RESUMEN
Spinal cord injury (SCI) severely affects the quality of life and autonomy of patients, and effective treatments are currently lacking. Autophagy, an essential cellular metabolic process, plays a crucial role in neuroprotection and repair after SCI. Glycoprotein non-metastatic melanoma protein B (GPNMB) has been shown to promote neural regeneration and synapse reconstruction, potentially through the facilitation of autophagy. However, the specific role of GPNMB in autophagy after SCI is still unclear. In this study, we utilized the spinal cord transection method to establish SCI rats model and overexpressed GPNMB using adenoviral vectors. We assessed tissue damage using hematoxylin and eosin (H&E) and Nissl staining, and observed cell apoptosis using TUNEL staining. We evaluated the inflammatory response by measuring inflammatory factors using enzyme-linked immunosorbent assay (ELISA). In addition, we measured reactive oxygen species (ROS) levels using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and assessed oxidative stress levels by measuring malondialdehyde (MDA) and glutathione (GSH) using ELISA. To evaluate autophagy levels, we performed immunofluorescence staining for the autophagy marker Beclin-1 and conducted Western blot analysis for autophagy-related proteins. We also assessed limb recovery through functional evaluation. Meanwhile, we induced cell injury using lipopolysaccharide (LPS) and added an autophagy inhibitor to verify the impact of GPNMB on SCI through autophagy modulation. The results demonstrated that GPNMB alleviated the inflammatory response, reduced oxidative stress levels, inhibited cell apoptosis, and promoted autophagy following SCI. Inhibiting autophagy reversed the effects of GPNMB. These findings suggest that GPNMB promotes neural injury repair after SCI, potentially through attenuating the inflammatory response, reducing oxidative stress, and inhibiting cell apoptosis.
Asunto(s)
Melanoma , Receptores Fc , Traumatismos de la Médula Espinal , Animales , Humanos , Ratas , Apoptosis , Autofagia , Glutatión/metabolismo , Glicoproteínas/farmacología , Melanoma/metabolismo , Melanoma/patología , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacología , Calidad de Vida , Ratas Sprague-Dawley , Recuperación de la Función/fisiología , Médula Espinal/patología , Traumatismos de la Médula Espinal/patologíaRESUMEN
Glycoprotein non-metastatic melanoma protein B (GPNMB) got its name from the first discovery in a cell line of non-metastatic melanoma. Later studies found that GPNMB is widely expressed in various tissues and cells of the human body, most abundant in neural tissue, epithelial tissue, bone tissue, and monocyte-macrophage system. GPNMB has been shown to have anti-inflammatory effects in a variety of neurological diseases, however, it has not been reported in subarachnoid hemorrhage (SAH). Male CD-1 mice were used and intra-arterial puncture method was applied to establish the SAH model. Exogenous recombinant GPNMB (rGPNMB) was injected intracerebroventricularly 1 h after SAH. SAH grading, brain edema and blood-brain barrier (BBB) integrity were quantified, and neurobehavioral tests were performed to evaluate the effect of GPNMB on the outcome. Dorsomorphin, the selective inhibitor on AMPK was introduced to study the downstream signaling through which the GPNMB works. Furthermore, western blot, immunofluorescence staining and ELISA were utilized to confirm the signaling. After SAH, GPNMB expression increased significantly as a result of the inflammatory response. GPNMB was expressed extensively in mouse microglia, astrocytes and neurons. The administration of rGPNMB could alleviate brain edema, restore BBB integrity and improve the neurological outcome of mice with SAH. GPNMB treatment significantly magnified the expression of p-AMPK while p-NFκB, IL-1ß, IL-6 and TNF-α were suppressed; in the meantime, the combined administration of GPNMB and AMPK inhibitor could decrease the intensity of p-AMPK and reverse the quantity of p-NFκB and the above inflammatory cytokines. GPNMB has the potential of ameliorating the brain edema and neuroinflammation, protecting the BBB and improving the neurological outcome, possibly via the AMPK/NFκB signaling pathway.
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Edema Encefálico , Melanoma , Hemorragia Subaracnoidea , Ratas , Ratones , Masculino , Humanos , Animales , Hemorragia Subaracnoidea/tratamiento farmacológico , Hemorragia Subaracnoidea/metabolismo , Hemorragia Subaracnoidea/patología , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/farmacología , Proteínas Quinasas Activadas por AMP/uso terapéutico , Edema Encefálico/tratamiento farmacológico , Enfermedades Neuroinflamatorias , Ratas Sprague-Dawley , Transducción de Señal , Glicoproteínas , Glicoproteínas de Membrana/farmacología , Glicoproteínas de Membrana/uso terapéuticoRESUMEN
Peritoneal metastasis is very common in gastrointestinal, reproductive, and genitourinary tract cancers in late stages or postsurgery, causing poor prognosis, so effective and nontoxic prophylactic strategies against peritoneal metastasis are highly imperative. Herein, we demonstrate the first gene transfection as a nontoxic prophylaxis preventing peritoneal metastasis or operative metastatic dissemination. Lipopolyplexes of TNF-related-apoptosis-inducing-ligand (TRAIL) transfected peritonea and macrophages to express TRAIL for over 15 days. The expressed TRAIL selectively induced tumor cell apoptosis while exempting normal tissue, providing long-term tumor surveillance. Therefore, tumor cells inoculated in the pretransfected peritoneal cavity quickly underwent apoptosis and, thus, barely formed tumor nodules, significantly prolonging the mouse survival time compared with chemotherapy prophylaxis. Furthermore, lipopolyplex transfection showed no sign of toxicity. Therefore, this peritoneal TRAIL-transfection is an effective and safe prophylaxis, preventing peritoneal metastasis.
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Proteínas Reguladoras de la Apoptosis , Neoplasias Peritoneales , Animales , Ratones , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/farmacología , Ligandos , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/prevención & control , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacología , Apoptosis/genética , Factor de Necrosis Tumoral alfa/genética , Transfección , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genéticaRESUMEN
Spinal cord injury (SCI) is characterized by a cascade of events that lead to sensory and motor disabilities. To date, this condition is irreversible, and no cure exists. To improve myelin repair and limit secondary degeneration, we developed a multitherapy based on nanomedicines (NMeds) loaded with the promyelinating agent triiodothyronine (T3), used in combination with systemic ibuprofen and mouse nerve growth factor (mNGF). Poly-L-lactic-co-glycolic acid (PLGA) NMeds were optimized and loaded with T3 to promote sustained release. In vitro experiments confirmed the efficacy of T3-NMeds to differentiate oligodendrocyte precursor cells. In vivo rat experiments were performed in contusion SCI to explore the NMed biodistribution and efficacy of combo drugs at short- and long-term post-lesion. A strong anti-inflammatory effect was observed in the short term with a reduction of type M1 microglia and glutamate levels, but with a subsequent increase of TREM2. In the long term, an improvement of myelination in NG2-IR, an increase in MBP content, and a reduction of the demyelination area were observed. These data demonstrated that NMeds can successfully be used to obtain more controlled local drug delivery and that this multiple treatment could be effective in improving the outcome of SCIs.
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Remielinización , Traumatismos de la Médula Espinal , Ratas , Ratones , Animales , Remielinización/fisiología , Distribución Tisular , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Vaina de Mielina/patología , Inflamación/tratamiento farmacológico , Inflamación/patología , Glicoproteínas de Membrana/farmacología , Receptores InmunológicosRESUMEN
INTRODUCTION: Microglia and macrophages participate in hematoma clearance after intracerebral hemorrhage (ICH), thereby facilitating tissue restoration and neurological recovery. Triggering receptor expressed on myeloid cells 2 (Trem2) has been indicated as a major pathology-induced immune signaling hub on the microglial/macrophage surface. Soluble Trem2 (sTrem2), the proteolytic form of Trem2, is abundant in the body fluid and is positively correlated with the pathological process. OBJECTIVES: In the present study, we aimed to investigate the potential role of sTrem2 in hematoma resolution after ICH and to elucidate its underlying mechanisms. METHODS: We explored the biological functions of sTrem2 in the murine ICH brain by stereotaxic injection of recombinant sTrem2 protein or by adeno-associated virus-mediated expression. Erythrocyte phagocytosis was assessed using flow cytometry and immunofluorescence. Western blotting was performed to evaluate protein expression. Changes in behavior, sTrem2-induced down-stream pathway, and microglia were examined. RESULTS: sTrem2 impedes hematoma resolution and impairs functional motor and sensory recovery. Interestingly, sTrem2 bypasses full-length Trem2, negatively regulating microglial/macrophage erythrophagocytosis, and promotes an inflammatory phenotype, which is associated with reduced retromer levels and impaired recycling of the pro-erythrophagocytic receptor CD36. Rescue of retromer Vps35 abolishes the phagocytosis-inhibiting effects and lysosome-dependent CD36 degradation caused by sTrem2. CONCLUSION: These findings indicate sTrem2 as a negative factor against microglia/macrophage-mediated hematoma and related neuronal damage clearance, provide insight into the mechanisms by which erythrophagocytosis is regulated and how it may be impaired after ICH, and suggest that the anti-proteolytic activity of Trem2 can be explored for ICH therapy.
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Hemorragia Cerebral , Linfohistiocitosis Hemofagocítica , Animales , Ratones , Hemorragia Cerebral/complicaciones , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/patología , Fagocitosis/fisiología , Macrófagos/metabolismo , Microglía/metabolismo , Microglía/patología , Hematoma/complicaciones , Hematoma/metabolismo , Linfohistiocitosis Hemofagocítica/complicaciones , Linfohistiocitosis Hemofagocítica/metabolismo , Linfohistiocitosis Hemofagocítica/patología , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/farmacología , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacología , Receptores Inmunológicos/metabolismoRESUMEN
Trastuzumab is a humanized monoclonal antibody, and has been clinical employed to treat human epidermal growth factor receptor 2 (HER2) positive breast cancer. However, drug resistance to trastuzumab remains a challenge due to the generally uncharacterized interactive immune responses within the tumor tissue. In this study, by means of single-cell sequencing, we identified a novel podoplanin-positive (PDPN+) cancer-associated fibroblasts (CAFs) subset, which was enriched in trastuzumab resistant tumor tissues. Furthermore, we found that PDPN+ CAFs promote resistance to trastuzumab in HER2+ breast cancer by secreting immunosuppressive factors indoleamine 2,3-dioxygenase 1 (IDO1) as well as tryptophan 2,3-dioxygenase 2 (TDO2), thereby suppressing antibody-dependent cell-mediated cytotoxicity (ADCC), which was mediated by functional NK cells. A dual inhibitor IDO/TDO-IN-3 simultaneously targeting IDO1 and TDO2 showed a promising effect on reversing PDPN+ CAFs-induced suppression of NK cells mediated ADCC. Collectively, a novel subset of PDPN+ CAFs was identified in this study, which induced trastuzumab resistance in breast cancer of HER2+ status via inhibiting ADCC immune response mediated by NK cells, hinting that PDPN+ CAFs could be a novel target of treatment to increase the sensitivity of HER2+ breast cancer to trastuzumab.
Asunto(s)
Neoplasias de la Mama , Fibroblastos Asociados al Cáncer , Humanos , Femenino , Trastuzumab/farmacología , Trastuzumab/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos , Receptor ErbB-2/genética , Células Asesinas Naturales/metabolismo , Línea Celular Tumoral , Glicoproteínas de Membrana/farmacología , Glicoproteínas de Membrana/uso terapéuticoRESUMEN
CONTEXT: Bazi Bushen capsule (BZBS) has anti-ageing properties and is effective in enhancing memory. OBJECTIVE: To find evidence supporting the mechanisms and biomarkers by which BZBS functions. MATERIALS AND METHODS: Male C57BL/6J mice were randomly divided into five groups: normal, ageing, ß-nicotinamide mononucleotide capsule (NMN), BZBS low-dose (LD-BZ) and BZBS high-dose (HD-BZ). The last four groups were subcutaneously injected with d-galactose (d-gal, 100 mg/kg/d) to induce the ageing process. At the same time, the LD-BZ, HD-BZ and NMN groups were intragastrically injected with BZBS (1 and 2 g/kg/d) and NMN (100 mg/kg/d) for treatment, respectively. After 60 days, the changes in overall ageing status, brain neuron morphology, expression of p16INK4a, proliferating cell nuclear antigen (PCNA), ionized calcium-binding adapter molecule 1 (Iba1), postsynaptic density protein 95 (PSD95), CD11b, Arg1, CD206, Trem2, Ym1 and Fizz1, and the senescence-associated secretory phenotype (SASP) factors were observed. RESULTS: Compared with the mice in the ageing group, the HD-BZ mice exhibited obvious improvements in strength, endurance, motor coordination, cognitive function and neuron injury. The results showed a decrease in p16INK4a, Iba1 and the upregulation of PCNA, PSD95 among brain proteins. The brain mRNA exhibited downregulation of Iba1 (p < 0.001), CD11b (p < 0.001), and upregulation of Arg1 (p < 0.01), CD206 (p < 0.05), Trem2 (p < 0.001), Ym1 (p < 0.01), Fizz1 (p < 0.05) and PSD95 (p < 0.01), as well as improvement of SASP factors. CONCLUSIONS: BZBS improves cognitive deficits via inhibition of cellular senescence and microglia activation. This study provides experimental evidence for the wide application of BZBS in clinical practice for cognitive deficits.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina , Galactosa , Animales , Masculino , Ratones , Calcio , Senescencia Celular , Cognición , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/farmacología , Homólogo 4 de la Proteína Discs Large , Glicoproteínas de Membrana/farmacología , Ratones Endogámicos C57BL , Microglía/metabolismo , Mononucleótido de Nicotinamida/farmacología , Antígeno Nuclear de Célula en Proliferación , Receptores Inmunológicos , ARN MensajeroRESUMEN
BACKGROUND: The transmembrane receptor podoplanin (PDPN) is a platelet aggregation-inducing factor, which is widely expressed in various malignant tumors such as squamous cell carcinomas, mesotheliomas, glioblastomas. Podoplanin regulates a pathway leading to cell invasion and migration. Glioblastoma multiforme (GBM) is the most aggressive and invasive tumor of the central nervous system. A high level of PDPN expression has been reported to be associated with reduced survival, cancer aggression and migration. Aim of study to determine the effect of anti-podoplanin antibody on cell-platelet aggregation, cell invasion and viability in Uppsala 87 malignant glioma (U87MG) cell lines. METHODS: The expression of podoplanin on U87MG cells was measured using flowcytometry. The dimethyl-thiazol diphenyl-tetrazolium bromide (MTT) assay was used to measure U87MG cell proliferation after treatment by anti-podoplanin monoclonal antibody (NZ-1.3). The invasion of cancer cells was assessed by using a novel microfluidic based assay. RESULTS: The results show reduction of cell viability and cell migration after treatment with anti-podoplanin antibody. After co- treatment of U87MG cells and platelets with and without anti-podoplanin antibody, the cell-platelet aggregation was significantly reduced in anti-podoplanin treated cell. CONCLUSIONS: Podoplanin is involved in aggregation of gliobastoma cells, and their viability and invasion and its neutralizing antibody can inhibit this process. So, blocking of podoplanin may be representing a promising therapeutic approach to glioblastoma multiform cancer therapy.
Asunto(s)
Glioblastoma , Glioma , Línea Celular Tumoral , Supervivencia Celular , Glioblastoma/patología , Humanos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacología , Agregación PlaquetariaRESUMEN
Alzheimer's disease-related cognition impairment is correlated with increased neuroinflammation. Studies show that physical exercises improve cognitive function and regulate neuroinflammation. However, no sufficient studies have been performed to directly observe the mechanism of exercise-related effects on microglia and neuroinflammation, in association with memory function under Alzheimer's disease. This study aims to explore the relationship of TREM2, microglia activation and neuroinflammation in the development of Alzheimer's disease, followed by investigating why physical exercises improve cognition in the Alzheimer's disease model by means of the adeno-associated virus (AAV) injection. We found that: 1) Recognition memory impairment in Aß-induced Alzheimer's disease model was associated with the reduction in TREM2 which induced microglial activation and neuroinflammation; 2) Exercise activated the TREM2 pathway, which was necessary for inhibiting microglial activation and neuroinflammation, leading to improved recognition memory in the Alzheimer's disease model. Together, the improvement of AD-associated recognition memory by exercises is associated with up-regulation of the TREM2 pathway which promotes the phenotypic conversion of microglia and decreases the level of neuroinflammation.
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Enfermedad de Alzheimer , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/metabolismo , Animales , Modelos Animales de Enfermedad , Ejercicio Físico , Hipocampo/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Transgénicos , Microglía/metabolismo , Enfermedades Neuroinflamatorias , Receptores Inmunológicos/metabolismoRESUMEN
Arsenic activates microglia and exerts bystander effects on neuron. The present study is focused to test whether minocycline, a second generation antibiotic, can reverse the effect of developmental arsenic exposure on microglial activation and function. Pregnant Balb/c dams were gavaged with sodium arsenite (0.38 mg/kg bd wt) from gestational day 5 (GD5) till post natal day 21 (PND21) and then one group of pups continued till PND59 with arsenic gavage. Minocycline (33 mg/kg bd wt) was administered intraperitoneally two weeks till sacrifice, every alternate day. Mice were sacrificed on PND22 and PND60 and used for various assays. Primary microglial were isolated (ex vivo microglia) from experimental animals and used to measure reactive oxygen species (ROS), nitric oxide (NO), cytokine production and phagocytosis. The whole brain lysate was used for western blot analysis of microglial marker CD68 and synaptic marker, post synaptic density protein 95 (PSD95). For real-time PCR analysis of triggering receptor expressed on myeloid cells 2 (TREM2) and PSD95, RNA isolated from whole brain was used. The study reveals that minocycline administration reversed arsenic-induced increased expression of CD68, ROS, NO, cytokine production, phagocytosis and TREM2 expression. Arsenic-induced reduced expression of PSD95 protein was reversed by minocycline, although the mRNA of PSD95 was unaltered among different groups. Finally, we have checked the learning and memory response of the experimental animals using Y-maze test to correlate the arsenic-induced altered level of synaptic protein. Taken together, the present study finds minocycline to reduce arsenic-induced microglial activation and function which in turn reverses the arsenic-induced impaired learning and memory response.
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Arsénico , Minociclina , Animales , Arsénico/metabolismo , Arsénico/toxicidad , Citocinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Endogámicos BALB C , Microglía , Minociclina/farmacología , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Inmunológicos/metabolismoRESUMEN
This study aimed to analyze the effects of fibrin constructs enhanced with laminin-nidogen, implanted in the wounded rat soft palate. Fibrin constructs with and without laminin-nidogen were implanted in 1 mm excisional wounds in the soft palate of 9-week-old rats and compared with the wounded soft palate without implantation. Collagen deposition and myofiber formation were analyzed at days 3, 7, 28 and 56 after wounding by histochemistry. In addition, immune staining was performed for a-smooth muscle actin (a-SMA), myosin heavy chain (MyHC) and paired homeobox protein 7 (Pax7). At day 56, collagen areas were smaller in both implant groups (31.25 ± 7.73% fibrin only and 21.11 ± 6.06% fibrin with laminin-nidogen)) compared to the empty wounds (38.25 ± 8.89%, p < 0.05). Moreover, the collagen area in the fibrin with laminin-nidogen group was smaller than in the fibrin only group (p Ë 0.05). The areas of myofiber formation in the fibrin only group (31.77 ± 10.81%) and fibrin with laminin-nidogen group (43.13 ± 10.39%) were larger than in the empty wounds (28.10 ± 11.68%, p Ë 0.05). Fibrin-based constructs with laminin-nidogen reduce fibrosis and improve muscle regeneration in the wounded soft palate. This is a promising strategy to enhance cleft soft palate repair and other severe muscle injuries.
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Fibrina/genética , Fibrosis/genética , Paladar Blando/lesiones , Cicatrización de Heridas/genética , Actinas/genética , Animales , Colágeno/genética , Fibrina/farmacología , Fibrosis/patología , Fibrosis/terapia , Humanos , Laminina/genética , Laminina/farmacología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/crecimiento & desarrollo , Miofibrillas/genética , Cadenas Pesadas de Miosina/genética , Factores de Transcripción Paired Box/genética , Paladar Blando/efectos de los fármacos , Paladar Blando/patología , Ratas , Regeneración/genéticaRESUMEN
We have previously demonstrated the ability of the human vaginal strain Lactobacillus crispatus 2029 (LC2029) for strong adhesion to cervicovaginal epithelial cells, expression of the surface layer protein 2 (Slp2), and antagonistic activity against urogenital pathogens. Slp2 forms regular two-dimensional structure around the LC2029 cells,which is secreted into the medium and inhibits intestinal pathogen-induced activation of caspase-9 and caspase-3 in the human intestinal Caco-2 cells. Here, we elucidated the effects of soluble Slp2 on adhesion of proteobacteria pathogens inducing necrotizing enterocolitis (NEC), such as Escherichia coli ATCC E 2348/69, E. coli ATCC 31705, Salmonella Enteritidis ATCC 13076, Campylobacter jejuni ATCC 29428, and Pseudomonas aeruginosa ATCC 27853 to Caco-2 cells, as well as on growth promotion, differentiation, vascular endothelial growth factor (VEGF) production, and intestinal barrier function of Caco-2 cell monolayers. Slp2 acts as anti-adhesion agent for NEC-inducing proteobacteria, promotes growth of immature Caco-2 cells and their differentiation, and enhances expression and functional activity of sucrase, lactase, and alkaline phosphatase. Slp2 stimulates VEGF production, decreases paracellular permeability, and increases transepithelial electrical resistance, strengthening barrier function of Caco-2 cell monolayers. These data support the important role of Slp2 in the early postnatal development of the human small intestine enterocytes.
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Diferenciación Celular , Enterocitos/metabolismo , Lactobacillus crispatus/química , Glicoproteínas de Membrana/farmacología , Vagina/microbiología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Adhesión Bacteriana/efectos de los fármacos , Células CACO-2 , Diferenciación Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Impedancia Eléctrica , Enterocitos/efectos de los fármacos , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Lactasa/genética , Lactasa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sacarasa/genética , Sacarasa/metabolismoRESUMEN
This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together In Vivo at neuromuscular junctions and in motor neurons.
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Glicoproteínas de Membrana/química , Metaloendopeptidasas/química , Fármacos Neuroprotectores/química , Oligopéptidos/química , Receptor trkB/química , Toxina Tetánica/química , Animales , Cristalografía por Rayos X , Humanos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacología , Metaloendopeptidasas/metabolismo , Metaloendopeptidasas/farmacología , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Dominios Proteicos , Ratas , Ratas Sprague-Dawley , Receptor trkB/metabolismo , Receptor trkB/farmacología , Toxina Tetánica/metabolismo , Toxina Tetánica/farmacologíaRESUMEN
Events mediated by the P-selectin/PSGL-1 pathway play a critical role in the initiation and propagation of venous thrombosis by facilitating the accumulation of leukocytes and platelets within the growing thrombus. Activated platelets and endothelium express P-selectin, which binds P-selectin glycoprotein ligand-1 (PSGL-1) that is expressed on the surface of all leukocytes. We developed a pegylated glycomimetic of the N terminus of PSGL-1, PEG40-GSnP-6 (P-G6), which proved to be a highly potent P-selectin inhibitor with a favorable pharmacokinetic profile for clinical translation. P-G6 inhibits human and mouse platelet-monocyte and platelet-neutrophil aggregation in vitro and blocks microcirculatory platelet-leukocyte interactions in vivo. Administration of P-G6 reduces thrombus formation in a nonocclusive model of deep vein thrombosis with a commensurate reduction in leukocyte accumulation, but without disruption of hemostasis. P-G6 potently inhibits the P-selectin/PSGL-1 pathway and represents a promising drug candidate for the prevention of venous thrombosis without increased bleeding risk.
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Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/uso terapéutico , Selectina-P/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Animales , Hemostasis/efectos de los fármacos , Humanos , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Endogámicos C57BL , Microcirculación/efectos de los fármacos , Selectina-P/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Polietilenglicoles/química , Polietilenglicoles/farmacología , Polietilenglicoles/uso terapéutico , Trombosis/metabolismoRESUMEN
Bile acids (BAs) are hydroxylated steroids derived from cholesterol that act at the intestinal level to facilitate the absorption of several nutrients and also play a role as signaling molecules. In the liver of various vertebrates, the trafficking of BAs is mediated by bile acid-binding proteins (L-BABPs). The ability to host hydrophobic or amphipathic molecules makes BABPs suitable for the distribution of a variety of physiological and exogenous substances. Thus, BABPs have been proposed as drug carriers, and more recently, they have also been employed to develop innovative nanotechnology and biotechnology systems. Here, we report an efficient protocol for the production, purification, and crystallization of chicken liver BABP (cL-BABP). By means of target expression as His6-tag cL-BABP, we obtained a large amount of pure and homogeneous proteins through a simple purification procedure relying on affinity chromatography. The recombinant cL-BABP showed a raised propensity to crystallize, allowing us to obtain its structure at high resolution and, in turn, assess the structural conservation of the recombinant cL-BABP with respect to the liver-extracted protein. The results support the use of recombinant cL-BABP for the development of drug carriers, nanotechnologies, and innovative synthetic photoswitch systems.
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Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/farmacología , Sistemas de Liberación de Medicamentos/métodos , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/farmacología , Secuencia de Aminoácidos/genética , Animales , Ácidos y Sales Biliares/metabolismo , Sitios de Unión/fisiología , Proteínas Portadoras/metabolismo , Pollos , Ácido Cólico/análisis , Ácido Cólico/química , Ácido Cólico/metabolismo , Cristalografía por Rayos X/métodos , Hígado/metabolismo , Hígado/patología , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Unión Proteica/fisiología , Proteínas Recombinantes/metabolismoRESUMEN
The abnormal expression of tropomyosin receptor kinase (Trk) serves an important role in the promotion of cancer progression. Homeobox C6 (HOXC6) and A disintegrin and metalloproteinase domaincontaining 8 (ADAM8) are associated with the invasiveness of cancer cells. However, the exact relationship between these molecules and their downstream signaling pathways in chemoresistant colon cancer cells are largely unknown. Therefore, the current study investigated the association between TrkB/C with HOXC6 and ADAM8 in the induction of drugresistant colon cancer cell metastasis. The results demonstrated that chemoresistant colon cancer cells exhibited upregulated TrkB/C, HOXC6 and ADAM8 expression. Additionally, but also chemoresistant colon cancer cells demonstrated higher migratory activities compared with parent colon cancer cells. The pharmacological inhibition of TrkB/C activity reduced the phosphorylation of mitogenactivated protein kinase kinase/ERK and subsequently suppressed HOXC6 and ADAM8 expression. In addition, gene silencing of HOXC6 inhibited ADAM8 and MMP activity, and inhibited the migration and invasion of drugresistant cancer cells. However, the targeted downregulation of ADAM8 using small interfering RNA failed to suppress TrkB/Cassociated ERKmediated HOXC6 signaling activity. Furthermore, pretreatment with ADAM10 and ADAM17specific inhibitors had no effect on attenuating the invasiveness of chemoresistant colon cancer cells. The results indicated that TrkB/Cmediated ERK activation serves an important role in the metastasis of drugresistant colon cancer cells through the regulation of HOXC6/ADAM8 activity.
Asunto(s)
Proteínas ADAM/metabolismo , Neoplasias del Colon/metabolismo , Genes Homeobox/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Receptor trkB/metabolismo , Receptor trkC/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Proteína ADAM17 , Antineoplásicos/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacología , Proteínas de la Membrana/genética , Receptor trkB/genética , Receptor trkB/farmacología , Receptor trkC/genética , Receptor trkC/farmacología , Transducción de Señal , Regulación hacia ArribaRESUMEN
BACKGROUND: Strategies to increase nucleic acid vaccine immunogenicity are needed to move towards clinical applications in oncology. In this study, we designed a new generation of DNA vaccines, encoding an engineered vesicular stomatitis virus glycoprotein as a carrier of foreign T cell tumor epitopes (plasmid to deliver T cell epitopes, pTOP). We hypothesized that pTOP could activate a more potent response compared with the traditional DNA-based immunotherapies, due to both the innate immune properties of the viral protein and the specific induction of CD4 and CD8 T cells targeting tumor antigens. This could improve the outcome in different tumor models, especially when the DNA-based immunotherapy is combined with a rational therapeutic strategy. METHODS: The ability of pTOP DNA vaccine to activate a specific CD4 and CD8 response and the antitumor efficacy were tested in a B16F10-OVA melanoma (subcutaneous model) and GL261 glioblastoma (subcutaneous and orthotopic models). RESULTS: In B16F10-OVA melanoma, pTOP promoted immune recognition by adequate processing of both MHC-I and MHC-II epitopes and had a higher antigen-specific cytotoxic T cell (CTL) killing activity. In a GL261 orthotopic glioblastoma, pTOP immunization prior to tumor debulking resulted in 78% durable remission and long-term survival and induced a decrease of the number of immunosuppressive cells and an increase of immunologically active CTLs in the brain. The combination of pTOP with immune checkpoint blockade or with tumor resection improved the survival of mice bearing, a subcutaneous melanoma or an orthotopic glioblastoma, respectively. CONCLUSIONS: In this work, we showed that pTOP plasmids encoding an engineered vesicular stomatitis virus glycoprotein, and containing various foreign T cell tumor epitopes, successfully triggered innate immunity and effectively promoted immune recognition by adequate processing of both MHC-I and MHC-II epitopes. These results highlight the potential of DNA-based immunotherapies coding for viral proteins to induce potent and specific antitumor responses.
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Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Vacunas contra el Cáncer/farmacología , Epítopos de Linfocito T/farmacología , Glioblastoma/tratamiento farmacológico , Inmunogenicidad Vacunal , Inmunoterapia , Glicoproteínas de Membrana/farmacología , Neoplasias/tratamiento farmacológico , Vacunas de ADN/farmacología , Proteínas del Envoltorio Viral/farmacología , Animales , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Terapia Combinada , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Glioblastoma/inmunología , Glioblastoma/metabolismo , Glioblastoma/patología , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunidad Innata/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
AIMS: The efficacy of anti-osteoporotic treatments is still limited. Our study aimed to investigate the effect of extracellular vesicles (EVs) derived from bone marrow-derived MSCs (BMSCs) overexpressing glycoprotein non-melanoma clone B (GPNMB) on osteoporosis (OP). MAIN METHODS: Lentiviral vector for GPNMB overexpression or its negative control was generated and transfected into BMSCs. EVs enriched with GPNMB (GPNMB-EVs) were extracted from GPNMB-modified BMSC-conditioned medium and then identified. Cellular uptake and proliferation were analyzed using the Dil-labeled assay and CCK-8 assay, respectively. Cytochemical staining, western blot, and RT-qPCR analysis were performed to assess the effect of GPNMB-EVs on osteogenic differentiation of BMSCs in vitro. Dickkopf-1 (DKK1) as the inhibitor was applied to explore the Wnt/ß-catenin signaling pathway involved in the GPNMB-EV-induced osteogenic differentiation. In vivo experiments were conducted using an ovariectomized (OVX) rat model of postmenopausal osteoporosis, and then assessed the effect of GPNMB-EVs by micro-CT, and histological and immunohistochemical assays. KEY FINDINGS: GPNMB-EVs were taken up by BMSCs, and they noticeably promoted the proliferation of BMSCs. Additionally, GPNMB-EVs activated the Wnt/ß-catenin signaling to stimulate osteogenesis in BMSCs. In vivo examination showed that GPNMB-EVs remarkably improved trabecular bone regeneration and alleviated the osteoporotic phenotype in the OVX-induced rat model of OP. SIGNIFICANCE: EVs derived from GPNMB-modified BMSCs significantly stimulated the proliferation and osteogenic differentiation of BMSCs via the activation of Wnt/ß-catenin signaling and attenuated the bone loss in the OVX-induced rat model of OP. Our findings suggest the promising potential of GPNMB-EVs as cell-free therapy for the treatment of OP.
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Vesículas Extracelulares/metabolismo , Glicoproteínas de Membrana/farmacología , Osteoblastos/metabolismo , Animales , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Huesos/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Femenino , Glicoproteínas de Membrana/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/fisiología , Osteogénesis/efectos de los fármacos , Osteoporosis/metabolismo , Ovariectomía , Ratas , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismoRESUMEN
P-glycoprotein (Pgp) detoxifies cells by exporting hundreds of chemically dissimilar hydrophobic and amphipathic compounds and is implicated in multidrug resistance (MDR) in the treatment of cancers. Photoaffinity labeling of plasma membrane vesicles of MDR CHO B30 cells with the anthracycline [125 I]-iodomycin, subsequent sequential cleavage with BNPS-skatol and endoproteinase Lys-C, and the Edman sequencing of the purified photoaffinity-labeled peptide identified the lysine residue at position 268 in the hamster Pgp primary sequence as the major photobinding site of iodomycin in CHO B30 cells. Lysine 268 is located adjacent to the cytosolic terminus of transmembrane 5. According to thermodynamic and kinetic analyses, this location should present the equilibrium binding site of ATP-free Pgp for daunomycin and iodomycin in B30 cells.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Sitios de Unión , Daunorrubicina/análogos & derivados , Lisina/química , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Daunorrubicina/química , Daunorrubicina/metabolismo , Humanos , Radioisótopos de Yodo/química , Radioisótopos de Yodo/metabolismo , Lisina/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/farmacología , Péptidos/química , Unión Proteica , Relación Estructura-ActividadRESUMEN
BACKGROUND: As a pattern recognition receptor, Toll-like receptor 7 (TLR7) widely presented in the endosomal membrane of various cells. However, the precise role and mechanism of TLR7 in septic cardiomyopathy remain unknown. This study aims to determine the role of TLR7 in cardiac dysfunction during sepsis and explore the mechanism of TLR7 in septic cardiomyopathy. METHODS: We generated a mouse model of septic cardiomyopathy by challenging with lipopolysaccharide (LPS). TLR7-knockout (TLR7-/- ), wild-type (WT) mice, cardiac-specific TLR7-transgenic (cTG-TLR7) overexpression, and littermates WT (LWT) mice were subjected to septic model. Additionally, to verify the role and mechanism of TLR7 in vitro, we transfected neonatal rat ventricular myocytes (NRVMs) with Ad-TLR7 and TLR7 siRNA before LPS administration. The effects of TLR7 were assessed by Ca2+ imaging, western blotting, immunostaining, and quantitative real-time polymerase chain reaction (qPCR). RESULTS: We found that TLR7 knockout markedly exacerbated sepsis-induced systolic dysfunction. Moreover, cardiomyocytes isolated from TLR7-/- mice displayed weaker Ca2+ handling than that in WT mice in response to LPS. Conversely, TLR7 overexpression alleviated LPS-induced systolic dysfunction, and loxoribine (TLR7-specific agonist) improved LPS-induced cardiac dysfunction. Mechanistically, these optimized effects were associated with enhanced the adenosine (cAMP)-protein kinase A (PKA) pathway, which upregulated phosphorylate-phospholamban (p-PLN) (Ser16) and promoted sarco/endoplasmic reticulum Ca2+ ATPase (Serca) and Ryanodine Receptor 2 (RyR2) expression in the sarcoplasmic reticulum (SR), and ultimately restored Ca2+ handling in response to sepsis. While improved Ca2+ handling was abrogated after H89 (a specific PKA inhibitor) pretreatment in cardiomyocytes isolated from cTG-TLR7 mice. Consistently, TLR7 overexpression improved LPS-induced Ca2+ -handling decrement in NRVMs. Nevertheless, TLR7 knockdown showed a deteriorative phenotype. CONCLUSIONS: Our data demonstrated that activation of TLR7 protected against sepsis-induced cardiac dysfunction through promoting cAMP-PKA-PLN pathway, and we revealed that TLR7 might be a novel therapeutic target to block the septic cardiomyopathy and support systolic function during sepsis.