RESUMEN
Astrocytes play many important functions in response to spinal cord injury (SCI) in an activated manner, including clearance of necrotic tissue, formation of protective barrier, maintenance of microenvironment balance, interaction with immune cells, and formation of the glial scar. More and more studies have shown that the astrocytes are heterogeneous, such as inflammatory astrocyte 1 (A1) and neuroprotective astrocyte 2 (A2) types. However, the subtypes of astrocyte resulting from SCI have not been clearly defined. In this study, using single-cell RNA sequencing, we constructed the transcriptomic profile of astrocytes from uninjured spinal cord tissue and injured tissue nearby the lesion epicenter at 0.5, 1, 3, 7, 14, 60, and 90 days after mouse hemisection spinal cord surgery. Our analysis uncovered six transcriptionally distinct astrocyte states, including Atp1b2+ , S100a4+ , Gpr84+ , C3+ /G0s2+ , GFAP+ /Tm4sf1+ , and Gss+ /Cryab+ astrocytes. We used these new signatures combined with canonical astrocyte markers to determine the distribution of morphologically and physiologically distinct astrocyte population at injured sites by immunofluorescence staining. Then we identified the dynamic evolution process of each astrocyte subtype following SCI. Finally, we also revealed the evolution of highly expressed genes in these astrocyte subtypes at different phases of SCI. Together, we provided six astrocyte subtypes at single-cell resolution following SCI. These data not only contribute to understand the heterogeneity of astrocytes during SCI but also help to find new astrocyte subtypes as a target for SCI repair.
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Proteínas de Transporte de Catión , Traumatismos de la Médula Espinal , Adenosina Trifosfatasas , Animales , Astrocitos/patología , Moléculas de Adhesión Celular Neuronal , Gliosis/patología , Ratones , Receptores Acoplados a Proteínas G , Médula Espinal/patología , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patologíaRESUMEN
Liver serine-threonine kinase B1 (LKB1) is a tumor suppressor that has been linked to many types of tumors. However, the role of LKB1 in cartilaginous tumorigenesis is still poorly understood. In this study, we find that cartilage-specific, tamoxifen-inducible Lkb1 knockout results in multiple enchondroma-like lesions adjacent to the disorganized growth plates. We showed that chondrocytes retain an immature status caused by loss of Lkb1, which may lead to the dramatic expansion of growth-plate cartilage and the formation of enchondroma-like lesions. Additionally, increased mammalian target of rapamycin complex 1 (mTORC1) activity is observed in the Lkb1 conditional knockout (cKO) chondrocytes, and rapamycin (mTORC1 inhibitor) treatment significantly alleviates the expansion of growth-plate cartilage and eliminates the enchondroma-like lesions in Lkb1 cKO mice. Thus, our findings indicate that loss of Lkb1 leads to the expansion of chondrocytes and the formation of enchondroma-like lesions during postnatal cartilage development, and that the up-regulated mTORC1-signaling pathway is implicated in this process. Our findings suggest that modulation of LKB1 and related signaling is a potential therapy in cartilaginous tumorigenesis.-Zhou, S., Li, Y., Qiao, L., Ge, Y., Huang, X., Gao, X., Ju, H., Wang, W., Zhang, J., Yan, J., Teng, H., Jiang, Q. Inactivation of Lkb1 in postnatal chondrocytes leads to epiphyseal growth-plate abnormalities and promotes enchondroma-like formation.
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Condrocitos/citología , Condrocitos/metabolismo , Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Animales , Western Blotting , Cartílago/efectos de los fármacos , Cartílago/metabolismo , Cartílago/patología , Células Cultivadas , Condrogénesis/efectos de los fármacos , Condroma/tratamiento farmacológico , Condroma/metabolismo , Condroma/patología , Femenino , Técnica del Anticuerpo Fluorescente , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Sirolimus/uso terapéutico , Tomografía Computarizada por Rayos XRESUMEN
Developmental dysplasia of the hip (DDH) is one of the most common congenital malformations and covers a spectrum of hip disorders from mild dysplasia to irreducible dislocation. The pathological mechanisms of DDH are poorly understood, which hampers the development of diagnostic tools and treatments. To gain insight into its disease mechanism, we explored the potential biological processes that underlie DDH by integrating pathway analysis tools and performing a genome-wide association study (GWAS). A total of 406 DDH-associated genes (P < 0.001) were identified by our GWAS using a Chinese Han cohort consisting of 386 DDH cases and 500 healthy controls (Set A). We verified the significant loci (P < 10-5 ) in another Chinese Han cohort consisting of 574 DDH patients and 569 healthy controls (Set B). An intronic Single Nucleotide Polymorphism (SNP) (rs61930502) showed significant association in Set A and Set B (P = 2.65 × 10-7 and 2.0 × 10-4 , respectively). The minor allele, rs61930502-A, which tended to prevent DDH showed a dominant effect. Heat shock 70 kDa protein 8 (HSPA8) showed the most direct interactions with other proteins which were coded by DDH-associated genes in the protein-protein interaction analysis. Interestingly, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested a relation between DDH and the genes involved in type II diabetes mellitus pathway (P = 0.0067). Our genetic and protein interaction evidence could open avenues for future studies of DDH.
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Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Luxación Congénita de la Cadera/diagnóstico , Luxación Congénita de la Cadera/genética , Alelos , Estudios de Casos y Controles , Biología Computacional/métodos , Perfilación de la Expresión Génica , Variación Genética , Humanos , Fenotipo , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , RadiografíaRESUMEN
The Polycomb group (PcG) proteins have an important role in controlling the expression of key genes implicated in embryonic development, differentiation, and decision of cell fates. Emerging evidence suggests that Polycomb repressive complexes 1 (PRC1) is defined by the six Polycomb group RING finger protein (Pcgf) paralogs, and Pcgf proteins can assemble into noncanonical PRC1 complexes. However, little is known about the precise mechanisms of differently composed noncanonical PRC1 in the maintenance of the pluripotent cell state. Here we disrupt the Pcgf genes in mouse embryonic stem cells by CRISPR-Cas9 and find Pcgf6 null embryonic stem cells display severe defects in self-renewal and differentiation. Furthermore, Pcgf6 regulates genes mostly involved in differentiation and spermatogenesis by assembling a noncanonical PRC1 complex PRC1.6. Notably, Pcgf6 deletion causes a dramatic decrease in PRC1.6 binding to target genes and no loss of H2AK119ub1. Thus, Pcgf6 is essential for recruitment of PRC1.6 to chromatin. Our results reveal a previously uncharacterized, H2AK119ub1-independent chromatin assembly associated with PRC1.6 complex.
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Células Madre Embrionarias/citología , Complejo Represivo Polycomb 1/fisiología , Proteínas del Grupo Polycomb/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ratones , Complejo Represivo Polycomb 1/genéticaRESUMEN
BACKGROUND: To explore the therapeutic effect and the biomechanical mechanism of 3D printing individualized heel cup in treating of plantar heel pain. METHODS: The clinical effect was evaluated by plantar pressure analysis and pain assessment in participants. Its biomechanical mechanism of protecting the plantar heel was explored using finite element simulation. RESULTS: The individualized heel cup could support and protect the osseous structure and soft tissue of plantar heel while walking and jogging, as well as significantly reduce the self-reported pain after being worn for 4 weeks. The nylon heel cup could alter the load concentration of the heel as well as decrease the load affected on plantar fascia and calcaneus bone. It also provided an obvious support for heel pad. CONCLUSION: To summarize, the 3D printed individualized heel cup can be used as an effective method for the treatment of plantar heel pain.
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Fascitis Plantar/patología , Talón/patología , Dolor/patología , Impresión Tridimensional , Autoinforme , Simulación por Computador , Fascitis Plantar/fisiopatología , Análisis de Elementos Finitos , Talón/fisiopatología , Humanos , Dolor/fisiopatología , Presión , Reproducibilidad de los Resultados , CaminataRESUMEN
Bone fracture healing is achieved through the proliferation and differentiation of stem cells, while bone marrow stem cells (BMSCs) contribute to endochondral ossification. During fracture healing, mesenchymal progenitor cells first form a cartilaginous blastema that becomes vascularized to recruit precursor cells of osteoblasts through the bone morphogenetic protein 2 (Bmp2)/Smad-dependent Runx2 pathway. Statins deplete geranylgeranyl diphosphate (GGPP), which participates in the regulation of BMSCs differentiation, through the inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, leading to impaired protein geranylgeranylation, which strongly impacts the bone synthesis induced by Bmp2. Accordingly, we would like to investigate the role of geranylgeranyl diphosphate synthase 1 (Ggps1) in bone fracture via endochondral ossification in mice. We used a Cre-loxP system, namely the tamoxifen-inducible Collagen 2-CreERT2 Ggps1 fl/fl, to eliminate specifically the Ggps1 activity in chondrocytes of 8-10-week-old mice. We found that the endochondral bone formation, calcification and vasculogenesis of the bony callus were accelerated in fractures in Ggps1-/-mice. Together, the results of this study confirm that the specific deletion of Ggps1, using the Collagen 2-CreERT2 mice, will accelerate the fracture healing process by activating the Bmp2/Smad-dependent Runx2 pathway. In addition, we managed to improve the fracture healing process by inhibiting the Ggps1 activity and its related products with statin drugs.
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Condrocitos/metabolismo , Farnesiltransferasa/metabolismo , Curación de Fractura , Técnicas de Silenciamiento del Gen , Complejos Multienzimáticos/metabolismo , Osteogénesis , Animales , Biomarcadores/metabolismo , Fenómenos Biomecánicos , Proteína Morfogenética Ósea 2/metabolismo , Callo Óseo/metabolismo , Callo Óseo/patología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Farnesiltransferasa/deficiencia , Fémur/irrigación sanguínea , Fémur/diagnóstico por imagen , Fémur/patología , Fémur/fisiopatología , Curación de Fractura/genética , Regulación de la Expresión Génica , Antígeno Ki-67/metabolismo , Masculino , Ratones Noqueados , Complejos Multienzimáticos/deficiencia , Neovascularización Fisiológica , Osteogénesis/genética , Transducción de Señal , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: This study is to investigate the effect of fenofibrate on the bone quality of Type 2 diabetes mellitus (T2DM) mouse model. METHODS: T2DM mouse model was induced by high-fat-diet, and the mice were treated with fenofibrate (100 mg/kg) (DIO-FENO) or PBS (DIO-PBS) for 4 weeks. The bone microstructure and biomechanical properties of femora were analyzed by micro-CT and 3-Point bending test. The protein expression was detected by immunohistochemical staining and Western blot. The cell apoptosis was evaluated by TUNEL staining. The Bcl2, caspase 3, and osteoblast marker genes were detected by RT-qPCR. RESULTS: The biomechanical properties of bones from DIO-FENO group were significantly lower than those in the control and DIO-PBS groups. Besides, the trabecular number was lower than those of the other groups, though the cortical porosity was decreased compared with that of DIO-PBS group because of the increase of apoptotic cells. The expression of osteocalcin and collagen I were decreased after treatment with fenofibrate in T2DM mice. Moreover, the cell viability was decreased after treated with different concentrations of fenofibrate, and the expression of Runx2 decreased after treated with high dose of fenofibrate. CONCLUSION: Fenofibrate decreases the bone quality of T2DM mice through decreasing the expression of collagen I and osteocalcin, which may be resulted from the down regulation of Runx2 expression.
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Densidad Ósea/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Diabetes Mellitus Tipo 2/genética , Dislipidemias/genética , Fenofibrato/efectos adversos , Hipolipemiantes/efectos adversos , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Dislipidemias/etiología , Dislipidemias/metabolismo , Dislipidemias/patología , Regulación de la Expresión Génica , Humanos , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteocalcina/genética , Osteocalcina/metabolismo , Porosidad/efectos de los fármacos , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal , Microtomografía por Rayos XRESUMEN
Oxygen-sensitive accumulation and degradation, two opposite but intrinsically linked events, of heme proteins in mitochondria affect mitochondrial functions, including bioenergetics and oxygen-sensing processes. Cystathionine ß-synthase (CBS) contains a prosthetic heme group and catalyzes the production of hydrogen sulfide in mammalian cells. Here we show that CBS proteins were present in liver mitochondria at a low level under normoxia conditions. Ischemia/hypoxia increased the accumulation of CBS proteins in mitochondria. The normalization of oxygen partial pressure accelerated the degradation of CBS proteins. Lon protease, a major degradation enzyme in mitochondrial matrix, recognized and degraded mitochondrial CBS by specifically targeting at the oxygenated heme group of CBS proteins. The accumulation of CBS in mitochondria increased hydrogen sulfide production, which prevented Ca(2+)-mediated cytochrome c release from mitochondria and decreased reactive oxygen species generation. Mitochondrial accumulation of heme oxygenase-1, another heme protein, was also regulated by oxygen level and Lon protease in the same mechanism as for CBS. Our findings provide a fundamental and general mechanism for oxygen-sensitive regulation of mitochondrial functions by linking oxygenation level to the accumulation/degradation of mitochondrial heme proteins.
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Cistationina betasintasa/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas Mitocondriales/metabolismo , Oxígeno/metabolismo , Proteasa La/metabolismo , Proteolisis , Animales , Células HEK293 , Hemo/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Humanos , Sulfuro de Hidrógeno/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
A growing body of work has shown that the highly homologous T-box transcription factors TBX2 and TBX3 play critical but distinct roles in embryonic development and cancer progression. For example, TBX2 and TBX3 are up-regulated in several cancers and recent evidence suggests that whereas TBX2 functions as a pro-proliferative factor, TBX3 inhibits cell proliferation but promotes cancer cell migration and invasion. While the molecular mechanisms regulating these functions of TBX2 and TBX3 are poorly understood we recently reported that the TGF-ß1 signaling pathway up-regulates TBX3 expression to mediate, in part, its well described anti-proliferative and pro-migratory roles. The TBX3 targets responsible for these functions were however not identified. Here we reveal for the first time that the TGF-ß1 signaling pathway represses TBX2 transcriptionally and we provide a detailed mechanism to show that this is mediated by TBX3. Furthermore, we implicate the down-regulation of TBX2 in the anti-proliferative function of the TGF-ß1-TBX3 axis. These findings have important implications for our understanding of the regulation of TBX2 and TBX3 and shed light on the mechanisms involved in the anti-proliferative and pro-migratory roles of TGF-ß1.
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Proliferación Celular/efectos de los fármacos , Proteínas de Dominio T Box/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Animales , Sitios de Unión/genética , Western Blotting , Línea Celular , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Mutación , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Proteínas de Dominio T Box/genéticaRESUMEN
BACKGROUND: Ischemic preconditioning (IPC) has many beneficial effects on the cardiovascular system. However, whether localized lower extremity IPC could be protective against the thrombogenic activity generated by lower extremity ischemia is unclear. MATERIAL AND METHODS: 41 male Sprague-Dawley rats were randomly assigned to either a IPC group or a sham group. The lower extremity blood inflow was previously treated with 4 cycles of 5 min ischemia followed by 5 min of reperfusion by clamping the abdominal aortic just before ligature of the left iliac vein(LIV) in the IPC group. Rats in the sham group had a 40-minute blank before left iliac vein ligation. The rats were euthanized at day 2 after ligation and the thrombosed LIV was carefully dissected out, while thrombi harvested from the LIV were measured with weight (g), length (mm) and weight/length (mg/mm). Influence of IPC on coagulation function was also tested. RESULTS: 21 and 20 rats were randomly assigned to einter the IPC group or the control group. Left iliac vein thrombosis was successfully generated in all 41 rats. IPC significantly protects the rats from experimental lower extremity thrombosis. Compared to control group, generated thrombus in rats in the IPC group showed significantly lower weight (2.73 ± 0.16 mg vs 1.82 ± 0.13 mg, P < 0.001), length (2.99 ± 0.17 mm vs 2.44 ± 0.08 mm, P < 0.009) and density (0.95 ± 0.05 mg/mm vs 0.75 ± 0.05 mg/mm, P = 0.01). Influence on coagulation function by IPC itself was not significant (P > 0.05). CONCLUSIONS: Our study demonstrated that localized lower extremity IPC could reduce DVT formation in rats in an in vivo experimental thrombosis model.
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Precondicionamiento Isquémico/métodos , Extremidad Inferior/irrigación sanguínea , Trombosis de la Vena/prevención & control , Animales , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Osteoarthritis (OA) is the most common degenerative joint disorder, and a major cause of pain and disability among the elderly. Histone deacetylase 4 (HDAC4) has been shown to be a key regulator of chondrocyte hypertrophy during skeletogenesis. The aims of present study were to investigate the expression of HDAC4 in normal and OA cartilage and its potential roles during OA pathogenesis. METHODS: The knee cartilage specimen (a total of 18, 12 female and 6 male) were obtained from primary OA patients undergoing total knee arthroplasty (TKA) and normal donors. By using immunohistochemistry staining, we detected the expression patterns of HDAC4 in OA cartilage and normal cartilage respectively. To assess the potential roles of HDAC4, HDAC4 expression in human chondrosarcoma cells (SW1353) was down-regulated by transfecting small interference RNA (siRNA), thereafter, cells were treated with IL-1ß or TNF-α, and the expressions of several matrix-degrading enzymes and anabolic factors were examined by using quantitative PCR. RESULTS: The expression of HDAC4 was observed in the OA cartilage, whereas it was barely detected in the normal cartilage. The extent of HDAC4 expression had a statistically negative correlation with OA severity. We further explored that the reduction of HDAC4 level led to a significant repression of proinflammation cytokines induced up-regulated expressions of matrix-degrading enzymes (MMP1 (Matrix metalloproteinase 1), MMP3 (Matrix metalloproteinase 3) , MMP13 (Matrix metalloproteinase 13), ADAMTS4 (aggrecanase 1) and ADAMTS5 (aggrecanase 2)) in SW1353 in vitro. Moreover, knockdown of HDAC4 inhibited the expression of some anabolic genes (such as aggrecan). CONCLUSIONS: In this study, our findings suggest that the abnormal expression of HDAC4 in osteoarthritic cartilage might be implicated in promoting catabolic activity of chondrocyte, which is associated with OA pathogenesis. Thus, our findings give a new insight into the mechanism of articular cartilage damage, and indicate that HDAC4 might be a potential target for the therapeutic interventions of OA.
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Cartílago Articular/enzimología , Histona Desacetilasas/biosíntesis , Homeostasis/fisiología , Osteoartritis de la Rodilla/enzimología , Proteínas Represoras/biosíntesis , Anciano , Anciano de 80 o más Años , Artroplastia de Reemplazo de Rodilla/tendencias , Cartílago Articular/patología , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoartritis de la Rodilla/diagnóstico , Osteoartritis de la Rodilla/cirugíaRESUMEN
Cystathionine γ-lyase (CSE) is the major enzyme in vascular smooth muscle cells (SMCs) that catalyzes the endogenous production of H(2)S. Phenotypic switching of SMCs is affected by endogenous H(2)S level and alterations of this switching may result in vascular disorders. To date, the mechanisms underlying the alteration of CSE expression and H(2)S production in vascular proliferative diseases have been unclear. In the present study, we found that serum deprivation induced SMC differentiation marker gene expressions and increased CSE expression and H(2)S production in cultured human aorta SMCs (HASMCs). Carotid artery ligation in mice resulted in enhanced neointima formation and down-regulation of CSE expression, suggesting an important role of CSE in SMC differentiation. Transient transfection of HASMCs with human CSE (hCSE) promoter/luciferase reporter revealed that the region between -226 to +140 base pair contains the core promoter for the hCSE gene. Deletion and mutation analysis demonstrated that two specificity protein-1 (Sp1) consensus binding sites were present in the core promoter region of the hCSE gene. Incubation of HASMCs with Sp1 binding inhibitor mithramycin inhibited CSE mRNA expression in a dose-dependent manner. Overexpression of Sp1 alone was sufficient to increase the activity of the hCSE core promoter and CSE protein expression. Chromatin immunoprecipitation assay showed that the binding of Sp1 to the hCSE promoter was increased in differentiated HASMCs compared with that in proliferated HASMCs. Exogenously applied H(2)S at 100 µM stimulated SMC differentiation, which was reversed by p38 MAPK inhibitor SB203580. These results suggest that transcript factor Sp1 is a critical regulator of the hCSE expression during SMC differentiation, and CSE/H(2)S system is essential for maintenance of SMC phenotype.
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Cistationina gamma-Liasa/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas Musculares/metabolismo , Miocitos del Músculo Liso/metabolismo , Elementos de Respuesta/fisiología , Factor de Transcripción Sp1/metabolismo , Contaminantes Atmosféricos/farmacología , Animales , Antibióticos Antineoplásicos/farmacología , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Cistationina gamma-Liasa/genética , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Sulfuro de Hidrógeno/farmacología , Imidazoles/farmacología , Ratones , Ratones Noqueados , Proteínas Musculares/genética , Miocitos del Músculo Liso/citología , Plicamicina/farmacología , Piridinas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factor de Transcripción Sp1/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
In the widely used Carbon tetrachloride (CCl4)-induced acute liver injury (ALI) mouse model, hepatocytes are known to die from programmed cell death (PCD) processes including apoptosis and necroptosis. Both in vivo and in vitro experiments showed that CCl4 treatment could induce both apoptosis and necroptosis. Treatment of mice with the apoptosis inducer SMAC mimetic reduced necroptosis, led to less pronounced liver damage, and improved overall liver function. By LC-MS/MS, we found that PP2Acα expression was increased in ALI mice liver, and we confirmed its high expression in subacute hepatitis patients. We observed that ALI severity (including aggravated fibrogenesis) was significantly alleviated in hepatocyte-specific PP2Acα conditional knockout (PP2Acα cKO) mice. Furthermore, the relative extent of apoptosis over necroptosis was increased in the PP2Acα cKO ALI mice. Pursuing the idea that biasing the type of PCD towards apoptosis may reduce liver damage, we found that treatment of PP2Acα cKO ALI mice with the apoptosis inhibitor z-Vad-fmk increased the extent of necroptosis and caused severer damage. Mechanistically, disruption of PP2Acα prevents the dephosphorylation of pASK1(Ser967), thereby preventing the sustained activation of JNK. Inhibition of PP2Acα prevents CCl4-induced liver injury and fibrogenesis by disrupting ASK/JNK pathway mediated PCD signaling, ultimately improving liver function by biasing hepatocytes towards an apoptotic rather than necroptotic cell fate. Thus, targeting PP2A and/or ASK1 to favor apoptotic over necroptotic hepatocyte fate may represent an attractive therapeutic strategy for treating ALI.
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Hepatopatías , Sistema de Señalización de MAP Quinasas , Ratones , Animales , Cromatografía Liquida , Espectrometría de Masas en Tándem , Necrosis/patología , Hepatocitos/metabolismo , Hepatopatías/metabolismo , Ratones Noqueados , FibrosisRESUMEN
Mechanical compression is a double-edged sword for cartilage remodeling, and the effect of mechanical compression on chondrogenic differentiation still remains elusive to date. Herein, we investigate the effect of mechanical dynamic compression on the chondrogenic differentiation of human synovium-derived mesenchymal stem cells (SMSCs). To this aim, SMSCs encapsulated in agarose hydrogels were cultured in chondrogenic-induced medium with or without dynamic compression. Dynamic compression was applied at either early time-point (day 1) or late time-point (day 21) during chondrogenic induction period. We found that dynamic compression initiated at early time-point downregulated the expression level of chondrocyte-specific markers as well as hypertrophy-specific markers compared with unloaded control. On the contrary, dynamic compression applied at late time-point not only enhanced the levels of cartilage matrix gene expression, but also suppressed the hypertrophic development of SMSCs compared with unloaded controls. Taken together, our findings suggest that dynamic mechanical compression loading not only promotes chondrogenic differentiation of SMSCs, but also plays a vital role in the maintenance of cartilage phenotype, and our findings also provide an experimental guide for stem cell-based cartilage repair and regeneration.
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OBJECTIVE: To investigate the relationship between leptin, osteopontin (OPN), sclerostin (SOST) and severity of knee osteoarthritis (KOA). METHODS: The study included 148 consecutive patients with knee OA and 101 non-KOA subjects enrolled in this cross-sectional study. All patients fulfilled the American College of Rheumatology criteria for primary knee OA. Severity of the disease was assessed using plain radiography of the affected knee, according to the Kellgren and Lawrence classification. Fasting blood samples were obtained from all patients and controls; the serum samples were kept at - 80 °C before assessment of leptin, OPN, and SOST using a multiplex particle-based flow cytometric assay. RESULTS: KOA patients group compared with the control group, serum leptin (KOA, 26581.7 ± 2011.5 pg/ml, vs control,6936.4 ± 702.2 pg/ml),OPN (KOA, 4908.3 ± 769.4 pg/ml, vs control, 2182.5 ± 217.8 pg/ml), and SOST (KOA, 2481.9 ± 543.5 pg/ml, vs control, 1288.9 ± 267.7 pg/ml) in the KOA group were higher than control group; there were also differences in three bone metabolic factors between male and female in the KOA group; meanwhile, there was correlation between each factor and the incidence of KOA. CONCLUSION: Our study of 249 serum samples was conducted. Serum leptin, OPN, and SOST were significantly increased in KOA patients, and there was an internal correlation; these findings could, at best, contribute to the identification of novel targets for medical interventions. Key Points ⢠The aim of this study was to assess the relationships of radiographic knee OA with altered serum levels of leptin, OPN, and SOST. Our study of 249 serum samples was conducted. Serum leptin, OPN, and SOST were significantly increased in KOA patients compared with control group. There were gender differences in the concentration of three serum bone turnover factors in KOA group and control group. Serum SOST concentration increased with Kellgren-Lawrence (K-L) grading. We found that serum leptin, OPN, and SOST were significantly increased in KOA patients, and there was an internal correlation. Leptin had a remarkable diagnostic value in the incidence of KOA.
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Osteoartritis de la Rodilla , Osteopontina , Estudios Transversales , Femenino , Humanos , Articulación de la Rodilla , Leptina , Masculino , Osteoartritis de la Rodilla/diagnóstico por imagenRESUMEN
Tendons and tendon interfaces have a very limited regenerative capacity, rendering their injuries clinically challenging to resolve. Tendons sense muscle-mediated load; however, our knowledge on how loading affects tendon structure and functional adaption remains fragmentary. Here, we provide evidence that the matricellular protein secreted protein acidic and rich in cysteine (SPARC) is critically involved in the mechanobiology of tendons and is required for tissue maturation, homeostasis, and enthesis development. We show that tendon loading at the early postnatal stage leads to tissue hypotrophy and impaired maturation of Achilles tendon enthesis in Sparc -/- mice. Treadmill training revealed a higher prevalence of spontaneous tendon ruptures and a net catabolic adaptation in Sparc -/- mice. Tendon hypoplasia was attenuated in Sparc -/- mice in response to muscle unloading with botulinum toxin A. In vitro culture of Sparc -/- three-dimensional tendon constructs showed load-dependent impairment of ribosomal S6 kinase activation, resulting in reduced type I collagen synthesis. Further, functional calcium imaging revealed that lower stresses were required to trigger mechanically induced responses in Sparc -/- tendon fascicles. To underscore the clinical relevance of the findings, we further demonstrate that a missense mutation (p.Cys130Gln) in the follistatin-like domain of SPARC, which causes impaired protein secretion and type I collagen fibrillogenesis, is associated with tendon and ligament injuries in patients. Together, our results demonstrate that SPARC is a key extracellular matrix protein essential for load-induced tendon tissue maturation and homeostasis.
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Predisposición Genética a la Enfermedad , Osteonectina , Tendones/fisiología , Animales , Homeostasis , Humanos , Ligamentos , Ratones , Ratones Noqueados , Osteonectina/genéticaRESUMEN
The mechanism(s) regulating the expression of the TBX2 gene, a key regulator of development, is poorly understood and thus limits an understanding of its function(s). Here we demonstrate that 12-O-tetradecanoylphorbol-13-acetate (TPA) induces TBX2 expression in normal human fibroblasts in a protein kinase C (PKC)-dependent and MAPK-independent manner. Our data further reveal that TPA activates transcription of TBX2 through activating MSK1, which leads to an increase in phosphorylated histone H3 and the recruitment of Sp1 to the TBX2 gene. In addition, TPA was shown to activate MSK1 in a PKC-dependent and MAPK-independent manner. This study is the first to provide evidence that phosphorylation of histone H3 leads to the transcriptional activation of the TBX2 gene and to link MSK1 to PKC.
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Histonas/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Proteínas de Dominio T Box/metabolismo , Sitios de Unión/genética , Western Blotting , Línea Celular , Inmunoprecipitación de Cromatina , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Flavonoides/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/farmacología , Indoles/farmacología , Maleimidas/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteína Quinasa C/antagonistas & inhibidores , Piridinas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Serina/metabolismo , Factor de Transcripción Sp1/metabolismo , Proteínas de Dominio T Box/genética , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The concept of in situ 3D bio-printing was previously reported, while its realization has still encountered with several difficulties. The present study aimed to report robotic-assisted in situ 3D bio-printing technology for cartilage regeneration, and explore its potential in clinical application. A six-degree-of-freedom (6-DOF) robot was introduced in this study, and a fast tool center point (TCP) calibration method was developed to improve printing accuracy. The bio-ink consisted of hyaluronic acid methacrylate and acrylate-terminated 4-armed polyethylene glycol was employed as well. The in vitro experiment was performed on a resin model to verify the printing accuracy. The in vivo experiment was conducted on rabbits to evaluate the cartilage treatment capability. According to our results, the accuracy of the robot could be notably improved, and the error of printed surface was less than 30⯵m. The osteochondral defect could be repaired during about 60â¯s, and the regenerated cartilage in hydrogel implantation and in situ 3D bio-printing groups demonstrated the same biomechanical and biochemical performance. We found that the cartilage injury could be treated by using this method. The robotic-assisted in situ 3D bio-printing is highly appropriate for improving surgical procedure, as well as promoting cartilage regeneration.
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BACKGROUND: To investigate the mechanism underlying the chondroprotective effect of estrogen in AMP-activated protein kinase (AMPK) deficiency mice. METHODS: Female cartilage-specific AMPKα double knockout (AMPKα cDKO) mice were generated and subjected to ovariectomy (OVX). The model of osteoarthritis (OA) was induced by destabilization of medial meniscus (DMM). Histopathological changes were evaluated by using OARSI scoring systems. Autophagy changes were analyzed by immunofluorescence staining. Human chondrocytes were subjected to mechanical stress to mimic OA development. and incubated in presence of or absence of 17ß-estradiol or/and compound C (AMPK inhibitor) or/and U0126 (ERK inhibitor). The expression levels of ERK1/2 phosphorylation, p70S6K phosphorylation and light chain 3 (LC3) were detected by Western blot. RESULTS: Compared with in OVX-sham AMPKα cDKO and OVX-sham WT mice, DMM-induced OA is more severe, and significantly low level of LC3 was observed in articular cartilage in OVX AMPK cDKO mice. Both mechanical stress and compound C were shown to induce an increase in phosphorylation of p70S6K, respectively. 17ß-estradiol stimulation led to a reduction in the basal level of p70S6K phosphorylation as well as in the compound C or mechanical stress-induced level of p70S6K phosphorylation. 17ß-estradiol stimulation not only led to an increase in LC3 conversion but also overrode the inhibitory effect of compound C on LC3 conversion. The effects of 17ß-estradiol were abrogated by blocking ERK signaling pathway. CONCLUSIONS: Our findings suggest that estrogen can protect articular cartilage from damage during OA development by promoting chondrocyte autophagy via ERK-mammalian target of rapamycin (mTOR) signaling, and give new insight into the mechanism of the chondroprotective effect of estrogen.
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BACKGROUND: Proinflammatory cytokines, which can upregulate the expression of matrix-degrading enzymes in chondrocytes, play important roles in the development of osteoarthritis. BET family proteins, acting as the "readers" of acetylated modifications on histones, have been linked to transcriptional regulation. And a BET protein inhibitor, I-BET151, has been shown to inhibit the induction of matrix-degrading enzymes by proinflammatory cytokines in chondrocytes. Our objective is to clarify the role and mechanism of BET proteins on matrix-degrading enzyme gene expression by using a human chondrosarcoma cell line (SW1353). METHODS: We pretreated SW1353 cells with I-BET151 prior to treatment with IL-1ß or TNF-α and then checked the expression of four matrix-degrading enzyme genes (MMP1, MMP3, MMP13, and ADAMTS4). We performed knockdown of BET protein family members (BRD2, BRD3, and BRD4) with corresponding siRNAs in SW1353 cells prior to treatment with IL-1ß or TNF-α and checked the expression of the matrix-degrading enzyme genes. We evaluated Brd-mediated transcriptional regulation on the matrix-degrading enzyme genes by ChIP assay. RESULTS: We confirmed that I-BET151 could suppress the IL-1ß- or TNF-α-induced expression of MMP1, MMP3, MMP13, and ADAMTS4 in SW1353 cells. Brd3 and Brd4 were required for the IL-1ß- or TNF-α-induced expression of matrix-degrading enzyme genes in SW1353 cells. We revealed that inducible acetylation of H4k5/8/12 and the recruitment of Brd3, Brd4, and p-TEFb to chromatin were involved in IL-1ß- or TNF-α-induced transcription. CONCLUSIONS: Our findings suggested that Brd3 and Brd4 were essential for the IL-1ß- or TNF-α-induced transcription of matrix-degrading enzyme genes, and recruitment of Brd3 and Brd4 to chromatin of these genes played the main role in this process.