestinal motor dysfunction. However, nobiletin reduced lipid accumulation and peroxidation in the colon; promoted Trem2, ß-tubulin 3, Nestin, P75NTR, SOX10 and Bcl2 expression; inhibited Bax and GFAP expression; reduced NOS2 mRNA transcription; and regulated the GDNF/AKT/FOXO3a/P21 pathway. Nobiletin also promoted PA-induced impairment of ENSCs. CONCLUSIONS: Nobiletin restored HFD-induced enteric nerve injury, which may be associated with inhibiting enteric nerve apoptosis, promoting enteric nerve survival and regulating the GDNF/AKT/FOXO3a/P21 pathway.
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Dieta Alta en Grasa , Sistema Nervioso Entérico , Flavonas , Proteína Forkhead Box O3 , Factor Neurotrófico Derivado de la Línea Celular Glial , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Animales , Proteína Forkhead Box O3/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Dieta Alta en Grasa/efectos adversos , Transducción de Señal/efectos de los fármacos , Masculino , Flavonas/farmacología , Flavonas/uso terapéutico , Sistema Nervioso Entérico/metabolismo , Sistema Nervioso Entérico/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/efectos de los fármacos , Ratones , Modelos Animales de Enfermedad , Ratas , Obesidad/metabolismo , Obesidad/tratamiento farmacológico , Apoptosis/efectos de los fármacosRESUMEN
Amyloid-peptide (Aß) monomeric forms (ABM) occurring in presymptomatic Alzheimer's disease (AD) brain are thought to be devoid of neurotoxicity while the transition/aggregation of ABM into oligomers is determinant for Aß-induced toxicity since Aß is predominantly monomeric up to 3 µM and aggregates over this concentration. However, recent imaging and/or histopathological investigations revealed alterations of myelin in prodromal AD brain in absence of aggregated Aß oligomers, suggesting that ABM may induce toxicity in myelin-producing cells in early AD-stages. To check this hypothesis, here we studied ABM effects on the viability of the Human oligodendrocyte cell line (HOG), a reliable oligodendrocyte model producing myelin proteins. Furthermore, to mimic closely interactions between oligodendrocytes and other glial cells regulating myelination, we investigated also ABM effects on mouse brain primary mixed-glial cell cultures. Various methods were combined to show that ABM concentrations (600 nM-1 µM), extremely lower than 3 µM, significantly decreased HOG cell and mouse brain primary mixed-glial cell survival. Interestingly, flow-cytometry studies using specific cell-type markers demonstrated that oligodendrocytes represent the most vulnerable glial cell population affected by ABM toxicity. Our work also shows that the neurosteroid 3α-O-allyl-allopregnanolone BR351 (250 and 500 nM) efficiently prevented ABM-induced HOG and brain primary glial cell toxicity. Bicuculline (50-100 nM), the GABA-A-receptor antagonist, was unable to block/reduce BR351 effect against ABM-induced HOG and primary glial cell toxicity, suggesting that BR351-evoked neuroprotection of these cells may not depend on GABA-A-receptor allosterically modulated by neurosteroids. Altogether, our results suggest that further exploration of BR351 therapeutic potential may offer interesting perspectives to develop effective neuroprotective strategies.
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Péptidos beta-Amiloides , Fármacos Neuroprotectores , Oligodendroglía , Pregnanolona , Animales , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Humanos , Péptidos beta-Amiloides/toxicidad , Fármacos Neuroprotectores/farmacología , Pregnanolona/farmacología , Ratones , Línea Celular , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Ratones Endogámicos C57BL , Fragmentos de Péptidos/toxicidad , Células Cultivadas , Relación Dosis-Respuesta a DrogaRESUMEN
Vitamin D deficiency (VDD) is widespread around the world and has been extensively documented to affect various health conditions, including the cognitive functioning of the brain. Serum 25-hydroxylated forms of vitamin D are traditionally used to determine vitamin D status. However, there is now evidence that cholecalciferol activation can occur and be controlled by locally expressed enzymes in the brain. This study aimed to investigate the effects of cholecalciferol supplementation on cognitive function in rats who underwent transient VDD in adulthood. Thirty-six adult Wistar rats were administered paricalcitol (seven doses of 32 ng injected every other day) along with a "vitamin D-free" diet to induce VDD, which was confirmed using a LC-MS/MS serum analysis of the cholecalciferol and 25-hydroxyvitamin D3 levels. Treatment was performed by including 1000 IU/kg and 10,000 IU/kg cholecalciferol in the diet. Cognitive performance was evaluated using the novel object recognition (NOR), Morris water maze (MWM), and radial arm maze (RAM) tests. An immunohistochemical analysis of the brain regions involved in learning and memory was performed by quantifying the neurons, astrocytes, and microglia labelled with anti-neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba-1) antibodies, respectively. The vitamin D deficient group showed the lowest performance in both the MWM and RAM tests. In contrast, the cholecalciferol-treated groups exhibited a faster learning curve. However, no difference was detected between the groups in the NOR test. On the other hand, differences in the cellular organization of the hippocampus and amygdala were observed between the groups. Cholecalciferol supplementation decreased the density of the Iba-1- and GFAP-labeled cells in the hilus and cornu Ammonis 3 (CA3) regions of the hippocampus and in the amygdala. These results support vitamin D's substantial role in learning and memory. They also highlight that subtle changes of cognitive function induced by transient VDD could be reversed by cholecalciferol supplementation. Further studies are needed to better understand VDD and cholecalciferol's effects on the brain structure and function.
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Colecalciferol , Suplementos Dietéticos , Hipocampo , Neuroglía , Ratas Wistar , Deficiencia de Vitamina D , Animales , Colecalciferol/farmacología , Deficiencia de Vitamina D/tratamiento farmacológico , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Masculino , Ratas , Cognición/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Ergocalciferoles/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Modelos Animales de Enfermedad , Vitamina D/farmacología , Vitamina D/sangreRESUMEN
BACKGROUND: Orthodontic pain affects the physical and mental health of patients. The spinal trigeminal subnucleus caudalis (SPVC) contributes to the transmission of pain information and serves as a relay station for integrating orofacial damage information. Recently, glial cells have been found to be crucial for both acute and maintenance phases of pain. It has also been demonstrated that rho kinase (ROCK) inhibitors can manage different pain models by inhibiting glial cell activation. Here, we hypothesized that orthodontic pain is related to glial cells in the SPVC, and Fasudil, a representative rho/rock kinase inhibitor, can relieve orthodontic pain by regulating the function of glial cells and the related inflammatory factors. In this study, we constructed a rat model of tooth movement pain and used immunofluorescence staining to evaluate the activation of microglia and astrocytes. Quantitative real-time PCR was used to detect the release of related cytokines and the expression of pain-related genes in the SPVC. Simultaneously, we investigated the effect of Fasudil on the aforementioned indicators. RESULTS: In the SPVC, the expression of c-Fos peaked on day 1 along with the expression of OX42 (related to microglial activation), CD16 (a pro-inflammatory factor), and CD206 (an anti-inflammatory factor) on day 3 after tooth movement, followed by a gradual decrease. GFAP-staining showed that the number of activated astrocytes was the highest on day 5 and that cell morphology became complex. After Fasudil treatment, the expression of these proteins showed a downward trend. The mRNA levels of pro-inflammatory factors (IL-1ß and TNF-α) peaked on day 3, and the mRNA expression of the anti-inflammatory factor TGF-ß was the lowest 3 days after tooth movement. Fasudil inhibited the mRNA expression of pain-related genes encoding CSF-1, t-PA, CTSS, and BDNF. CONCLUSION: This study shows that tooth movement can cause the activation of glial cells in SPVC, and ROCK inhibitor Fasudil can inhibit the activation of glial cells and reduce the expression of the related inflammatory factors. This study presents for the first time the potential application of Fasudil in othodontic pain.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Neuroglía , Técnicas de Movimiento Dental , Animales , Técnicas de Movimiento Dental/métodos , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Ratas , Neuroglía/efectos de los fármacos , Ratas Sprague-Dawley , Masculino , Microglía/efectos de los fármacos , Núcleo Caudal del Trigémino/efectos de los fármacos , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Modelos Animales de Enfermedad , Citocinas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Astrocitos/efectos de los fármacosRESUMEN
Clostridioides difficile (C. difficile) is responsible for a spectrum of nosocomial/antibiotic-associated gastrointestinal diseases that are increasing in global incidence and mortality rates. The C. difficile pathogenesis is due to toxin A and B (TcdA/TcdB), both causing cytopathic and cytotoxic effects and inflammation. Recently, we demonstrated that TcdB induces cytopathic and cytotoxic (apoptosis and necrosis) effects in enteric glial cells (EGCs) in a dose/time-dependent manner and described the underlying signaling. Despite the role played by lipids in host processes activated by pathogens, to counter infection and/or induce cell death, to date no studies have investigated lipid changes induced by TcdB/TcdA. Here, we evaluated the modification of lipid composition in our in vitro model of TcdB infection. Apoptosis, cell cycle, cell viability, and lipidomic profiles were evaluated in EGCs treated for 24 h with two concentrations of TcdB (0.1 ng/mL; 10 ng/mL). In EGCs treated with the highest concentration of TcdB, not only an increased content of total lipids was observed, but also lipidome changes, allowing the separation of TcdB-treated cells and controls into different clusters. The statistical analyses also allowed us to ascertain which lipid classes and lipid molecular species determine the clusterization. Changes in lipid species containing inositol as polar head and plasmalogen phosphatidylethanolamine emerged as key indicators of altered lipid metabolism in TcdB-treated EGCs. These results not only provide a picture of the phospholipid profile changes but also give information regarding the lipid metabolism pathways altered by TcdB, and this might represent an important step for developing strategies against C. difficile infection.
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Proteínas Bacterianas , Toxinas Bacterianas , Neuroglía , Fosfolípidos , Neuroglía/metabolismo , Neuroglía/efectos de los fármacos , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Toxinas Bacterianas/farmacología , Fosfolípidos/metabolismo , Proteínas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Lipidómica , HumanosRESUMEN
Glutamate excitotoxicity is involved in retinal ganglion cell (RGC) death in various retinal degenerative diseases, including ischemia-reperfusion injury and glaucoma. Excitotoxic RGC death is caused by both direct damage to RGCs and indirect damage through neuroinflammation of retinal glial cells. Omidenepag (OMD), a novel E prostanoid receptor 2 (EP2) agonist, is a recently approved intraocular pressure-lowering drug. The second messenger of EP2 is cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). In this study, we investigated the neuroprotective effects of OMD on excitotoxic RGC death by focusing on differences in cAMP downstream signaling from the perspective of glia-neuron interactions. We established a glutamate excitotoxicity model in vitro and NMDA intravitreal injection model in vivo. In vitro, rat primary RGCs were used in an RGC survival rate assay. MG5 cells (mouse microglial cell line) and A1 cells (astrocyte cell line) were used for immunocytochemistry and Western blotting to evaluate the expressions of COX-1/2, PKA, Epac1/2, pCREB, cleaved caspase-3, inflammatory cytokines, and neurotrophic factors. Mouse retinal specimens underwent hematoxylin and eosin staining, flat-mounted retina examination, and immunohistochemistry. OMD significantly suppressed excitotoxic RGC death, cleaved caspase-3 expression, and activated glia both in vitro and in vivo. Moreover, it inhibited Epac1 and inflammatory cytokine expression and promoted COX-2, pCREB, and neurotrophic factor expression. OMD may have neuroprotective effects through inhibition of the Epac pathway and promotion of the COX-2-EP2-cAMP-PKA pathway by modulating glia-neuron interaction.
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Proteínas Quinasas Dependientes de AMP Cíclico , AMP Cíclico , Ciclooxigenasa 2 , Neuroglía , Fármacos Neuroprotectores , Células Ganglionares de la Retina , Animales , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Fármacos Neuroprotectores/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclooxigenasa 2/metabolismo , AMP Cíclico/metabolismo , Ratones , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP2 de Receptores de Prostaglandina E/agonistas , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Ratas Sprague-Dawley , Ratas , Ácido Glutámico/metabolismo , Ácido Glutámico/toxicidad , Ratones Endogámicos C57BL , Masculino , N-Metilaspartato/farmacología , N-Metilaspartato/toxicidad , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
Hypertension causes platelet activation and adhesion in the brain resulting in glial activation and neuroinflammation. Further, activation of Angiotensin-Converting Enzyme 2/Angiotensin (1-7)/Mas Receptor (ACE2/Ang (1-7)/MasR) axis of central Renin-Angiotensin System (RAS), is known to reduce glial activation and neuroinflammation, thereby exhibiting anti-hypertensive and anti-neuroinflammatory properties. Therefore, in the present study, the role of ACE2/Ang (1-7)/MasR axis was studied on platelet-induced glial activation and neuroinflammation using Diminazene Aceturate (DIZE), an ACE2 activator, in astrocytes and microglial cells as well as in rat model of hypertension. We found that the ACE2 activator DIZE, independently of its BP-lowering properties, efficiently prevented hypertension-induced glial activation, neuroinflammation, and platelet CD40-CD40L signaling via upregulation of ACE2/Ang (1-7)/MasR axis. Further, DIZE decreased platelet deposition in the brain by reducing the expression of adhesion molecules on the brain endothelium. Activation of ACE2 also reduced hypertension-induced endothelial dysfunction by increasing eNOS bioavailability. Interestingly, platelets isolated from hypertensive rats or activated with ADP had significantly increased sCD40L levels and induced significantly more glial activation than platelets from DIZE treated group. Therefore, injection of DIZE pre-treated ADP-activated platelets into normotensive rats strongly reduced glial activation compared to ADP-treated platelets. Moreover, CD40L-induced glial activation, CD40 expression, and NFкB-NLRP3 inflammatory signaling are reversed by DIZE. Furthermore, the beneficial effects of ACE2 activation, DIZE was found to be significantly blocked by MLN4760 (ACE2 inhibitor) as well as A779 (MasR antagonist) treatments. Hence, our study demonstrated that ACE2 activation reduced the platelet CD40-CD40L induced glial activation and neuroinflammation, hence imparted neuroprotection.
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Enzima Convertidora de Angiotensina 2 , Ligando de CD40 , Diminazeno , Modelos Animales de Enfermedad , Hipertensión , Peptidil-Dipeptidasa A , Transducción de Señal , Animales , Diminazeno/análogos & derivados , Diminazeno/farmacología , Diminazeno/uso terapéutico , Enzima Convertidora de Angiotensina 2/metabolismo , Masculino , Transducción de Señal/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Ligando de CD40/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Ratas , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Proto-Oncogenes Mas , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Fragmentos de Péptidos , Angiotensina I , Células Cultivadas , Microglía/efectos de los fármacos , Microglía/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Ratas Wistar , Sistema Renina-Angiotensina/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Antígenos CD40/metabolismo , Humanos , Activación Plaquetaria/efectos de los fármacos , Antihipertensivos/farmacología , Antihipertensivos/uso terapéuticoRESUMEN
Pro-inflammatory polarization of microglia and astrocytes results in neuroinflammation and blood-brain barrier (BBB) disruption after a primary traumatic brain injury (TBI). Herein, we demonstrate that the dual-ligand functionalized lipid nanoparticles (AM31 LNPs) were actively and specifically internalized by microglia and astrocytes via mannose receptor (MR)- and adenosine receptor (AR)-mediated endocytosis, respectively, in a mouse model of TBI. Systemic administration of AM31 LNPs carrying siRNA against p65 resulted in internalization by the glial cells in the peri-infarct region and a robust knockdown of p65 at both mRNA and protein levels in these cells, leading to significant down-regulation of key pro-inflammatory cytokines and up-regulation of key anti-inflammatory cytokines. AM31 LNP-mediated silencing of p65 ameliorated TBI-induced BBB disruption. Our data proved that AM 31 LNP is a promising vehicle for RNA therapeutics for targeting microglia and astrocytes in neural disorder.
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Barrera Hematoencefálica , Lípidos , Nanopartículas , Animales , Barrera Hematoencefálica/metabolismo , Nanopartículas/química , Ligandos , Ratones , Lípidos/química , ARN Interferente Pequeño/metabolismo , Ratones Endogámicos C57BL , Interferencia de ARN , Microglía/metabolismo , Microglía/efectos de los fármacos , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/terapia , Lesiones Traumáticas del Encéfalo/patología , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Masculino , Neuroglía/metabolismo , Neuroglía/efectos de los fármacosRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Jiawei Bai-Hu-Decoction (JWBHD), a prescription formulated with seven traditional Chinese medicinal material has demonstrated clinical efficacy in mitigating brain injury among heat stroke (HS) patients. AIM OF THE STUDY: This study aimed to evaluate the therapeutic efficacy of JWBHD on rat model of HS and to explore its therapeutic mechanisms by integrating network pharmacology and pharmacodynamic methodologies, which major components were analyzed by using UPLC-MS/MS. MATERIALS AND METHODS: The network pharmacology analysis was firstly conducted to predict the potential active ingredients and therapeutic targets of JWBHD. The anti-HS effectiveness of JWBHD was then evaluated on rats experienced HS. Rat brain tissues were harvested for a comprehensive array of experiments, including Western blot, PCR, H&E staining, Nissl staining, ELISA, transmission electron microscope, flow cytometry and immunofluorescence to validate the protective effects of JWBHD against HS-induced brain damage. Furthermore, the inhibitory effects of JWBHD on TLR4/NF-κB signal and mitophagy of glial were further verified on HS-challenged F98 cell line. Finally, the chemical compositions of the water extract of JWBHD were analyzed by using UPLC-MS/MS. RESULTS: Network pharmacology has identified fifty core targets and numerous HS-related signaling pathways as potential therapeutic targets of JWBHD. Analysis of protein-protein interaction (PPI) and GO suggests that JWBHD may suppress HS-induced inflammatory signals. In experiments conducted on HS-rats, JWBHD significantly reduced the core temperature, restored blood pressure and alleviated neurological defect. Furthermore, JWBHD downregulated the counts of white blood cells and monocytes, decreased the levels of inflammatory cytokines such as IL-1ß, IL-6 and TNF-α in peripheral blood, and suppressed the expression of TLR4 and NF-κB in the cerebral cortex of HS-rats. Besides, JWBHD inhibited the apoptosis of cortical cells and mitigated the damage to the cerebral cortex in HS group. Conversely, overactive mitophagy was observed in the cerebral cortex of HS-rats. However, JWBHD restored the mitochondrial membrane potential and downregulated expressions of mitophagic proteins including Pink1, Parkin, LC3B and Tom20. JWBHD reduced the co-localization of Pink1 and GFAP, a specific marker of astrocytes in the cerebral cortex of HS-rats. In addition, the inhibitory effect of JWBHD on TLR4/NF-κB signaling and overactive mitophagy were further confirmed in F98 cells. Finally, UPLC-MS/MS analysis showed that the main components of JWBHD include isoliquiritigenin, liquiritin, dipotassium glycyrrhizinate, ginsenoside Rb1, ginsenoside Re, etc. CONCLUSIONS: JWBHD protected rats from HS and prevented HS-induced damage in the cerebral cortex by suppressing TLR4/NF-κB signaling and mitophagy of glial.
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Medicamentos Herbarios Chinos , Golpe de Calor , Mitofagia , FN-kappa B , Neuroglía , Ratas Sprague-Dawley , Transducción de Señal , Receptor Toll-Like 4 , Animales , Receptor Toll-Like 4/metabolismo , Mitofagia/efectos de los fármacos , FN-kappa B/metabolismo , Masculino , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/química , Transducción de Señal/efectos de los fármacos , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Ratas , Golpe de Calor/tratamiento farmacológico , Golpe de Calor/complicaciones , Fármacos Neuroprotectores/farmacología , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/prevención & control , Farmacología en Red , Modelos Animales de EnfermedadRESUMEN
The brain is especially vulnerable to environmental influences during the perinatal period. While the effects of environmental factors are usually studied in isolation, it is more typical to be exposed to multiple influences during early development, necessitating study of synergistic actions on the developing brain. Both maternal infection and endocrine disrupting phthalates can decrease cell number in the medial prefrontal cortex (mPFC), a region critical for executive functioning. In the present study, groups of pregnant Long Evans rats were treated with either (1) 100 µg/kg (i.p.) lipopolysaccharide (LPS) on embryonic days 15 and 16 combined with a low-dose (1 mg/kg) phthalate mixture throughout gestation and the neonatal period, (2) LPS alone, (3) phthalates alone, or (4) neither phthalates nor LPS (control). Neurons and glial cells were stereologically quantified in the mPFC. The adult offspring previously exposed to LPS or phthalates alone had reduced mPFC neuron number in exposed males, but not females, while the combination treatment did not produce significant effects. In males, LPS alone also reduced the number of glia in the mPFC. Additionally, the combination of LPS and phthalates resulted in fewer pregnancies to term and decreased litter size. These results provide insight into how common environmental factors can interact to alter the developmental trajectory of the mPFC.
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Lipopolisacáridos , Neuronas , Ácidos Ftálicos , Corteza Prefrontal , Efectos Tardíos de la Exposición Prenatal , Ratas Long-Evans , Animales , Corteza Prefrontal/efectos de los fármacos , Femenino , Embarazo , Lipopolisacáridos/toxicidad , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/patología , Masculino , Ratas , Neuronas/efectos de los fármacos , Ácidos Ftálicos/toxicidad , Recuento de Células , Neuroglía/efectos de los fármacos , Exposición Materna/efectos adversosRESUMEN
Neural precursor cells (NPCs) that persist in the postnatal/adult subventricular zone (SVZ) express connexins that form hemichannels and gap junctions. Gap junctional communication plays a role in NPC proliferation and differentiation during development, but its relevance on postnatal age remains to be elucidated. In this work we aimed to evaluate the effect of the blockade of gap junctional communication on proliferation and cell fate of NPCs obtained from the SVZ of postnatal rats. NPCs were isolated and expanded in culture as neurospheres. Electron microscopy revealed the existence of gap junctions among neurosphere cells. Treatment of cultures with octanol, a broad-spectrum gap junction blocker, or with Gap27, a specific blocker for gap junctions formed by connexin43, produced a significant decrease in bromodeoxyuridine incorporation. Octanol treatment also exerted a dose-dependent antiproliferative effect on glioblastoma cells. To analyze possible actions on NPC fate, cells were seeded in the absence of mitogens. Treatment with octanol led to an increase in the percentage of astrocytes and oligodendrocyte precursors, whereas the percentage of neurons remained unchanged. Gap27 treatment, in contrast, did not modify the differentiation pattern of SVZ NPCs. Our results indicate that general blockade of gap junctions with octanol induces significant effects on the behavior of postnatal SVZ NPCs, by reducing proliferation and promoting glial differentiation.
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Diferenciación Celular , Proliferación Celular , Uniones Comunicantes , Células-Madre Neurales , Neuroglía , Octanoles , Animales , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Proliferación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Ratas , Octanoles/farmacología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/citología , Células Cultivadas , Ventrículos Laterales/citología , Ventrículos Laterales/metabolismo , Ventrículos Laterales/efectos de los fármacos , Conexina 43/metabolismo , Ratas Wistar , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/citología , Animales Recién Nacidos , HumanosRESUMEN
BACKGROUND: Retinopathy of Prematurity (ROP) is a proliferative retinal vascular disease occurring in the retina of premature infants and is the main cause of childhood blindness. Nowadays anti-VEGF and retinal photocoagulation are mainstream treatments for ROP, but they develop a variety of complications. Hydrogen (H2) is widely considered as a useful neuroprotective and antioxidative therapeutic method for hypoxic-ischemic disease without toxic effects. However, whether H2 provides physiological angiogenesis promotion, neovascularization suppression and glial protection in the progression of ROP is largely unknown.This study aims to investigate the effects of H2 on retinal angiogenesis, neovascularization and neuroglial dysfunction in the retinas of oxygen-induced retinopathy (OIR) mice. METHODS: In this study, mice that were seven days old and either wild-type (WT) or Nrf2-deficient (Nrf2-/-) were exposed to 75% oxygen for 5 days and then returned to normal air conditions. Different stages of hydrogen gas (H2) inhalation were administered. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. To count the number of neovascularization endothelial nuclei, routine HE staining of retinal sections was conducted. Immunohistochemistry was performed using DyLight 594 labeled GSL I-isolectin B4 (IB4), as well as primary antibodies against proliferating cell nuclear antigen (PCNA), glial fibrillary acidic protein (GFAP), and Iba-1. Western blots were used to measure the expression of NF-E2-related factor 2 (Nrf2), vascular endothelial growth factor (VEGF), Notch1, Dll4, and HIF-1α. Additionally, the expression of target genes such as NQO1, HO-1, Notch1, Hey1, Hey2, and Dll4 was measured. Human umbilical vein endothelial cells (HUVECs) treated with H2 under hypoxia were used as an in vitro model. RT-PCR was used to evaluate the mRNA expression of Nrf2, Notch/Dll4, and the target genes. The expression of reactive oxygen species (ROS) was observed using immunofluorescence staining. RESULTS: Our results indicate that 3-4% H2 does not disturb retinal physiological angiogenesis, but ameliorates vaso-obliteration and neovascularization in OIR mice. Moreover, H2 prevents the decreased density and reverses the morphologic and functional changes in retinal astrocytes caused by oxygen-induced injury. In addition, H2 inhalation reduces microglial activation, especially in the area of neovascularization in OIR mice. H2 plays a protective role in vascular regeneration by promoting Nrf2 activation and suppressing the Dll4-induced Notch signaling pathway in vivo. Also, H2 promotes the proliferation of HUVECs under hypoxia by negatively regulating the Dll4/Notch pathway and reducing ROS levels through Nrf2 pathway aligning with our findings in vivo.Moreover, the retinal oxygen-sensing mechanisms (HIF-1α/VEGF) are also involved in hydrogen-mediated retinal revascularization and neovascularization suppression. CONCLUSIONS: Collectively, our results indicate that H2 could be a promising therapeutic agent for POR treatment and that its beneficial effect in human ROP might involve the activation of the Nrf2-Notch axis as well as HIF-1α/VEGF pathways.
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Modelos Animales de Enfermedad , Hidrógeno , Neuroglía , Oxígeno , Neovascularización Retiniana , Retinopatía de la Prematuridad , Animales , Hidrógeno/farmacología , Neovascularización Retiniana/tratamiento farmacológico , Neuroglía/efectos de los fármacos , Ratones , Retinopatía de la Prematuridad/tratamiento farmacológico , Ratones Endogámicos C57BL , Retina/efectos de los fármacos , Animales Recién Nacidos , Regeneración/efectos de los fármacos , Inmunohistoquímica , Vasos Retinianos/efectos de los fármacosRESUMEN
A major Streptococcus pneumoniae pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, binding membrane cholesterol and producing permanent lytic or transient pores. During brain infections, vascular damage with variable ischemia occurs. The role of ischemia on pneumolysin's pore-forming capacity remains unknown. In acute brain slice cultures and primary cultured glia, we studied acute toxin lysis (via propidium iodide staining and LDH release) and transient pore formation (by analyzing increases in the intracellular calcium). We analyzed normal peripheral tissue glucose conditions (80 mg%), normal brain glucose levels (20 mg%), and brain hypoglycemic conditions (3 mg%), in combinations either with normoxia (8% oxygen) or hypoxia (2% oxygen). At 80 mg% glucose, hypoxia enhanced cytolysis via pneumolysin. At 20 mg% glucose, hypoxia did not affect cell lysis, but impaired calcium restoration after non-lytic pore formation. Only at 3 mg% glucose, during normoxia, did pneumolysin produce stronger lysis. In hypoglycemic (3 mg% glucose) conditions, pneumolysin caused a milder calcium increase, but restoration was missing. Microglia bound more pneumolysin than astrocytes and demonstrated generally stronger calcium elevation. Thus, our work demonstrated that the toxin pore-forming capacity in cells continuously diminishes when oxygen is reduced, overlapping with a continuously reduced ability of cells to maintain homeostasis of the calcium influx once oxygen and glucose are reduced.
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Proteínas Bacterianas , Colesterol , Glucosa , Oxígeno , Streptococcus pneumoniae , Estreptolisinas , Estreptolisinas/toxicidad , Estreptolisinas/metabolismo , Glucosa/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/toxicidad , Oxígeno/metabolismo , Colesterol/metabolismo , Streptococcus pneumoniae/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Calcio/metabolismo , Células Cultivadas , Neuroglía/efectos de los fármacos , Neuroglía/metabolismoRESUMEN
Despite antiretroviral therapy (ART), HIV-associated peripheral neuropathy remains one of the most prevalent neurologic manifestations of HIV infection. The spinal cord is an essential component of sensory pathways, but spinal cord sampling and evaluation in people with HIV has been very limited, especially in those on ART. The SIV/macaque model allows for assessment of the spinal cord at key time points throughout infection with and without ART. In this study, RNA was isolated from the spinal cord of uninfected, SIV+, and SIV + ART animals to track alterations in gene expression using global RNA-seq. Next, the SeqSeek platform was used to map changes in gene expression to specific cell types. Pathway analysis of differentially expressed genes demonstrated that highly upregulated genes in SIV-infected spinal cord aligned with interferon and viral response pathways. Additionally, this upregulated gene set significantly overlapped with those expressed in myeloid-derived cells including microglia. Downregulated genes were involved in cholesterol and collagen biosynthesis, and TGF-b regulation of extracellular matrix. In contrast, enriched pathways identified in SIV + ART animals included neurotransmitter receptors and post synaptic signaling regulators, and transmission across chemical synapses. SeqSeek analysis showed that upregulated genes were primarily expressed by neurons rather than glia. These findings indicate that pathways activated in the spinal cord of SIV + ART macaques are predominantly involved in neuronal signaling rather than proinflammatory pathways. This study provides the basis for further evaluation of mechanisms of SIV infection + ART within the spinal cord with a focus on therapeutic interventions to maintain synaptodendritic homeostasis.
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Neuroglía , Neuronas , Síndrome de Inmunodeficiencia Adquirida del Simio , Médula Espinal , Animales , Síndrome de Inmunodeficiencia Adquirida del Simio/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/virología , Neuroglía/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/virología , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/virología , Antirretrovirales/uso terapéutico , Antirretrovirales/farmacología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Macaca mulatta , Expresión Génica/efectos de los fármacos , Masculino , Regulación de la Expresión Génica/efectos de los fármacosRESUMEN
The damage to the mechanical barrier of the intestinal mucosa is the initiating factor and the core link of the progression of ulcerative colitis (UC). Protecting the mechanical barrier of the intestinal mucosa is of great significance for improving the health status of UC patients. ZO-1 is a key scaffold protein of the mechanical barrier of the intestinal mucosa, and its fusion with the membrane of the intestinal epithelium is a necessary condition to maintain the integrity of the mechanical barrier of the intestinal mucosa. Enteric glial cells (EGCs) play an important role in the maintenance of intestinal homeostasis and have become a new target for regulating intestinal health in recent years. In this study, we found that glycyrol (GC), a representative coumarin compound isolated from Licorice (Glycyrrhiza uralensis Fisch, used for medicine and food), can alleviate UC by promoting the production of neurotrophic factor GDNF in mice EGCs. Specifically, we demonstrated that GC promotes the production of GDNF, then activates its receptor RET, promotes ZO-1 fusion with cell membranes, and protects the intestinal mucosal mechanical barrier. The results of this study can provide new ideas for the prevention and treatment of UC.
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Colitis Ulcerosa , Factor Neurotrófico Derivado de la Línea Celular Glial , Mucosa Intestinal , Neuroglía , Proteína de la Zonula Occludens-1 , Animales , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/metabolismo , Ratones , Humanos , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genética , Masculino , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Proteínas Proto-Oncogénicas c-ret/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Ratones Endogámicos C57BL , Cumarinas/farmacología , Cumarinas/química , Transducción de Señal/efectos de los fármacos , Glycyrrhiza/químicaRESUMEN
Whey derived peptides have shown potential activity improving brain function in pathological condition. However, there is little information about their mechanism of action on glial cells, which have important immune functions in brain. Astrocytes and microglia are essential in inflammatory and oxidative defense that take place in neurodegenerative disease. In this work we evaluate antioxidant and anti-inflammatory potential bioactivity of whey peptide in glial cells. Peptides were formed during simulated gastrointestinal digestion (Infogest protocol), and low molecular weight (<5kDA) peptides (WPHf) attenuated reactive oxygen species (ROS) production induced by hydrogen peroxide stimulus in both cells in dose-dependent manner. WPHf induced an increase in the antioxidant glutathione (GSH) content and prevented GSH reduction induced by lipopolysaccharides (LPS) stimulus in astrocytes cells in a cell specific form. An increase in cytokine mRNA expression (TNFα and IL6) and nitric oxide secretion induced by LPS was attenuated by WPHf pre-treatment in both cells. The inflammatory pathway was dependent on NFκB activation. Bioactive peptide ranking analysis showed positive correlation with hydrophobicity and negative correlation with high molecular weights. The sequence identification revealed 19 peptides cross-referred with bioactive database. Whey peptides were rich in leucine, valine and tyrosine in the C-terminal region and lysine in the N-terminal region. The anti-inflammatory and antioxidant potential of whey peptides were assessed in glia cells and its mechanisms of action were related, such as modulation of antioxidant enzymes and anti-inflammatory pathways. Features of the peptide structure, such as molecular size, hydrophobicity and types of amino acids present in the terminal region are associated to bioactivity.
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Antiinflamatorios , Antioxidantes , Neuroglía , Proteína de Suero de Leche , Antioxidantes/farmacología , Antiinflamatorios/farmacología , Proteína de Suero de Leche/farmacología , Proteína de Suero de Leche/química , Proteína de Suero de Leche/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Animales , Especies Reactivas de Oxígeno/metabolismo , Lipopolisacáridos/farmacología , Glutatión/metabolismo , Péptidos/farmacología , Óxido Nítrico/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismoRESUMEN
Despite widespread use of combination antiretroviral therapy (cART), there remains a subset of individuals who display cognitive impairment broadly known as HIV-associated neurocognitive disorder (HAND). Interestingly, HIV-infected cells continuously release the HIV-1 protein Tat even in the presence of cART. Persistent exposure to Tat is proposed to increase both neuroinflammation and neurotoxicity. In vitro evidence shows that matrix metalloproteinases (MMPs) are among the neuroinflammatory molecules induced by Tat, which are known to disrupt specialized neuronal extracellular matrix structures called perineuronal nets (PNNs). PNNs predominantly surround parvalbumin interneurons and help to buffer these cells from oxidant stress and to independently increase their excitability. In order to better understand the link between short-term exposure to Tat, neuroinflammation, and PNNs, we explored the direct effects of Tat on glial cells and neurons. Herein, we report that in mixed glial cultures, Tat directly increases the expression of proinflammatory molecules, including MMP-9. Moreover, direct injection of Tat protein into mouse hippocampus increases the expression of astrocyte and microglia markers as well as MMP-9. The number of PNNs is decreased following Tat exposure, followed later by decreased numbers of hippocampal parvalbumin-expressing neurons. In older mice, Tat induced significant increases in the gene expression of proinflammatory molecules including markers of gliosis, MMPs and complement system proteins. Taken together, these data support a direct effect of Tat on glial-derived MMP expression subsequently affecting PNNs and neuronal health, with older mice more susceptible to Tat-induced inflammation.
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Neuroglía , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Animales , Ratones , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Neuroglía/metabolismo , Neuroglía/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Ratones Endogámicos C57BL , Masculino , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/virología , Red Nerviosa/efectos de los fármacos , Red Nerviosa/metabolismo , Células Cultivadas , Humanos , Parvalbúminas/metabolismoRESUMEN
Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with the loss of dopaminergic neurons and neuroinflammation. Recent studies have identified a role of T cells in the pathogenesis of PD. Additionally, these studies suggested that α-synuclein (α-Syn) is related to abnormal T-cell responses and may act as an epitope and trigger autoimmune T-cell responses. However, it is unclear whether the α-Syn-mediated autoimmune response occurs and whether it is related to neuronal cell death and glial cell activation. In this study, we investigated the autoimmune T-cell response induced by α-Syn peptides and evaluated the neurotoxic effect of the α-Syn peptide-mediated autoimmune response. The immunization of mice with α-Syn peptides resulted in enhanced autoimmune responses, such as the peptide recall response, polarization toward Th1/Th17 cells, and regulatory T cell imbalance. Furthermore, the α-Syn autoimmune response led to the death of primary neurons cocultured with splenocytes. Treatment with conditioned media from α-Syn peptide-immunized splenocytes induced microglia and toxic A1-type astrocyte activation. Taken together, our results provide evidence of the potential role of the α-Syn-initiated autoimmune response and its contribution to neuronal cell death and glial cell activation.
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Autoinmunidad , Muerte Celular , Neuronas , alfa-Sinucleína , Animales , alfa-Sinucleína/inmunología , alfa-Sinucleína/metabolismo , Ratones , Muerte Celular/efectos de los fármacos , Neuronas/inmunología , Neuronas/metabolismo , Neuronas/patología , Neuroglía/inmunología , Neuroglía/metabolismo , Neuroglía/efectos de los fármacos , Enfermedad de Parkinson/inmunología , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/metabolismo , Ratones Endogámicos C57BL , Humanos , Activación de Linfocitos/inmunología , Activación de Linfocitos/efectos de los fármacos , Péptidos/inmunología , Células Cultivadas , Femenino , Linfocitos T Reguladores/inmunologíaRESUMEN
Neurodegenerative diseases (NDDs) are progressive multifactorial disorders of the nervous system sharing common pathogenic features, including intracellular misfolded protein aggregation, mitochondrial deficit, and inflammation. Taking into consideration the multifaceted nature of NDDs, development of multitarget-directed ligands (MTDLs) has evolved as an attractive therapeutic strategy. Compounds that target the cannabinoid receptor type II (CB2R) are rapidly emerging as novel effective MTDLs against common NDDs, such as Alzheimer's disease (AD). We recently developed the first CB2R bitopic/dualsteric ligand, namely FD22a, which revealed the ability to induce neuroprotection with fewer side effects. To explore the potential of FD22a as a multitarget drug for the treatment of NDDs, we investigated here its ability to prevent the toxic effect of ß-amyloid (Aß25-35 peptide) on human cellular models of neurodegeneration, such as microglia (HMC3) and glioblastoma (U87-MG) cell lines. Our results displayed that FD22a efficiently prevented Aß25-35 cytotoxic and proinflammatory effects in both cell lines and counteracted ß-amyloid-induced depression of autophagy in U87-MG cells. Notably, a quantitative proteomic analysis of U87-MG cells revealed that FD22a was able to potently stimulate the autophagy-lysosomal pathway (ALP) by activating its master transcriptional regulator TFEB, ultimately increasing the potential of this novel CB2R bitopic/dualsteric ligand as a multitarget drug for the treatment of NDDs.
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Péptidos beta-Amiloides , Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Proteómica , Receptor Cannabinoide CB2 , Humanos , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Proteómica/métodos , Receptor Cannabinoide CB2/metabolismo , Ligandos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Autofagia/efectos de los fármacos , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Línea Celular TumoralRESUMEN
Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.