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Brain-targeted drug delivery poses a great challenge due to the blood-brain barrier (BBB). In a previous study, we have developed a peptide-modified stealth liposome (SP-sLip) to enhance BBB penetration via the adsorption of apolipoproteins in plasma. SP is an 11-amino acid peptide derived from 25 to 35 of the Amyloid ß peptide (Aß1-42), which is a nature ligand of apolipoproteins. Although freshly prepared SP-sLip exhibited efficient brain targeting performance, it occured self-aggregation and instability in storage. In this study, we developed a D-peptide ligand according to the reverse sequence of SP with D-amino acids, known as DSP, to improve the stability in storage. Notably, DSP exhibited a reduced tendency for self-aggregation and improved stability in comparison to the SP peptide. Furthermore, compared to SP-sLip, DSP-modified sLip (DSP-sLip) demonstrated enhanced stability (>2 weeks), prolonged blood circulation (AUC increased 44.4%), reduced liver and spleen accumulation (reduced by 2.23 times and 1.86 times) with comparable brain-targeting efficiency. Similar to SP-sLip, DSP-sLip selectively adsorbed apolipoprotein A1, E, and J in the blood to form functionalized protein corona, thus crossing BBB via apolipoprotein receptor-mediated transcytosis. These findings underscored the importance of ligand stability in the in vitro and in vivo performance of brain-targeted liposomes, therefore paving the way for the design and optimization of efficient and stable nanocarriers.
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Polyethylene glycol (PEG) plays important roles in stabilizing and lengthening circulation time of lipid nanoparticle (LNP) vaccines. Nowadays various levels of PEG antibodies have been detected in human blood, but the impact and mechanism of PEG antibodies on the in vivo performance of LNP vaccines has not been clarified thoroughly. By illustrating the distribution characteristics of PEG antibodies in human, the present study focused on the influence of PEG antibodies on the safety and efficacy of LNP-mRNA vaccine against COVID-19 in animal models. It was found that PEG antibodies led to shortened blood circulation duration, elevated accumulation and mRNA expression in liver and spleen, enhanced expression in macrophage and dendritic cells, while without affecting the production of anti-Spike protein antibodies of COVID-19 LNP vaccine. Noteworthily, PEG antibodies binding on the LNP vaccine increased probability of complement activation in animal as well as in human serum and led to lethal side effect in large dosage via intravenous injection of mice. Our data suggested that PEG antibodies in human was a risky factor of LNP-based vaccines for biosafety concerns but not efficacy.
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COVID-19 , Nanopartículas , Vacunas , Humanos , Animales , Ratones , Polietilenglicoles , Vacunas de ARNm , Vacunas contra la COVID-19 , AnticuerposRESUMEN
PEGylated nanocarriers have gained increasing attention due to reduced toxicity and enhanced circulation compared with free drugs. According to guidances of drug regulatory departments worldwide, it is crucial to determine free and liposomal drug concentrations; however, the conventional used separation methods including dialysis, ultrafiltration, and solid-phase extraction (SPE) have drawbacks of time-consuming, drug leakage, environmental pollution or error bias of trace level drug. Here we developed a facile PEG-scFv-based separation method combined with HPLC to quantify free doxorubicin (DOX) and liposomal DOX in plasma. Anti-PEG single chain variable fragment antibody (PEG-scFv) was adopted to sediment PEGylated liposomes by simple incubation and low speed centrifugation. Compared to SPE, it demonstrated sufficient accuracy and sensitivity to evaluate free and liposomal DOX with intact liposomes. Therefore, it can serve as an alternative approach of SPE, which is suitable for quality assessment and pharmacokinetics evaluation of PEGylated liposomal drugs and possible other PEGylated nanocarriers.
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Liposomas , Anticuerpos de Cadena Única , Doxorrubicina/farmacocinética , PolietilenglicolesRESUMEN
Glucose transporter (GLUT)-mediated transcytosis has been validated as an efficient method to cross the blood-brain barrier and enhance brain transport of nanomedicines. However, the transcytosis process remains elusive. Glycopeptide-modified nanodisks (Gly-A7R-NDs), which demonstrated high capacity of brain targeting via GLUT-mediated transcytosis in our previous reports, were utilized to better understand the whole transcytosis process. Gly-A7R-NDs internalized brain capillary endothelial cells mainly via GLUT-mediated/clathrin dependent endocytosis and macropinocytosis. The intracellular Gly-A7R-NDs remained intact, and the main excretion route of Gly-A7R-NDs was lysosomal exocytosis. Glycosylation of nanomedicine was crucial in GLUT-mediated transcytosis, while morphology did not affect the efficiency. This study highlights the pivotal roles of lysosomal exocytosis in the process of GLUT-mediated transcytosis, providing a new impetus to development of brain targeting drug delivery by accelerating lysosomal exocytosis.
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It remains challenging to precisely decipher the structural and functional characteristics of protein coronas. To overcome the drawbacks frequently occurring in the traditional separation methods, an anti-PEG single-chain variable fragment (PEG-scFv) based affinity chromatography (AfC) was developed to achieve precise and efficient separation of protein coronas on PEGylated liposomes (sLip). His-tagged PEG-scFv could readily capture sLip without affecting protein corona compositions, and separate sLip/protein complex from plasma protein aggregates and endogenous vesicles through the Ni-NTA column. AfC demonstrated 43-fold higher protein corona collecting efficiency than centrifugation, which was extremely crucial for separation of in vivo protein coronas due to the limitation of sample size. AfC evaded contamination by endogenous vesicles and protein aggregates occurring in centrifugation, and reserved the loosely bound proteins, providing an unprecedented approach to deeply decipher protein coronas. The scFv-based AfC also paves new avenues for the separation of protein coronas formed on other nanomedicines.
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Corona de Proteínas , Anticuerpos de Cadena Única , Cromatografía de Afinidad , Liposomas , Nanomedicina , Anticuerpos de Cadena Única/genéticaRESUMEN
Anti-PEG antibodies have been witnessed in patients and experimental animals, accelerating the blood clearance (termed ABC phenomenon) of PEGylated nanomedicines by activating complement after absorption on the nano-surface. The ABC phenomenon presents an obstacle to the clinical translation of PEGylated nanomedicines. Herein, an anti-PEG single-chain variable fragment (PEG-scFv) that possesses a low molecule weight (30 kDa) and high PEG binding affinity was exploited to ameliorate the ABC phenomenon of PEGylated liposomes (sLip). Pre-deposition of PEG-scFv on the surface of sLip was incompetent to activate complement due to the lack of Fc chains, exhibiting negligible influence on in vivo performance of sLip in naïve rats (without anti-PEG antibodies). However, PEG-scFv effectively competed the binding of anti-PEG IgM in rats that were pre-stimulated with low dose of sLip, thus ameliorated the ABC phenomenon of sLip. PEG- scFv was also effective to inhibit the binding of anti-PEG antibodies with sLip in human plasma and the consequent complement activation, presenting a promising tool to improve the performance of PEGylated nanomedicines and to mitigate individual difference occurred by the varying levels of anti-PEG antibodies in the clinic. The application of anti-PEG scFv paves a new avenue for the development of nanocarriers to achieve precise medication.
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Nanomedicina , Anticuerpos de Cadena Única , Animales , Humanos , Inmunoglobulina M , Liposomas , Polietilenglicoles , RatasRESUMEN
Glioblastoma (GBM) is an aggressive malignancy with poor prognosis. New therapeutic strategies for GBM are urgently needed. Although clinical studies have demonstrated the feasibility and safety of chimeric antigen receptor (CAR) T cell therapy for GBM, its efficacy has not been that impressive. The major limitation for anti-tumor efficacy of CAR-Ts is the immunosuppressive milieu of the GBM tumor microenvironment (TME). TGFß, a substantial component in GBM, compromises the immune response and contributes to immune evasion and tumor progression. To overcome this limitation and improve the efficacy of CAR-T cells for GBM, we optimized an EGFRvIII-specific CAR construct with TGFRII ectodomain as a TGFß-trap and generated TGFß-resistant CAR-Ts for GBM therapy. We demonstrated that this TGFß-trapped architecture enhanced anti-tumor efficacy of EGFRvIII-specific CAR-T and prolonged the survival of mice bearing GBM. In addition, the GBM-infiltrated microglia, typically considered tumorigenic, showed increased expression of M1 polarization markers after treatment with the TGFß-trap CAR-Ts group, indicating that these microglia were polarized toward a pro-inflammatory and anti-tumorigenic phenotype. Overall, these results indicated that arming CAR-T cells with a TGFß-trap diminishes the immunosuppressive effect and is a potential strategy to improve CAR-T efficacy for GBM therapy.
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Protein corona presents a major obstacle to bench-to-bedside translation of targeted drug delivery systems, severely affecting targeting yields and directing unfavorable biodistribution. Corona-mediated targeting provides a new impetus for specific drug delivery by precisely manipulating interaction modes of functional plasma proteins on nano-surface. Here bio-inspired liposomes (SP-sLip) were developed by modifying liposomal surface with a short nontoxic peptide derived from Aß1-42 that specifically interacts with the lipid-binding domain of exchangeable apolipoproteins. SP-sLip absorb plasma apolipoproteins A1, E and J, consequently exposing receptor-binding domain of apolipoproteins to achieve brain-targeted delivery. Doxorubicin loaded SP-sLip (SP-sLip/DOX) show significant enhancement of brain distribution and anti-brain cancer effect in comparison to doxorubicin loaded plain liposomes. SP-sLip preserve functions of the absorbed human plasma ApoE, and the corona-mediated targeting strategy works in SP modified PLGA nanoparticles. The present study may pave a new avenue to facilitate clinical translation of targeted drug delivery systems.
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Neoplasias Encefálicas/tratamiento farmacológico , Doxorrubicina/administración & dosificación , Nanopartículas/química , Corona de Proteínas/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Animales , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Doxorrubicina/farmacocinética , Humanos , Liposomas , Masculino , Ratones , Nanopartículas/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Ratas , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Depression is a common disease that endangers people's physical and mental health. Traditional Chinese medicine has advantages in treating the emotional and cognitive symptoms of depressive disorders. OBJECTIVE: To study the effects of baicalin on the behavior and to clarify the underlying mechanism through evaluation of the Rac1-LIMK1-cofilin pathway. METHODS: A chronic mild stress (CMS) model of depression was used. Baicalin was administered to the mice for the intervention, and the positive control group was treated with fluoxetine. Behavioral tests were conducted to observe the degree of depressive disorders. Synaptophysin (SYP), postsynaptic density protein-95 (PSD95), brain-derived neurotrophic factor (BDNF), tyrosine kinase receptors (TrkB), Rac1 and cofilin expression was determined using Western blot analysis, and mRNA was quantified using real-time PCR. RESULTS: Mice in the CMS group showed an increase in depression-like behavior (p < 0.01), while mice in the baicalin and fluoxetine groups showed a decrease in depression-like behavior (p < 0.01), compared with the control group. Electron microscopy showed ultrastructural changes in the hippocampal CA3 area of the CMS group, which were alleviated by baicalin treatment. SYP, PSD95, BDNF, TrkB, Rac1 and cofilin protein expression levels were decreased in the CMS group compared with the control group, while these levels were increased in the baicalin and fluoxetine groups (p < 0.01). There was no significant difference among the baicalin and fluoxetine groups (p > 0.05). CONCLUSION: Baicalin markedly alleviated depression-like behavioral changes, exerted effects on SYP, PSD95, BDNF, and TrkB expression, activated the Rac1-cofilin pathway, and subsequently improve synaptic plasticity.
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Factores Despolimerizantes de la Actina/metabolismo , Conducta Animal , Depresión/tratamiento farmacológico , Flavonoides/uso terapéutico , Quinasas Lim/metabolismo , Transducción de Señal , Estrés Psicológico/tratamiento farmacológico , Proteínas de Unión al GTP rac/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Enfermedad Crónica , Depresión/complicaciones , Homólogo 4 de la Proteína Discs Large/genética , Homólogo 4 de la Proteína Discs Large/metabolismo , Flavonoides/química , Flavonoides/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/patología , Hipocampo/ultraestructura , Masculino , Ratones , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor trkB/metabolismo , Transducción de Señal/efectos de los fármacos , Estrés Psicológico/complicaciones , Natación , Sinaptofisina/genética , Sinaptofisina/metabolismoRESUMEN
Keratan sulfate (KS) is a carbohydrate side chain covalently attached to extracellular proteoglycans. KS is composed of disaccharide units of 6-sulfated N-acetylglucosamine (GlcNAc) and galactose. We have previously shown that GlcNAc-6-O-sulfotransferase (GlcNAc6ST) 1 encoded by Chst2 is an enzyme necessary for the synthesis of GlcNAc-6-sulfated KS chains that are required for neuronal plasticity in the visual cortex of the mouse brain during the critical period, but not in adulthood. Here, we show that GlcNAc-6-sulfated KS recognized by the R-10G anti-KS antibody, of which the minimum epitope structure is Galß1-4GlcNAc(6S)ß1-3Galß1-4GlcNAc(6S), distributes diffusely in neuropils and presents densely in close proximity to the perineuronal region of the perineuronal net (PNN)-positive neurons in the adult visual cortex. Surprisingly, GlcNAc6ST3, which was discovered as an intestinal GlcNAc6ST encoded by Chst5, is a major brain KS sulfotransferase expressed in oligodendrocytes in adulthood. Moreover, we identified an isoform of the protein-tyrosine phosphatase PTPRZ as a R-10G-reactive KS proteoglycan. These results indicate that GlcNAc6ST3 may play a role in synthesis of a component of PNN in the adult brain, and that the KS-modified isoform of PTPRZ encoded by Ptprz1 could be an extracellular molecule associated with PNNs.
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Encéfalo/enzimología , Encéfalo/metabolismo , Sulfato de Queratano/metabolismo , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Sulfotransferasas/metabolismo , Animales , Femenino , Inmunoprecipitación , Masculino , Ratones , Microscopía Confocal , Proteínas Tirosina Fosfatasas , Proteoglicanos/metabolismo , Sinaptofisina/metabolismo , Carbohidrato SulfotransferasasRESUMEN
Glycosaminoglycans (GAG) from the velvet antlers of Sika deer (Cervus nippon) at the different growing stages (Fukurozuno, Anshi, and Santajo) of bred and wild deer were isolated and their concentrations and sulfation patterns were analyzed. GAG were digested with chondroitinase ABC, ACI, heparinase-I and -III, and keratanase-II into the corresponding repeating disaccharides of chondroitin sulfate (CS), dermatan sulfate (DS), hyaluronan, heparan sulfate (HS), and keratan sulfate. Cartilaginous tissues contained CS-DS at high concentrations with an almost equal ratio of 4- and 6-sulfates, while 4-sulfate-type CS-DS predominantly occupied ossified tissues, but at low concentrations. High O- and N-sulfation degrees of HS correspond to high ossification. Dynamic quantitative changes in CS-DS and compositional changes in CS-DS and HS were closely associated with the mineralization of deer antlers.
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Cuernos de Venado/química , Glicosaminoglicanos/análisis , Animales , Cuernos de Venado/crecimiento & desarrollo , Cuernos de Venado/metabolismo , Ciervos , Glicosaminoglicanos/metabolismo , MasculinoRESUMEN
Targeting ligands are anticipated to facilitate the precise delivery of therapeutic agents to diseased tissues; however, they may also severely affect the interaction of nanocarriers with plasma proteins. Here, we study the immunocompatibility of brain-targeted liposomes, which inversely correlates with absorbed natural IgM. Modification of long, stable positively charged peptide ligands on liposomes is inclined to absorb natural IgM, leading to rapid clearance and enhanced immunogenicity. Small peptidomimetic D8 developed by computer-aided peptide design exhibits improved immunocompatibility by attenuating natural IgM absorption. The present study highlights the effects of peptide ligands on the formed protein corona and in vivo fate of liposomes. Stable positively charged peptide ligands play double-edged roles in targeted delivery, preserving in vivo bioactivities for binding receptors and long-term unfavorable interactions with the innate immune system. The development of D8 provides insights into how to rationally design immunocompatible drug delivery systems by modulating the protein corona composition.
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Sistemas de Liberación de Medicamentos , Inmunoglobulina M/química , Ligandos , Liposomas/química , Animales , Encéfalo/metabolismo , Línea Celular Tumoral , Células Dendríticas/citología , Inmunidad Innata , Inmunoglobulina G/química , Masculino , Ratones , Ratones Endogámicos BALB C , Péptidos/química , Polietilenglicoles/química , Unión Proteica , Células RAW 264.7 , Electricidad EstáticaRESUMEN
Glioma is among the most formidable brain cancers due to location in the brain. Cholera toxin subunit B (CTB) is investigated to facilitate multifunctional glioma-targeted drug delivery by targeting the glycosphingolipid GM1 expressed in the blood-brain barrier (BBB), neovasulature, and glioma cells. When modified on the surface of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (CTB-NPs), CTB fully retains its bioactivity after 24 h incubation in the fresh mouse plasma. The formed protein corona (PC) of CTB-NP and plain PLGA nanoparticles (NP) after incubation in plasma is analyzed using liquid chromatography tandem massspectrometry (nano-LC-MS/MS). CTB modification does not alter the protein components of the formed PC, macrophage phagocytosis, or pharmacokinetic profiles. CTB-NP can efficiently penetrate the in vitro BBB model and target glioma cells and human umbilical vascular endothelial cells. Paclitaxel is loaded in NP (NP/PTX) and CTB-NP (CTB-NP/PTX), and their antiglioma effects are assessed in nude mice bearing intracranial glioma. CTB-NP/PTX can efficiently induce apoptosis of intracranial glioma cells and ablate neovasulature in vivo, resulting in significant prolongation of survival of nude mice bearing intracranial glioma (34 d) in comparison to those treated with NP/PTX (29 d), Taxol (24 d), and saline (21 d). The present study suggests a potential multifunctional glioma-targeted drug delivery system enabled by cholera toxin subunit B.
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Toxina del Cólera , Sistemas de Liberación de Medicamentos/métodos , Glioma/tratamiento farmacológico , Nanopartículas , Paclitaxel , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Línea Celular Tumoral , Toxina del Cólera/química , Toxina del Cólera/farmacocinética , Toxina del Cólera/farmacología , Glioma/metabolismo , Glioma/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ácido Láctico/química , Ácido Láctico/farmacocinética , Ácido Láctico/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Nanopartículas/química , Nanopartículas/uso terapéutico , Paclitaxel/química , Paclitaxel/farmacocinética , Paclitaxel/farmacología , Ácido Poliglicólico/química , Ácido Poliglicólico/farmacocinética , Ácido Poliglicólico/farmacología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Células RAW 264.7 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We previously showed that microglial keratan sulfate (KS) was induced in amyotrophic lateral sclerosis. However, the functional roles of the glycan and its synthetic enzyme in neurodegenerative diseases, such as Alzheimer's disease (AD), a progressive disorder, are unclear. In our study, KS modified with sialic acids having a molecular mass of 125-220 kDa and the carbohydrate sulfotransferase GlcNAc6ST1 were up-regulated in the brains of two transgenic mouse models (J20 and Tg2576) and the brains of patients with AD. GlcNAc6ST1-deficient J20 (J20/GlcNAc6ST1-/-) mice demonstrated a complete absence of the microglial sialylated KS. J20/GlcNAc6ST1-/- primary microglia showed an increased level of amyloid-ß phagocytosis and were hyperresponsive to interleukin 4, a potent antiinflammatory cytokine. Moreover, J20/GlcNAc6ST1-/- mice manifested reduced cerebral amyloid-ß deposition. GlcNAc6ST1-synthesizing sialylated KS thus modulates AD pathology. Inhibition of KS synthesis by targeting GlcNAc6ST1 may therefore be beneficial for controlling AD pathogenesis.
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Ácido N-Acetilneuramínico/metabolismo , Sulfotransferasas/metabolismo , Anciano de 80 o más Años , Precursor de Proteína beta-Amiloide/genética , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Sulfato de Queratano/metabolismo , Masculino , Ratones Transgénicos , Microglía/metabolismo , Microglía/patología , Fagocitosis , Placa Amiloide/metabolismo , Placa Amiloide/patología , Polisacáridos/metabolismo , Carbohidrato SulfotransferasasRESUMEN
Alzheimer's disease (AD) is a progressive disorder leading to cognitive impairment and neuronal loss. Cerebral extracellular accumulation and deposition of amyloid ß plaques is a pathological hallmark of AD. Chondroitin sulfate (CS) is an extracellular component abundant in the brain. CS is a sulfated glycosaminoglycan covalently attached to a core protein, forming chondroitin sulfate proteoglycan. The structure of CS is heterogeneous with sulfation modification and elongation of the chain. The structural diversity of CS allows it to play various roles in the brain. Increasing evidence has shown that CS promotes aggregation of amyloid ß peptides into higher-order species such as insoluble amyloid ß fibrils. Difficulties in the structural analysis of brain CS, as well as its heterogeneity, limit the study of potential roles of CS in AD pathology. Here we established a microanalysis method with reversed-phase ion-pair high performance liquid chromatography and found that CS in the brains of Tg2576 AD model mice show a lower molecular size and an increased ratio of CS-B motif di-sulfated disaccharide. Our findings provide insight into the structural changes of cerebral CS upon Alzheimer's pathogenesis.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Animales , Sulfatos de Condroitina , Disacáridos , Ratones , Ratones Endogámicos C57BL , Placa AmiloideRESUMEN
The functional role of 5D4 antibody-reactive keratan sulfate (KS) in the pathogenesis of neurodegenerative diseases is unknown. We therefore studied the expression of 5D4-reactive KS in amyotrophic lateral sclerosis (ALS), a motor neuron-degenerative disease, with the use of SOD1(G93A) ALS model mice and patients with ALS. Histochemical and immunoelectron microscopic characterizations showed that the 5D4-reactive KS is expressed in Mac2/galectin-3-positive activated or proliferating microglia of SOD1(G93A) ALS model mice at disease end stage and that the KS is an O-linked glycan modified with sialic acid and fucose, which was thus far shown to exist in cartilage. Intriguingly, microglial KS was detected in the spinal cord and brainstem but not in the cerebral cortex of SOD1(G93A) mice. We found that KSGal6ST, a galactose-6-sulfotransferase, is required for biosynthesis of the microglial 5D4-reactive KS by generating SOD1(G93A)/KSGal6ST(-/-) mice. The requirement of GlcNAc6ST1 for this synthesis was corroborated by analyzing SOD1(G93A)/GlcNAc6ST1(-/-) mice. These results indicate that both galactose-6- and N acteylglucosamine-6-sulfated KS elicited in the spinal cord and brainstem are associated with the degeneration of spinal and bulbar lower motor neurons in ALS pathology and may play a role in disease progression via microglial activation and proliferation.
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Esclerosis Amiotrófica Lateral/inmunología , Sulfato de Queratano/inmunología , Sulfotransferasas/inmunología , Superóxido Dismutasa/genética , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/mortalidad , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Epítopos/inmunología , Femenino , Galectina 3/metabolismo , Humanos , Sulfato de Queratano/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/inmunología , Microglía/metabolismo , Persona de Mediana Edad , Neuronas Motoras/metabolismo , Mutación Missense , Médula Espinal/metabolismo , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Superóxido Dismutasa/inmunología , Carbohidrato SulfotransferasasRESUMEN
Considerable evidence indicates that methionine sulfoxide (MetO) reductase A (MsrA) plays an important role in cytoprotection against oxidative stress and serves as a potential drug target. To screen for MsrA regulators, a rapid and specific assay to monitor MsrA activity is required. Most of current assays for MsrA activity are based on the reduction of radioactive substrates such as [3H]-N-acetyl-MetO or fluorescent derivatives such as dimethylaminoazo-benzenesulfonyl-MetO. However, these assays require extraction procedures and special instruments. Here, we developed a specific colorimetric microplate assay for testing MsrA activity quickly, which was based on the fact that MsrA can catalyze the reduction of methyl sulfoxides and simultaneously oxidize dithiothreitol (DTT), whose color can be produced by reacting with Ellman's reagent (dithio-bis-nitrobenzoic acid, DTNB). The corresponding absorbance change at 412nm was recorded with a microplate reader as the reaction proceeded. This method to monitor MsrA activity is easy to handle. Our findings may serve as a rapid method for the characterization of recombinant enzyme and for the screening of enzyme inhibitors, pharmacological activators, gene expression regulators and novel substrates.
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Colorimetría/métodos , Oxidorreductasas/metabolismo , Animales , Ácido Ditionitrobenzoico , Ditiotreitol/metabolismo , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Estrés Oxidativo , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/genética , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometría , Especificidad por Sustrato , Sulfóxidos/metabolismoRESUMEN
Hypothalamic AMP-activated protein kinase (AMPK) and orexins/hypocretins are both involved in the control of feeding behavior, but little is known about the interaction between these two signaling systems. Here, we demonstrated that orexin-A elicited significant activation of AMPK in the arcuate nucleus (ARC) of the hypothalamus by elevating cytosolic free Ca²âº involving extracellular calcium influx. Electrophysiological results revealed that orexin-A increased the L-type calcium current via the orexin receptor-phospholipase C-protein kinase C signaling pathway in ARC neurons that produce neuropeptide Y, an important downstream effector of orexin-A's orexigenic effect. Furthermore, the L-type calcium channel inhibitor nifedipine attenuated orexin-A-induced AMPK activation in vitro and in vivo. We found that inhibition of AMPK by either compound C (6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine) or the ATP-mimetic 9-ß-D-arabinofuranoside prevented the appetite-stimulating effect of orexin-A. This action can be mimicked by nifedipine, the blocker of the L-type calcium channel. Our results indicated that orexin-A activates hypothalamic AMPK signaling through a Ca²âº-dependent mechanism involving the voltage-gated L-type calcium channel, which may serve as a potential target for regulating feeding behavior.
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Proteínas Quinasas Activadas por AMP/metabolismo , Núcleo Arqueado del Hipotálamo/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/farmacología , Neuropéptidos/farmacología , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Células Cultivadas , Conducta Alimentaria , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Activación del Canal Iónico , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuropéptido Y/metabolismo , Neuropéptidos/metabolismo , Receptores de Orexina/metabolismo , Orexinas , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Fosfolipasas de Tipo C/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Sinomenine (SN), a bioactive alkaloid, has been utilized clinically to treat rheumatoid arthritis in China. Our preliminary experiments indicated that it could protect PC12 cells from oxygen-glucose deprivation-reperfusion (OGD-R), we thus investigated the possible effects of SN on cerebral ischaemia and the related mechanism. EXPERIMENTAL APPROACH: Middle cerebral artery occlusion in rats was used as an animal model of ischaemic stroke in vivo. The mechanisms of the effects of SN were investigated in vitro using whole-cell patch-clamp recording, calcium imaging in PC12 cells and rat cortical neurons subjected to OGD-R. KEY RESULTS: Pretreatment with SN (10 and 30 mg·kg(-1) , i.p.) significantly decreased brain infarction and the overactivation of calcium-mediated events in rats subjected to 2 h ischaemia followed by 24 h reperfusion. Extracellular application of SN inhibited the currents mediated by acid-sensing ion channel 1a and L-type voltage-gated calcium channels, in the rat cultured neurons, in a concentration-dependent manner. These inhibitory effects contribute to the neuroprotection of SN against OGD-R and extracellular acidosis-induced cytotoxicity. More importantly, administration of SN (30 mg·kg(-1) , i.p.) at 1 and 2 h after cerebral ischaemia also decreased brain infarction and improved functional recovery. CONCLUSION AND IMPLICATIONS: SN exerts potent protective effects against ischaemic brain injury when administered before ischaemia or even after the injury. The inhibitory effects of SN on acid-sensing ion channel 1a and L-type calcium channels are involved in this neuroprotection.
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Isquemia Encefálica/tratamiento farmacológico , Canales de Calcio Tipo L/metabolismo , Morfinanos/uso terapéutico , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Fármacos Neuroprotectores/uso terapéutico , Canales Iónicos Sensibles al Ácido , Animales , Animales Recién Nacidos , Western Blotting , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Células CHO , Técnicas de Cultivo de Célula , Cricetinae , Cricetulus , Citoprotección/efectos de los fármacos , Modelos Animales de Enfermedad , Glucosa/metabolismo , Masculino , Morfinanos/administración & dosificación , Morfinanos/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacología , Oxígeno/metabolismo , Células PC12 , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Canales de SodioRESUMEN
Protein-bound methionine (Met) oxidation has been associated with normal aging and a variety of age-related diseases, including Alzheimer's disease and Parkinson's disease. Monitoring the changes of protein-bound methionine content in the brain in response to normal aging and oxidative stress is of great interest and could be used as an indicator of oxidative stress of rats in pathological conditions. We have developed a rapid analytical method for the determination of oxidized products of protein-bound methionine in rat brain. The assay involved rapid acid proteolysis with microwave irradiation and solid-phase extraction of the free amino acids followed by LC-ESI-ITMS analysis. Detection was achieved in positive ionization with an ion trap mass spectrometer operating in multiple-reaction monitoring mode. The calibration curves of the analytes were linear (r(2) > 0.99) in the range between 0.098 and 1.560 µg/mL. Intra- and inter-day relative standard deviation percentages were <9% and <8%, respectively. The assay performance was sufficient to support a rapid analytical tool for monitoring brain protein-bound methionine oxidation levels. The content of protein-bound Met and methionine sulfoxide (MetO) in the hippocampus of adult and old rats with or without H(2)O(2) treatment was determined by employing the new method. The content of protein-bound MetO was significantly increased in old rats after exposure to H(2)O(2). This result indicates increased sensitivity to Met oxidation in the hippocampus of old rats.