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BACKGROUND: Facilitating the healing process of injured anterior cruciate ligament (ACL) tissue is crucial for patients to safely return to sports. Stem cell derived exosomes have shown positive effects on enhancing the regeneration of injured tendons/ligaments. However, clinical application of exosomes in terms of storage and pre-assembly is challenging. We hypothesized that lyophilized exosomes derived from human umbilical cord stem cells (hUSC-EX) could enhance the cell activity of chronically injured ACL cells. MATERIALS AND METHODS: We harvested the 8 weeks injured ACL cells from rabbit under IACUC (No. 110232) approval. The studied exosomes were purified from the culture medium of human umbilical cord stem cells (IRB approval No. A202205014), lyophilized to store, and hydrated for use. We compared exosome treated cells with non-exosome treated cells (control group) from the same rabbits. We examined the cell viability, proliferation, migration capability and gene expression of type I and III collagen, TGFß, VEGF, and tenogenesis in the 8 weeks injured ACL cells after hUSC-EX treatment. RESULTS: After hydration, the average size of hUSC-EX was 84.5 ± 70.6 nm, and the cells tested positive for the Alix, TSG101, CD9, CD63, and CD81 proteins but negative for the α-Tubulin protein. After 24 h of treatment, hUSC-EX significantly improved the cell viability, proliferation and migration capability of 8 weeks injured ACL cells compared to that of no exosome treatment group. In addition, the expression of collagen synthesis, TGFß, VEGF, and tenogenesis gene were all significantly increased in the 8 weeks injured ACL cells after 24 h hUSC-EX delivery. DISCUSSION: Lyophilized exosomes are easily stored and readily usable after hydration, thereby preserving their characteristic properties. Treatment with lyophilized hUSC-EX improved the activity and gene expression of 8 weeks injured ACL cells. CONCLUSION: Lyophilized hUSC-EX preserve the characteristics of exosomes and can improve chronically injured (8 weeks) ACL cells. Lyophilized hUSC-EX could serve as effective and safe biomaterials that are ready to use at room temperature to enhance cell activity in patients with partial ACL tears and after remnant preservation ACL reconstruction.
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Lesiones del Ligamento Cruzado Anterior , Exosomas , Animales , Conejos , Exosomas/metabolismo , Lesiones del Ligamento Cruzado Anterior/terapia , Humanos , Liofilización , Proliferación Celular , Cordón Umbilical/citología , Células Cultivadas , Supervivencia Celular/fisiología , Movimiento Celular/fisiología , Enfermedad CrónicaRESUMEN
BACKGROUND: Spermatogonial stem cells (SSCs) are essential for the maintenance and initiation of male spermatogenesis. Despite the advances in understanding SSC biology in mouse models, the mechanisms underlying human SSC development remain elusive. RESULTS: Here, we analyzed the signaling pathways involved in SSC regulation by testicular somatic cells using single-cell sequencing data (GEO datasets: GSE149512 and GSE112013) and identified that Leydig cells communicate with SSCs through pleiotrophin (PTN) and its receptor syndecan-2 (SDC2). Immunofluorescence, STRING prediction, and protein immunoprecipitation assays confirmed the interaction between PTN and SDC2 in spermatogonia, but their co-localization was observed only in approximately 50% of the cells. The knockdown of SDC2 in human SSC lines impaired cell proliferation, DNA synthesis, and the expression of PLZF, a key marker for SSC self-renewal. Transcriptome analysis revealed that SDC2 knockdown downregulated the expression of GFRA1, a crucial factor for SSC proliferation and self-renewal, and inhibited the HIF-1 signaling pathway. Exogenous PTN rescued the proliferation and GFRA1 expression in SDC2 knockdown SSC lines. In addition, we found downregulation of PTN and SDC2 as well as altered localization in non-obstructive azoospermia (NOA) patients, suggesting that downregulation of PTN and SDC2 may be associated with impaired spermatogenesis. CONCLUSIONS: Our results uncover a novel mechanism of human SSC regulation by the testicular microenvironment and suggest a potential therapeutic target for male infertility.
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Proteínas Portadoras , Proliferación Celular , Citocinas , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Células Intersticiales del Testículo , Sindecano-2 , Masculino , Humanos , Proliferación Celular/fisiología , Células Intersticiales del Testículo/metabolismo , Citocinas/metabolismo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Sindecano-2/metabolismo , Sindecano-2/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Supervivencia Celular/fisiología , Espermatogonias/metabolismo , Transducción de Señal/fisiología , Células Madre Germinales Adultas/metabolismo , Células Madre Germinales Adultas/fisiologíaRESUMEN
The Ribosomal S6 Kinase (RSK) family of serine/threonine kinases function as key downstream effectors of the MAPK signaling cascade. In the nervous system, RSK signaling plays crucial roles in neuronal development and contributes to activity-dependent neuronal plasticity. This study examined the role of RSK signaling in cell viability during neuronal development and in neuroprotection in the mature nervous system. Using neuronal cell-culture-based profiling, we found that suppressing RSK signaling led to significant cell death in developing primary neuronal cultures. To this end, treatment with the RSK inhibitors BiD1870 or SL0101 on the first day of culturing resulted in over 80% cell death. In contrast, more mature cultures showed attenuated cell death upon RSK inhibition. Inhibition of RSK signaling during early neuronal development also disrupted neurite outgrowth and cell growth. In maturing hippocampal explant cultures, treatment with BiD1870 had minimal effects on cell viability, but led to a striking augmentation of NMDA-induced cell death. Finally, we used the endothelin 1 (ET-1) model of ischemia to examine the neuroprotective effects of RSK signaling in the mature hippocampus in vivo. Notably, in the absence of RSK inhibition, the granule cell layer (GCL) was resistant to the effects of ET-1; However, disruption of RSK signaling (via the microinjection of BiD1870) prior to ET-1 injection triggered cell death within the GCL, thus indicating a neuroprotective role for RSK signaling in the mature nervous system. Together these data reveal distinct, developmentally-defined, roles for RSK signaling in the nervous system.
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Muerte Celular , Supervivencia Celular , Neuronas , Proteínas Quinasas S6 Ribosómicas , Transducción de Señal , Animales , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Células Cultivadas , Endotelina-1/farmacología , Endotelina-1/metabolismo , N-Metilaspartato/farmacología , Ratas Sprague-Dawley , Ratas , Neurogénesis/fisiología , Neurogénesis/efectos de los fármacos , PteridinasRESUMEN
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
The experimental study and transplantation of pancreatic islets requires their isolation from the surrounding tissue, and therefore, from the vasculature. Under these conditions, avascular islets rely on the diffusion of peripheral oxygen and nutrients to comply with the requirements of islet cells while responding to changes in body glucose. As a complement to the experimental work, computational models have been widely used to estimate how avascular islets would be affected by the hypoxic conditions found both in culture and transplant sites. However, previous models have been based on simplified representations of pancreatic islets which has limited the reach of the simulations performed. Aiming to contribute with a more realistic model of avascular human islets, in this work we used architectures of human islets reconstructed from experimental data to simulate the availability of oxygen for α, ß and δ-cells, emulating culture and transplant conditions at different glucose concentrations. The modeling approach proposed allowed us to quantitatively estimate how the loss of cells due to severe hypoxia would impact interactions between islet cells, ultimately segregating the islet into disconnected subnetworks. According to the simulations performed, islet encapsulation, by reducing the oxygen available within the islets, could severely compromise cell viability. Moreover, our model suggests that even without encapsulation, only microislets composed of less than 100 cells would remain viable in oxygenation conditions found in transplant sites. Overall, in this article we delineate a novel modeling methodology to simulate detailed avascular islets in experimental and transplant conditions with potential applications in the field of islet encapsulation.
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Supervivencia Celular , Simulación por Computador , Glucosa , Islotes Pancreáticos , Modelos Biológicos , Oxígeno , Humanos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/irrigación sanguínea , Oxígeno/metabolismo , Glucosa/metabolismo , Supervivencia Celular/fisiología , Biología Computacional , Trasplante de Islotes Pancreáticos/métodos , Hipoxia de la Célula/fisiologíaRESUMEN
Traumatic brain injury (TBI) presents a significant public health challenge, necessitating innovative interventions for effective treatment. Recent studies have challenged conventional perspectives on neurogenesis, unveiling endogenous repair mechanisms within the adult brain following injury. However, the intricate mechanisms governing post-TBI neurogenesis remain unclear. The microenvironment of an injured brain, characterized by astrogliosis, neuroinflammation, and excessive cell death, significantly influences the fate of newly generated neurons. Adenosine kinase (ADK), the key metabolic regulator of adenosine, emerges as a crucial factor in brain development and cell proliferation after TBI. This study investigates the hypothesis that targeting ADK could enhance brain repair, promote neuronal survival, and facilitate differentiation. In a TBI model induced by controlled cortical impact, C57BL/6 male mice received intraperitoneal injections of the small molecule ADK inhibitor 5-iodotubercidin (ITU) for three days following TBI. To trace the fate of TBI-associated proliferative cells, animals received intraperitoneal injections of BrdU for seven days, beginning immediately after TBI. Our results show that ADK inhibition by ITU improved brain repair 14 days after injury as evidenced by a diminished injury size. Additionally, the number of mature neurons generated after TBI was increased in ITU-treated mice. Remarkably, the TBI-associated pathological events including astrogliosis, neuroinflammation, and cell death were arrested in ITU-treated mice. Finally, ADK inhibition modulated cell death by regulating the PERK signaling pathway. Together, these findings demonstrate a novel therapeutic approach to target multiple pathological mechanisms involved in TBI. This research contributes valuable insights into the intricate molecular mechanisms underlying neurogenesis and gliosis after TBT.
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Adenosina Quinasa , Lesiones Traumáticas del Encéfalo , Diferenciación Celular , Supervivencia Celular , Ratones Endogámicos C57BL , Neurogénesis , Neuronas , Animales , Lesiones Traumáticas del Encéfalo/patología , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/metabolismo , Neurogénesis/efectos de los fármacos , Neurogénesis/fisiología , Masculino , Ratones , Adenosina Quinasa/antagonistas & inhibidores , Adenosina Quinasa/metabolismo , Diferenciación Celular/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Tubercidina/análogos & derivadosRESUMEN
Retinitis pigmentosa (RP), an inherited retinal disease, affects 1,5 million people worldwide. The initial mutation-driven photoreceptor degeneration leads to chronic inflammation, characterized by Müller cell activation and upregulation of CD44. CD44 is a cell surface transmembrane glycoprotein and the primary receptor for hyaluronic acid. It is involved in many pathological processes, but little is known about CD44's retinal functions. CD44 expression is also increased in Müller cells from our Pde6bSTOP/STOP RP mouse model. To gain a more detailed understanding of CD44's role in healthy and diseased retinas, we analyzed Cd44-/- and Cd44-/-Pde6bSTOP/STOP mice, respectively. The loss of CD44 led to enhanced photoreceptor degeneration, reduced retinal function, and increased inflammatory response. To understand the underlying mechanism, we performed proteomic analysis on isolated Müller cells from Cd44-/- and Cd44-/-Pde6bSTOP/STOP retinas and identified a significant downregulation of glutamate transporter 1 (SLC1A2). This downregulation was accompanied by higher glutamate levels, suggesting impaired glutamate homeostasis. These novel findings indicate that CD44 stimulates glutamate uptake via SLC1A2 in Müller cells, which in turn, supports photoreceptor survival and function.
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Células Ependimogliales , Receptores de Hialuranos , Retinitis Pigmentosa , Transducción de Señal , Animales , Receptores de Hialuranos/metabolismo , Receptores de Hialuranos/genética , Ratones , Células Ependimogliales/metabolismo , Transducción de Señal/fisiología , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patología , Retinitis Pigmentosa/genética , Ratones Noqueados , Ratones Endogámicos C57BL , Células Fotorreceptoras de Vertebrados/metabolismo , Supervivencia Celular/fisiología , Ratones Transgénicos , Retina/metabolismo , Retina/patologíaRESUMEN
Mesenchymal stem cells (MSCs) are involved in tissue repair and anti-inflammatory activities and have shown promising therapeutic efficiency in different animal models of neurodegenerative disorders. Microvesicles (MVs), implicated in cellular communication, are secreted from MSCs and play a key role in determining the fate of cell differentiation. Our study examines the effect of human umbilical cord MSC-derived MVs (hUC-MSC MVs) on the proliferation and differentiation potential of adult neural stem cells (NSCs). Results showed that 0.2 µg MSC derived MVs significantly increased the viability of NSCs and their proliferation, as demonstrated by an increase in the number of neurospheres and their derived cells, compared to controls. In addition, all hUC-MSC MVs concentrations (0.1, 0.2 and 0.4 µg) induced the differentiation of NSCs toward precursors (Olig2 + ) and mature oligodendrocytes (MBP+). This increase in mature oligodendrocytes was inversely proportional to the dose of MVs. Moreover, hUC-MSC MVs induced the differentiation of NSCs into neurons (ß-tubulin + ), in a dose-dependent manner, but had no effect on astrocytes (GFAP+). Furthermore, treatment of NSCs with hUC-MSC MVs (0.1 and 0.2 µg) significantly increased the expression levels of the proliferation marker Ki67 gene, compared to controls. Finally, hUC-MSC MVs (0.1 µg) significantly increased the expression level of Sox10 transcripts; but not Pax6 gene, demonstrating an increased NSC ability to differentiate into oligodendrocytes. In conclusion, our study showed that hUC-MSC MVs increased NSC proliferation in vitro and induced NSC differentiation into oligodendrocytes and neurons, but not astrocytes.
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Diferenciación Celular , Proliferación Celular , Micropartículas Derivadas de Células , Células Madre Mesenquimatosas , Células-Madre Neurales , Neurogénesis , Oligodendroglía , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Proliferación Celular/fisiología , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/fisiología , Células Cultivadas , Oligodendroglía/citología , Oligodendroglía/fisiología , Diferenciación Celular/fisiología , Células Madre Adultas/fisiología , Células Madre Adultas/citología , Animales , Factor de Transcripción PAX6/metabolismo , Supervivencia Celular/fisiologíaRESUMEN
Discrete components of tea possess multitude of health advantages. Escalating evidence advocate a consequential association between habitual tea consumption and a subsided risk of Parkinson's disease (PD). l-theanine is a non-protein amino acid inherent in tea plants, which exhibits structural resemblance with glutamate, the copious excitatory neurotransmitter in brain. Neuromodulatory effects of l-theanine are evident from its competency in traversing the blood brain barrier, promoting a sense of calmness beyond enervation, and enhancing cognition and attention. Despite the multifarious reports on antioxidant properties of l-theanine and its potential to regulate brain neurotransmitter levels, it is obligatory to understand its exact contribution in ameliorating the pathophysiology of PD. In this study, MPTP-induced mouse model was established and PD-like symptoms were developed in test animals where an increasing dosage of l-theanine (5, 25, 50, 100 and 250 mg/kg) was intraperitoneally administered for 23 days. 50 and 100 mg/kg dosage of l-theanine alleviated motor impairment and specific non-motor symptoms in Parkinsonian mice. The dosage of 100 mg/kg of l-theanine also improved striatal dopamine and serotonin level and tyrosine-hydroxylase positive cell count in the substantia nigra. Most crucial finding of the study is the proficiency of l-theanine to diminish astroglial injury as well as nitric oxide synthesis, which suggests its possible credential to prevent neurodegeneration by virtue of its anti-inflammatory attribute.
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Glutamatos , Té , Animales , Glutamatos/farmacología , Ratones , Té/química , Masculino , Ratones Endogámicos C57BL , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
BACKGROUND: Effective bone formation relies on osteoblast differentiation, a process subject to intricate post-translational regulation. Ubiquitin-specific proteases (USPs) repress protein degradation mediated by the ubiquitin-proteasome pathway. Several USPs have been documented to regulate osteoblast differentiation, but whether other USPs are involved in this process remains elusive. METHODS: In this study, we conducted a comparative analysis of 48 USPs in differentiated and undifferentiated hFOB1.19 osteoblasts, identifying significantly upregulated USPs. Subsequently, we generated USP knockdown hFOB1.19 cells and evaluated their osteogenic differentiation using Alizarin red staining. We also assessed cell viability, cell cycle progression, and apoptosis through MTT, 7-aminoactinomycin D staining, and Annexin V/PI staining assays, respectively. Quantitative PCR and Western blotting were employed to measure the expression levels of osteogenic differentiation markers. Additionally, we investigated the interaction between the USP and its target protein using co-immunoprecipitation (co-IP). Furthermore, we depleted the USP in hFOB1.19 cells to examine its effect on the ubiquitination and stability of the target protein using immunoprecipitation (IP) and Western blotting. Finally, we overexpressed the target protein in USP-deficient hFOB1.19 cells and evaluated its impact on their osteogenic differentiation using Alizarin red staining. RESULTS: USP36 is the most markedly upregulated USP in differentiated hFOB1.19 osteoblasts. Knockdown of USP36 leads to reduced viability, cell cycle arrest, heightened apoptosis, and impaired osteogenic differentiation in hFOB1.19 cells. USP36 interacts with WD repeat-containing protein 5 (WDR5), and the knockdown of USP36 causes an increased level of WDR5 ubiquitination and accelerated degradation of WDR5. Excessive WDR5 improved the impaired osteogenic differentiation of USP36-deficient hFOB1.19 cells. CONCLUSIONS: These observations suggested that USP36 may function as a key regulator of osteoblast differentiation, and its regulatory mechanism may be related to the stabilization of WDR5.
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Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Osteoblastos , Osteogénesis , Osteoblastos/metabolismo , Osteoblastos/citología , Diferenciación Celular/fisiología , Diferenciación Celular/genética , Humanos , Supervivencia Celular/fisiología , Supervivencia Celular/genética , Proliferación Celular/fisiología , Proliferación Celular/genética , Osteogénesis/fisiología , Osteogénesis/genética , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Línea Celular , Apoptosis/genética , Apoptosis/fisiología , Ubiquitinación , Técnicas de Silenciamiento del GenRESUMEN
Posttraumatic osteoarthritis (PTOA) is closely related to the inflammatory response caused by mechanical injury and leads to joint degeneration. Herein, we aimed to evaluate the role and underlying mechanism of NUMB in PTOA progression. Anterior cruciate ligament transection (ACLT)-induced rats and interleukin (IL)-1ß-treated chondrocytes were used as in vivo and in vitro models of PTOA, respectively. The NUMB overexpression plasmid (pcDNA-NUMB) was administered by intra-articular injection to PTOA model rats, and safranin O-fast green staining, the Osteoarthritis Research Society International (OARSI) scoring system, and HE staining were used to evaluate the severity of cartilage damage. The secretion of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) and chondrocyte-specific markers (MMP13 and COL2A1) was detected via ELISA. Cell viability and apoptosis were evaluated by MTT and TUNEL assays. NUMB was expressed at lower levels in ACLT-induced PTOA rats and in IL-1ß-treated chondrocytes than in control rats and cells. NUMB overexpression enhanced cell viability and reduced cell apoptosis, inflammation and cartilage degradation in chondrocytes stimulated by IL-1ß. NUMB bound to BTRC to promote p-IκBα expression, resulting in NF-κB pathway inactivation. BTRC overexpression reversed the promoting effect of NUMB overexpression on cell viability and the inhibitory effects of NUMB overexpression on apoptosis, inflammation and cartilage degradation in IL-1ß-induced chondrocytes. In addition, overexpression of NUMB alleviated articular cartilage damage by repressing inflammation and cartilage degradation in ACLT-induced PTOA rats. Our data indicated that NUMB regulated PTOA progression through the BTRC/NF-κB pathway, which may be a viable therapeutic target in PTOA.
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Condrocitos , Péptidos y Proteínas de Señalización Intracelular , FN-kappa B , Osteoartritis , Transducción de Señal , Animales , Masculino , Ratas , Lesiones del Ligamento Cruzado Anterior/complicaciones , Apoptosis , Supervivencia Celular/fisiología , Células Cultivadas , Condrocitos/metabolismo , Interleucina-1beta/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , FN-kappa B/metabolismo , Osteoartritis/etiología , Osteoartritis/metabolismo , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
OBJECTIVE: Brain tumors display remarkable cellular and molecular diversity, significantly impacting the progression and outcomes of the disease. The utilization of tumor tissue acquired through surgical handheld devices for tumor characterization raises important questions regarding translational research. This study seeks to evaluate the integrity of tissue resected using a microdebrider (MD) in the context of establishing tumor organoids from glioblastomas (GBM). METHODS: Tumor samples were collected from patients with GBM using both tumor forceps (en bloc) and a MD. The time required to protocol completion and cell viability of paired samples was measured. H&E staining was performed to examine histologic morphology. RESULTS: Ten paired samples were obtained from GBM patients using tumor forceps and the MD. Samples collected with the MD demonstrated significantly shorter processing times compared to those obtained through en bloc resection, with overall means of 31.7 ± 2.4 mins and 38.8±3 mins, respectively (P < 0.001). Cell viability measured at the end of protocol completion was comparable between tissues obtained using both the MD and en bloc, with mean viabilities of 80.2 ± 12.4% and 79.1 ± 12.5%, respectively (P = 0.848). H&E examination of tissues revealed no significant differences in the cellular and histologic characteristics of paired samples obtained using both methods across GBM tumors, nor in the corresponding established organoids. CONCLUSIONS: Tumor tissues obtained using the MD and en bloc methods demonstrate a high success rate in establishing GBM organoids, with the MD offering the advantage of significantly reduced processing time. Both methods display comparable cell viability and maintain consistent histologic characteristics in the resected tissue and the corresponding organoids.
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Neoplasias Encefálicas , Glioblastoma , Organoides , Humanos , Glioblastoma/patología , Glioblastoma/cirugía , Organoides/patología , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/cirugía , Femenino , Masculino , Persona de Mediana Edad , Recolección de Tejidos y Órganos/métodos , Supervivencia Celular/fisiología , Anciano , AdultoRESUMEN
Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by mutant ataxin-3 with an abnormally expanded polyQ tract and is the most common dominantly inherited ataxia worldwide. There are no suitable therapeutic options for this disease. Autophagy, a defense mechanism against the toxic effects of aggregation-prone misfolded proteins, has been shown to have beneficial effects on neurodegenerative diseases. Thus, trehalose, which is an autophagy inducer, may have beneficial effects on SCA3. In the present study, we examined the effects of trehalose on an SCA3 cell model. After trehalose treatment, aggregate formation, soluble ataxin-3 protein levels and cell viability were evaluated in HEK293T cells overexpressing ataxin-3-15Q or ataxin-3-77Q. We also explored the mechanism by which trehalose affects autophagy and stress pathways. A filter trap assay showed that trehalose decreased the number of aggregates formed by mutant ataxin-3 containing an expanded polyQ tract. Western blot and Cell Counting Kit-8 (CCK-8) results demonstrated that trehalose also reduced the ataxin-3 protein levels and was safe for ataxin-3-expressing cells, respectively. Western blot and total antioxidant capacity assays suggested that trehalose had great therapeutic potential for treating SCA3, likely through its antioxidant activity. Our data indicate that trehalose plays a neuroprotective role in SCA3 by inhibiting the aggregation and reducing the protein level of ataxin-3, which is also known to protect against oxidative stress. These findings provide a new insight into the possibility of treating SCA3 with trehalose and highlight the importance of inducing autophagy in SCA3.
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Ataxina-3 , Enfermedad de Machado-Joseph , Trehalosa , Trehalosa/farmacología , Humanos , Ataxina-3/metabolismo , Ataxina-3/genética , Enfermedad de Machado-Joseph/metabolismo , Enfermedad de Machado-Joseph/tratamiento farmacológico , Células HEK293 , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Autofagia/efectos de los fármacos , Autofagia/fisiología , Agregado de Proteínas/efectos de los fármacos , Mutación , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Agregación Patológica de Proteínas/metabolismo , PéptidosRESUMEN
Glucocorticoids (GCs) are widely used as powerful anti-inflammatory and immunosuppressive therapeutics in multiple pathological conditions. However, compelling evidence indicates that they might promote neurodegeneration by altering mitochondrial homeostatic processes. Although the effect of dexamethasone on cell survival and homeostasis has been widely investigated, the effect of other glucocorticoids needs to be explored in more detail. In this report, we have compared the neurotoxicity induced by dexamethasone, prednisolone, betamethasone, and hydrocortisone in cultured neuroblastoma cells, through the analysis of several parameters such as cell viability, ER stress, oxidative stress, and mitochondrial fusion and fission markers. Interestingly, we have found that synthetic glucocorticoids may impact neuronal viability by affecting different cellular responses, suggesting that their therapeutic use should be consciously decided after careful consideration of benefits and detrimental effects.
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Supervivencia Celular , Glucocorticoides , Neuroblastoma , Estrés Oxidativo , Glucocorticoides/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Neuroblastoma/patología , Línea Celular Tumoral , Humanos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/fisiología , Dexametasona/farmacología , Hidrocortisona/farmacología , Prednisolona/farmacología , Betametasona/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
BACKGROUND: Oxidative stress is a well-known pathological factor driving neuronal loss and age-related neurodegenerative diseases. Melatonin, coenzyme Q10 and lecithin are three common nutrients with an antioxidative capacity. Here, we examined the effectiveness of them administrated individually and in combination in protecting against oxidative stress-induced neuronal death in vitro, and neurodegenerative conditions such as Alzheimer's disease and associated deficits in vivo. METHODS: Mouse neuroblastoma Neuro-2a (N2a) cells were exposed with H2O2 for 6 h, and subsequently treated with melatonin, coenzyme Q10, and lecithin alone or in combination for further 24 h. Cell viability was assessed using the CCK-8 assay. Eight-week-old male mice were intraperitoneally injected with D-(+)-galactose for 10 weeks and administrated with melatonin, coenzyme Q10, lecithin, or in combination for 5 weeks starting from the sixth week, followed by behavioral tests to assess the effectiveness in mitigating neurological deficits, and biochemical assays to explore the underlying mechanisms. RESULTS: Exposure to H2O2 significantly reduced the viability of N2a cells and increased oxidative stress and tau phosphorylation, all of which were alleviated by treatment with melatonin, coenzyme Q10, lecithin alone, and, most noticeably, by combined treatment. Administration of mice with D-(+)-galactose-induced oxidative stress and tau phosphorylation, brain aging, impairments in learning and memory, anxiety- and depression-like behaviors, and such detrimental effects were mitigated by melatonin, coenzyme Q10, lecithin alone, and, most consistently, by combined treatment. CONCLUSIONS: These results suggest that targeting oxidative stress via supplementation of antioxidant nutrients, particularly in combination, is a better strategy to alleviate oxidative stress-mediated neuronal loss and brain dysfunction due to age-related neurodegenerative conditions.
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Antioxidantes , Peróxido de Hidrógeno , Neuronas , Estrés Oxidativo , Ubiquinona , Animales , Estrés Oxidativo/efectos de los fármacos , Ratones , Ubiquinona/análogos & derivados , Ubiquinona/farmacología , Ubiquinona/administración & dosificación , Masculino , Antioxidantes/farmacología , Peróxido de Hidrógeno/toxicidad , Neuronas/efectos de los fármacos , Neuronas/patología , Línea Celular Tumoral , Melatonina/farmacología , Melatonina/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Proteínas tau/metabolismo , Fármacos Neuroprotectores/farmacología , Galactosa/toxicidad , Quimioterapia CombinadaRESUMEN
BACKGROUND: The pathogenesis of acute lung injury (ALI) involves a severe inflammatory response, leading to significant morbidity and mortality. N6-methylation of adenosine (m6A), an abundant mRNA nucleotide modification, plays a crucial role in regulating mRNA metabolism and function. However, the precise impact of m6A modifications on the progression of ALI remains elusive. METHODS: ALI models were induced by either intraperitoneal injection of lipopolysaccharide (LPS) into C57BL/6 mice or the LPS-treated alveolar type II epithelial cells (AECII) in vitro. The viability and proliferation of AECII were assessed using CCK-8 and EdU assays. The whole-body plethysmography was used to record the general respiratory functions. M6A RNA methylation level of AECII after LPS insults was detected, and then the "writer" of m6A modifications was screened. Afterwards, we successfully identified the targets that underwent m6A methylation mediated by METTL3, a methyltransferase-like enzyme. Last, we evaluated the regulatory role of METTL3-medited m6A methylation at phosphatase and tensin homolog (Pten) in ALI, by assessing the proliferation, viability and inflammation of AECII. RESULTS: LPS induced marked damages in respiratory functions and cellular injuries of AECII. The m6A modification level in mRNA and the expression of METTL3, an m6A methyltransferase, exhibited a notable rise in both lung tissues of ALI mice and cultured AECII cells subjected to LPS treatment. METTL3 knockdown or inhibition improved the viability and proliferation of LPS-treated AECII, and also reduced the m6A modification level. In addition, the stability and translation of Pten mRNA were enhanced by METTL3-mediated m6A modification, and over-expression of PTEN reversed the protective effect of METTL3 knockdown in the LPS-treated AECII. CONCLUSIONS: The progression of ALI can be attributed to the elevated levels of METTL3 in AECII, as it promotes the stability and translation of Pten mRNA through m6A modification. This suggests that targeting METTL3 could offer a novel approach for treating ALI.
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Lesión Pulmonar Aguda , Células Epiteliales Alveolares , Proliferación Celular , Metiltransferasas , Ratones Endogámicos C57BL , Fosfohidrolasa PTEN , ARN Mensajero , Animales , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Ratones , Proliferación Celular/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/patología , Masculino , ARN Mensajero/metabolismo , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Metilación , Adenosina/análogos & derivados , Adenosina/metabolismo , Lipopolisacáridos/toxicidad , Estabilidad del ARN , Células CultivadasRESUMEN
Prolactin has been recognized as neuroprotective hormone against various types of neuronal damage. This study was aimed to determine if prolactin protects against streptozotocin injury. A series of experiments were performed to determine neuronal survival by counting total neurons in medial hippocampus cortex and cerebellum. Astrogliosis was determined by immunofluorescence assays using GFAP, and behavioral improvement by prolactin after neuronal damage was determined by open-field and light-dark box tests. Results demonstrated that prolactin induced significant neuronal survival in both the hippocampus and cortex, but not in the cerebellum. No increase in astrogliosis was identified, but a significant reduction in anxiety levels was observed. Overall data indicate that prolactin may protect against a complex form of cell damage including oxidant stress and metabolic disruption by streptozotocin. Prolactin may be helpful strategy in the treatment of neuronal damage in neurological diseases.
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Hipocampo , Neuronas , Fármacos Neuroprotectores , Prolactina , Estreptozocina , Animales , Prolactina/metabolismo , Masculino , Fármacos Neuroprotectores/farmacología , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Ratas , Neuroprotección/fisiología , Neuroprotección/efectos de los fármacos , Ratas Sprague-Dawley , Gliosis/metabolismo , Cerebelo/metabolismo , Cerebelo/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiologíaRESUMEN
As an element of the cellular signaling systems, extracellular vesicles (EVs) exhibit many desirable traits for usage as targeted delivery vehicles. When administered, EVs cause little to no toxic or immune response, stay in circulation for longer periods compared to synthetic carriers, preferentially accumulate in tissues that are the same or similar to their cell-of-origin and can pass through the blood-brain barrier. Combined, these traits make neural EVs a particularly promising tool for delivering drugs to the brain. This study aims to combine tissue and EVs engineering to prepare neural differentiated cells derived EVs that exhibit neural properties, to develop an effective, tissue-homing drug and gene delivery platform for the brain. Early neural differentiated cell-derived EVs were produced with neural characteristics from neural differentiated human neonatal dermal fibroblasts. The EVs carried key neural proteins such as Nestin, Sox2 and Doublecortin. The cellular uptake of early neural differentiated cell-derived EVs was higher compared to non-neural EVs during in vitro uptake assays on neuroblastoma cells. Moreover, eND-EVs were significantly decreased the viability of neuroblastoma cells. In conclusion, this study revealed that early neural differentiated cell-derived EVs have potential as a promising drug carrier for the treatment of various neural disorders.
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Vesículas Extracelulares , Células-Madre Neurales , Neuroblastoma , Vesículas Extracelulares/metabolismo , Humanos , Células-Madre Neurales/metabolismo , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Fibroblastos/metabolismo , Factores de Transcripción SOXB1RESUMEN
Background: Ferroptosis is extremely relevant to the progression of neurodegenerative pathologies such as Alzheimer's disease (AD). Ubiquitin-specific proteases (USP) can affect the NADPH oxidase family. Objective: Our study aimed to elucidate the potential role and molecular basis of a certain USP19 in reducing ferroptosis and mitochondrial injury in AD cells by targeting NOX4 stability. Methods: The deubiquitinase USP family gene USP19, which affects the stability of NOX4 protein, was first screened. The cell model of AD was constructed after interfering with SH-SY5Y cells by Aß1-40, and then SH-SY5Y cells were infected with lentiviral vectors to knock down USP19 and overexpress NOX4, respectively. Finally, the groups were tested for cell viability, changes in cellular mitochondrial membrane potential, lipid reactive oxygen species, intracellular iron metabolism, and NOX4, Mf1, Mf2, and Drp1 protein expression. Results: 5 µmol/L Aß1-40 intervened in SH-SY5Y cells for 24âh to construct a cell model of AD. Knockdown of USP19 decreased the expression of NOX4 protein, promoted the expression of mitochondrial fusion proteins Mnf1 and Mnf2, and inhibited the expression of the splitting protein Drp1. Furthermore, USP19 knockdown decreased mitochondrial membrane potential, SOD, MDA, intracellular iron content and increased GSH/GSSG ratio in SH-SY5Y cells. Our study revealed that NOX4 protein interacts with USP19 and knockdown of USP19 enhanced ubiquitination to maintain NOX4 protein stability. Conclusions: USP19 attenuates mitochondrial damage in SH-SY5Y cells by targeting NOX4 protein with Aß1-40.
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Péptidos beta-Amiloides , Ferroptosis , Mitocondrias , NADPH Oxidasa 4 , Humanos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Ferroptosis/fisiología , Ferroptosis/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , NADPH Oxidasa 4/metabolismo , NADPH Oxidasa 4/genética , Fragmentos de Péptidos/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Morphine (Mor) has exhibited efficacy in safeguarding neurons against ischemic injuries by simulating ischemic/hypoxic preconditioning (I/HPC). Concurrently, autophagy plays a pivotal role in neuronal survival during IPC against ischemic stroke. However, the involvement of autophagy in Mor-induced neuroprotection and the potential mechanisms remain elusive. Our experiments further confirmed the effect of Mor in cellular and animal models of ischemic stroke and explored its potential mechanism. The findings revealed that Mor enhanced cell viability in a dose-dependent manner by augmenting autophagy levels and autophagic flux in neurons subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Pretreatment of Mor improved neurological outcome and reduced infarct size in mice with middle cerebral artery occlusion/reperfusion (MCAO/R) at 1, 7 and 14 days. Moreover, the use of autophagy inhibitors nullified the protective effects of Mor, leading to reactive oxygen species (ROS) accumulation, increased loss of mitochondrial membrane potential (MMP) and neuronal apoptosis in OGD/R neurons. Results further demonstrated that Mor-induced autophagy activation was regulated by mTOR-independent activation of the c-Jun NH2- terminal kinase (JNK)1/2 Pathway, both in vitro and in vivo. Overall, these findings suggested Mor-induced neuroprotection by activating autophagy, which were regulated by JNK1/2 pathway in ischemic stroke.