RESUMO
Atherosclerosis (AS) is a chronic inflammatory disease with high morbidity and mortality rates worldwide. This study aimed to investigate the role of circular RNA protein tyrosine phosphatase receptor type A (circRNA_PTPRA) in oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cell (HUVECs) injury and its underlying molecular mechanism. The expression of circRNA-PTPRA and microRNA (miR)-671-5p was assessed by quantitative reverse transcription PCR (qRT-PCR). The interaction between circRNA-PTPRA and miR-671-5p was predicted using bioinformatic analysis. Cell viability and apoptosis were determined using the Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. Inflammation in HUVECs was analyzed by measuring the secretion of tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1ß), and IL-6 using enzyme-linked immunosorbent assay (ELISA). Cleaved-caspase-3 expression was assessed using western blotting. The results indicated that circRNA-PTPRA expression was significantly increased and miR-671-5p expression was decreased in the serum of patients with AS and in ox-LDL-treated HUVECs. The interaction between circRNA-PTPRA and miR-671-5p was verified by dual luciferase reporter and RNA pull-down assays. In HUVECs, downregulation of circRNA-PTPRA reversed ox-LDL-induced reduction in cell viability, increase in apoptosis, and enhanced inflammation, whereas all these effects mediated by circRNA-PTPRA downregulation in ox-LDL-treated HUVECs were abolished by miR-671-5p downregulation. In conclusion, circRNA-PTPRA downregulation protects against ox-LDL-induced HUVECs injury by upregulating miR-671-5p, thereby providing potential therapeutic targets for AS.
Assuntos
Aterosclerose , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Circular/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Lipoproteínas LDL/farmacologia , Apoptose , Inflamação/genética , Inflamação/metabolismo , Aterosclerose/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/farmacologiaRESUMO
Cyclic nucleotide-gated (CNG) channels are important mediators in the transduction pathways of rod and cone photoreceptors. Native CNG channels are heterotetramers composed of homologous A and B subunits. Biallelic mutations in CNGA1 or CNGB1 genes result in autosomal recessive retinitis pigmentosa (RP). To investigate the pathogenic mechanism of CNG channel-associated retinal degeneration, we developed a mouse model of CNGA1 knock-out using CRISPR/Cas9 technology. We observed progressive retinal thinning and a concomitant functional deficit in vivo as typical phenotypes for RP. Immunofluorescence and TUNEL staining showed progressive degeneration in rods and cones. Moreover, microglial activation and oxidative stress damage occurred in parallel. RNA-sequencing analysis of the retinae suggested down-regulated synaptic transmission and phototransduction as early as 9 days postnatal, possibly inducing later photoreceptor degeneration. In addition, the down-regulated PI3K-AKT-mTOR pathway indicated upregulation of autophagic process, and chaperone-mediated autophagy was further shown to coincide with the time course of photoreceptor death. Taken together, our studies add to a growing body of research exploring the mechanisms of photoreceptor death during RP progression and provide a novel CNGA1 knockout mouse model for potential development of therapies.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/deficiência , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Deleção de Genes , Degeneração Retiniana/genética , Animais , Caspases , Autofagia Mediada por Chaperonas , Feminino , Luz , Camundongos , Camundongos Knockout , Microglia , Neuroglia , Estresse Oxidativo/efeitos da radiação , Fenótipo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , RNA-Seq , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina , TranscriptomaRESUMO
Retinal detachment (RD) results in disruption of retinal physiology and visual function. Although surgical intervention has been well-developed to restore the retinal anatomic structure, post-op progression of visual function decline is prominent in a large proportion of patients. Therefore, the establishment of a disease model that accurately mimics RD pathogenesis is crucial to mechanistic study and drug screening. General protocols to induce RD in mice are frequently associated with complications leading to model instability and reduced reproducibility. In this study, we established a stable and reproducible mice RD model with a detached area of over 90% and rare complications. Briefly, the modified method was realized by vitreous humor extraction to reduce intraocular pressure, followed by directly-visible hyaluronic acid injection into subretinal space. The detachment of retina was confirmed by fundus photography, and progressive thinning of the outer nuclear layer (ONL) was determined by HE staining. Apoptotic signals were prominent in the ONL. Consistently, visual function was significantly compromised as determined by ERG. Moreover, retinal vasculature appeared to remodel and acquired winding, twisted and dilated structures illustrated by 3D reconstruction. In addition, activation of Müller cells and microglia, and infiltration of blood-derived macrophages were detected locally. Collectively, we have established a modified protocol to model RD with increased stability, reproducibility and fewer complications, and 3D high-resolution imaging and reconstruction of vasculature could provide new tools to evaluate this model.
Assuntos
Eletrorretinografia/métodos , Imageamento Tridimensional/métodos , Retina/diagnóstico por imagem , Descolamento Retiniano/diagnóstico , Vasos Retinianos/diagnóstico por imagem , Tomografia de Coerência Óptica/métodos , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BLRESUMO
VEGF is a critical driver of ocular neovascularization under disease conditions. Current therapeutic strategies rely on intraocular delivery of VEGF-antagonizing reagents, which results in sustained suppression of pathogenic vascularization. Although significant advancement has been achieved in VEGF antagonism, substantial adverse effects have been reported in retrospective clinical studies. To study mechanisms for VEGF antagonism-associated adverse effects in visual system, we intravitreally delivered recombinant adeno-associated virus-mediated expression of soluble Fms-related tyrosine kinase-1 (rAAV.sFLT-1), the extracellular domain of VEGF receptor, and analyzed the morphology and functions of retinal tissue. Here, we confirmed that intraocular VEGF antagonism induced retinal degeneration and gliosis. The functional deficit in retinal response to visual stimulation was also demonstrated in rAAV.sFLT-1-treated eyes by electroretinogram. Moreover, high-throughput RNA sequencing analysis suggests that VEGF antagonism activates retinal degeneration, inflammation, and other adverse effects. Taken together, our findings have shed light on pathogenic mechanisms for VEGF antagonism-associated adverse effects and potential therapeutic targets.-Xiao, M., Liu, Y., Wang, L., Liang, J., Wang, T., Zhai, Y., Wang, Y., Liu, S., Liu, W., Luo, X., Wang, F., Sun, X. Intraocular VEGF deprivation induces degeneration and fibrogenic response in retina.
Assuntos
Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Neovascularização de Coroide/metabolismo , Dependovirus/metabolismo , Vetores Genéticos/metabolismo , Gliose/metabolismo , Células HEK293 , Humanos , Inflamação/metabolismo , Camundongos , Retina , Degeneração Retiniana/metabolismo , Estudos Retrospectivos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Photoreceptor degeneration is a leading cause of visual impairment worldwide. Separation of neurosensory retina from the underlying retinal pigment epithelium is a prominent feature preceding photoreceptor degeneration in a variety of retinal diseases. Although ophthalmic surgical procedures have been well developed to restore retinal structures, postoperative patients usually experience progressive photoreceptor degeneration and irreversible vision loss that is incurable at present. Previous studies point to a critical role of mitochondria-mediated apoptotic pathway in photoreceptor degeneration, but the upstream triggers remain largely unexplored. In this study, we show that after experimental retinal detachment induction, photoreceptors activate dynamin-related protein 1 (Drp1)-dependent mitochondrial fission pathway and subsequent apoptotic cascades. Mechanistically, endogenous reactive oxygen species (ROS) are necessary for Drp1 activation in vivo, and exogenous ROS insult is sufficient to activate Drp1-dependent mitochondrial fission in cultured photoreceptors. Accordingly, inhibition of Drp1 activity effectively preserves mitochondrial integrity and rescues photoreceptors. Collectively, our data delineate an ROS-Drp1-mitochondria axis that promotes photoreceptor degeneration in retinal diseased models.
Assuntos
Dinaminas/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneração Retiniana/patologia , Descolamento Retiniano/patologia , Animais , Apoptose , Masculino , Mitocôndrias/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Degeneração Retiniana/metabolismo , Descolamento Retiniano/metabolismoRESUMO
PURPOSE: To characterize two patients with macular and rod-cone dystrophy and identify the genetic basis for disease. METHOD: Ophthalmic examinations were performed for the family and the peripheral blood samples were collected for whole exome sequencing. The mutated sequences of PROM1 gene were cloned and expressed in cultured cell lines after transient transfection followed by analysis with confocal microscopy and bridge-PCR. RESULT: We reported that two patients, brothers in a family, were diagnosed with macular and rod-cone dystrophy. Phenotypically, both patients experience progressive visual impairment and nyctalopia. The fundus examination showed macular and choroid dystrophy with pigment deposits in the macular region. Functionally, photoreceptor response to electrophysiological stimulation was significantly compromised with more severe decline in rods. Genetic analysis by whole exome sequencing revealed two novel compound heterogeneous point mutations in PROM1 gene that co-segregate with patients in an autosomal recessive manner. Specifically, the c.C1902G(p.Y634X) nonsense mutation results in a truncated, labile, and mislocalized protein, while the c.C1682+3A>G intronic mutation disrupts messenger RNA splicing. CONCLUSION: Our findings have identified two novel deleterious mutations in PROM1 gene that are associated with hereditary macular and rod-cone dystrophy in human.
Assuntos
Antígeno AC133/genética , Distrofias de Cones e Bastonetes/genética , DNA/genética , Mutação , Células Fotorreceptoras Retinianas Bastonetes/patologia , Tomografia de Coerência Óptica/métodos , Antígeno AC133/metabolismo , Adulto , Western Blotting , Células Cultivadas , Distrofias de Cones e Bastonetes/diagnóstico , Distrofias de Cones e Bastonetes/metabolismo , Análise Mutacional de DNA , Eletrorretinografia , Feminino , Genes Recessivos , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Linhagem , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
Age-associated dysfunction of retinal pigment epithelial cells (RPEs) is considered to be the initial trigger of retinal diseases such as age-related macular degeneration. Although autophagy is upregulated in RPEs during the course of aging, little is known about how autophagy is regulated and its functional role in RPEs. In this study, we found that expression of Sirtuin 6 (SIRT6) and autophagic markers are upregulated in RPEs of aged mice where subretinal deposition of amyloid-ß is accumulated and in amyloid-ß stimulated RPEs. In addition, gain and loss-of-function studies confirmed the positive role of SIRT6 in regulating autophagy. Interesting, inhibition of autophagy attenuates amyloid-ß stimulated inflammatory response in RPEs. Collectively, our findings uncover the autophagy modulated by SIRT6 may be a proinflammatory mechanism for amyloid-ß induced RPE dysfunction.
Assuntos
Peptídeos beta-Amiloides/imunologia , Autofagia/imunologia , Células Epiteliais/imunologia , Mediadores da Inflamação/imunologia , Epitélio Pigmentado da Retina/imunologia , Retinite/imunologia , Sirtuínas/imunologia , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Retinite/induzido quimicamente , Retinite/patologiaRESUMO
Recent studies indicate that histone deacetylases (HDACs) activity is associated with the development and progression of cardiac hypertrophy. In this study, we investigated the effects of a HDACs inhibitor, valproic acid sodium (VPA), on cardiac remodeling and the differential expression of HDACs in left ventricles (LVs) of renovascular hypertensive rats. Renovascular hypertension was induced in rats by the two-kidney two-clip (2K2C) method. Cardiac remodeling, heart function and the differential expression of HDACs were examined at different weeks after 2K2C operation. The effects of VPA on cardiac remodeling, the expressions of HDACs, transforming growth factor-beta 1 (TGF-ß1) and connective tissue growth factor (CTGF) in LV were investigated. The expressions of atrial natriuretic factor, ß-myosin heavy chain, HDAC2 and HDAC8 increased in LV of 2K2C rats at 4, 8, 12 weeks after operation. Cardiac dysfunction, cardiac hypertrophy and fibrosis were markedly attenuated by VPA treatment in 2K2C rats. Further studies revealed that VPA inhibited the expressions of HDAC2, HDAC8, TGF-ß1 and CTGF in LV of 2K2C rats. In summary, these data indicate that HDAC2 and HDAC8 play a key role in cardiac remodeling in renovascular hypertensive rats and that VPA attenuates hypertension and cardiac remodeling. The effect of VPA is possibly exerted via decreasing HDAC2, HDAC8, TGF-ß1 and CTGF expressions in LV of 2K2C rats.
Assuntos
Histona Desacetilase 2/fisiologia , Histona Desacetilases/fisiologia , Hipertensão Renovascular/tratamento farmacológico , Hipertensão Renovascular/enzimologia , Ácido Valproico/uso terapêutico , Remodelação Ventricular/efeitos dos fármacos , Animais , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Histona Desacetilase 2/antagonistas & inibidores , Hipertensão Renovascular/patologia , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Ácido Valproico/farmacologia , Remodelação Ventricular/fisiologiaRESUMO
PURPOSE: Retinal pigment epithelium (RPE) cell dysfunction is essential to the development of retinal degenerative disease. This study was designed to investigate how spliced X-box-binding protein 1 (XBP1s) regulates different modes of RPE cell death in vitro. METHODS: Human ARPE19 cells were incubated with 25 µM N-retinylidene-N-retinylethanolamine (A2E) and irradiated with blue light. Expressions of glucose-regulated protein 78 (GRP78) and XBP1s were detected by real-time quantitative PCR and Western blot. STF-083010 was used to suppress XBP1s expression. ARPE19 cell apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling and flow cytometry. Receptor-interacting protein kinase-3 (RIP3) was detected by Western blot. Changes in the morphology of ARPE19 cells were identified by transmission electron microscopy. RESULTS: Blue light-induced A2E-containing ARPE19 cell damage caused a transient elevation of GRP78 and XBP1s, while RIP3 rose in the late stage. STF-083010 effectively inhibited XBP1s expression and brought about the aggravation of apoptosis together with an alleviation of RIP3 expression. Most of the dying cells exhibited apoptotic morphology. CONCLUSION: A2E, along with blue light, brought about apoptosis and necroptosis of ARPE19 cells, and XBP1s was transiently elevated. The suppression of XBP1s induced ARPE19 cell death by promoting apoptosis rather than necroptosis. XBP1s might play a role in the pathogenesis of retinal degenerative diseases.
Assuntos
Apoptose/genética , Regulação da Expressão Gênica , RNA/genética , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina/metabolismo , Proteína 1 de Ligação a X-Box/genética , Western Blotting , Sobrevivência Celular , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/ultraestrutura , Proteína 1 de Ligação a X-Box/biossínteseRESUMO
As axon damage and retinal ganglion cell (RGC) loss lead to blindness, therapies that increase RGC survival and axon regrowth have direct clinical relevance. Given that NFκB signaling is critical for neuronal survival and may regulate neurite growth, we investigated the therapeutic potential of NFκB signaling in RGC survival and axon regeneration. Although both NFκB subunits (p65 and p50) are present in RGCs, p65 exists in an inactive (unphosphorylated) state when RGCs are subjected to neurotoxic conditions. In this study, we used a phosphomimetic approach to generate DNA coding for an activated (phosphorylated) p65 (p65mut), then employed an adeno-associated virus serotype 2 (AAV2) to deliver the DNA into RGCs. We tested whether constitutive p65mut expression prevents death and facilitates neurite outgrowth in RGCs subjected to transient retinal ischemia or optic nerve crush (ONC), two models of neurotoxicity. Our data indicate that RGCs treated with AAV2-p65mut displayed a significant increase in survival compared to controls in ONC model (77 ± 7% vs. 25 ± 3%, P-value = 0.0001). We also found protective effect of modified p65 in RGCs of ischemic retinas (55 ± 12% vs. 35 ± 6%), but not to a statistically significant degree (P-value = 0.14). We did not detect a difference in axon regeneration between experimental and control animals after ONC. These findings suggest that increased NFκB signaling in RGCs attenuates retinal damage in animal models of neurodegeneration, but insignificantly impacts axon regeneration.
Assuntos
Axônios/metabolismo , Regeneração Nervosa , Traumatismos do Nervo Óptico/metabolismo , Células Ganglionares da Retina/metabolismo , Fator de Transcrição RelA/genética , Animais , Axônios/fisiologia , Linhagem Celular , Células Cultivadas , Dependovirus/genética , Terapia Genética , Camundongos , Camundongos Endogâmicos C57BL , Crescimento Neuronal , Traumatismos do Nervo Óptico/terapia , Fator de Transcrição RelA/metabolismoRESUMO
Mammalian target of rapamycin (mTOR) functions as a master sensor of nutrients and energy, and controls protein translation and cell growth. Deletion of phosphatase and tensin homolog (PTEN) in adult CNS neurons promotes regeneration of injured axons in an mTOR-dependent manner. However, others have demonstrated mTOR-independent axon regeneration in different cell types, raising the question of how broadly mTOR regulates axonal regrowth across different systems. Here we define the role of mTOR in promoting collateral sprouting of spared axons, a key axonal remodeling mechanism by which functions are recovered after CNS injury. Using pharmacological inhibition, we demonstrate that mTOR is dispensable for the robust spontaneous sprouting of corticospinal tract axons seen after pyramidotomy in postnatal mice. In contrast, moderate spontaneous axonal sprouting and induced-sprouting seen under different conditions in young adult mice (i.e., PTEN deletion or degradation of chondroitin proteoglycans; CSPGs) are both reduced upon mTOR inhibition. In addition, to further determine the potency of mTOR in promoting sprouting responses, we coinactivate PTEN and CSPGs, and demonstrate that this combination leads to an additive increase in axonal sprouting compared with single treatments. Our findings reveal a developmental switch in mTOR dependency for inducing axonal sprouting, and indicate that PTEN deletion in adult neurons neither recapitulates the regrowth program of postnatal animals, nor is sufficient to completely overcome an inhibitory environment. Accordingly, exploiting mTOR levels by targeting PTEN combined with CSPG degradation represents a promising strategy to promote extensive axonal plasticity in adult mammals.
Assuntos
Axônios/fisiologia , Lesões Encefálicas/fisiopatologia , Regeneração Nervosa/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Lesões Encefálicas/patologia , Condroitina ABC Liase/farmacologia , Proteoglicanas de Sulfatos de Condroitina/antagonistas & inibidores , Proteoglicanas de Sulfatos de Condroitina/fisiologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Regeneração Nervosa/efeitos dos fármacos , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/fisiologia , Tratos Piramidais/efeitos dos fármacos , Tratos Piramidais/lesões , Tratos Piramidais/fisiologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
Injury to the CNS leads to formation of scar tissue, which is important in sealing the lesion and inhibiting axon regeneration. The fibrotic scar that comprises a dense extracellular matrix is thought to originate from meningeal cells surrounding the CNS. However, using transgenic mice, we demonstrate that perivascular collagen1α1 cells are the main source of the cellular composition of the fibrotic scar after contusive spinal cord injury in which the dura remains intact. Using genetic lineage tracing, light sheet fluorescent microscopy, and antigenic profiling, we identify collagen1α1 cells as perivascular fibroblasts that are distinct from pericytes. Our results identify collagen1α1 cells as a novel source of the fibrotic scar after spinal cord injury and shift the focus from the meninges to the vasculature during scar formation.
Assuntos
Cicatriz/etiologia , Fibroblastos/patologia , Pericitos/patologia , Traumatismos da Medula Espinal/complicações , Análise de Variância , Animais , Antígenos/genética , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Antígenos CD13/metabolismo , Contagem de Células , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Modelos Animais de Doenças , Progressão da Doença , Feminino , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Lectinas , Antígenos Comuns de Leucócito , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pericitos/metabolismo , Piperidinas/metabolismo , Proteoglicanas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Traumatismos da Medula Espinal/patologia , Fatores de Tempo , Uracila/análogos & derivados , Uracila/metabolismoRESUMO
The translational repressor Nanos is expressed in the germline and stem cell populations of jellyfish as well as humans. Surprisingly, we observed that unlike other mRNAs, synthetic nanos1 RNA translates very poorly if at all after injection into Xenopus oocytes. The current model of simple sequestration of nanos1 within germinal granules is insufficient to explain this observation and suggests that a second level of repression must be operating. We find that an RNA secondary structural element immediately downstream of the AUG start site is both necessary and sufficient to prevent ribosome scanning in the absence of a repressor. Accordingly, repression is relieved by small in-frame insertions before this secondary structure, or translational control element (TCE), that provide the 15 nucleotides required for ribosome entry. nanos1 is translated shortly after fertilization, pointing to the existence of a developmentally regulated activator. Oocyte extracts were rendered fully competent for nanos1 translation after the addition of a small amount of embryo extract, confirming the presence of an activator. Misexpression of Nanos1 in oocytes from unlocalized RNA results in abnormal development, highlighting the importance of TCE-mediated translational repression. Although found in prokaryotes, steric hindrance as a mechanism for negatively regulating translation is novel for a eukaryotic RNA. These observations unravel a new mode of nanos1 regulation at the post-transcriptional level that is essential for normal development.
Assuntos
RNA Mensageiro/química , Proteínas de Xenopus/genética , Animais , Western Blotting , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Imunoprecipitação , Microscopia Confocal , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Relação Estrutura-Atividade , XenopusRESUMO
BACKGROUND: Cerebral ischemia-reperfusion injury (I/R) can affect patient outcomes and can even be life-threatening. This study aimed to explore the role of Shionone in cerebral I/R and reveal its mechanism of action through the cerebral I/R in vitro model. METHODS: SH-SY5Y cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to induce cerebral I/R in vitro model. SH-SY5Y cells were treated with different concentrations of Shionone. Cell counting kit-8 and flow cytometry assays were used to detect cell viability and apoptosis levels. The levels of superoxide dismutase, catalase, and malondialdehyde were determined using their corresponding kits to examine the level of oxidative stress. The inflammation response was detected by IL-6, IL-1ß, and TNF-α levels, using enzyme-linked-immunosorbent-assay. RT-qPCR was performed to measure the mRNA levels of p38 and NF-κB. Western blotting was used to quantify the apoptosis-related proteins and p38MAPK/NF-κB signaling pathway proteins. RESULTS: Shionone exhibited no toxic effects on SH-SY5Y cells. Shionone inhibited OGD/R-induced cell apoptosis, improved the inflammatory response caused by OGD/R, and reduced the level of oxidative stress in cells. Western blot assay results showed that Shionone alleviated OGD/R-induced injury by inhibiting the activity of the p38 MAPK/NF-κB signaling pathway. The p38/MAPK agonist P79350 reversed the beneficial effects of Shionone. CONCLUSION: Shionone alleviates cerebral I/R and may thus be a novel therapeutic strategy for treating cerebral I/R.
Assuntos
Apoptose , Glucose , NF-kappa B , Oxigênio , Traumatismo por Reperfusão , Proteínas Quinases p38 Ativadas por Mitógeno , Humanos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Glucose/deficiência , NF-kappa B/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Apoptose/efeitos dos fármacos , Oxigênio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Linhagem Celular TumoralRESUMO
The Alpinia oxyphylla fruit (AOF) is a popular condiment and traditional Chinese medicine in Asia, known for its neuroprotective compound nootkatone. However, there has not been a comprehensive study of its flavor or the relationship between sensory and bioactive compounds. To address this issue, we examined AOF's microstructure, flavor, and metabolomic profiles during fruit maturation. The key markers used to distinguish samples included fruit expansion, testa pigmentation, aril liquefaction, oil cell expansion, peel spiciness, aril sweetness, and seed bitterness. A full-spectrum metabolomic analysis, combining a nontargeted metabolomics approach for volatile compounds and a widely targeted metabolomics approach for nonvolatile compounds, identified 1,448 metabolites, including 1,410 differentially accumulated metabolites (DAMs). Notably, 31 DAMs, including nootkatone, were associated with spicy peel, sweet aril, and bitter seeds. Correlational analysis indicated that bitterness intensity is an easy-to-use biomarker for nootkatone content in seeds. KEGG enrichment analysis linked peel spiciness to phenylpropanoid and capsaicin biosynthesis, seed bitterness to terpenoid (especially nootkatone) biosynthesis, and aril sweetness to starch and sucrose metabolism. This investigation advances the understanding of AOF's complex flavor chemistry and underlying bioactive principle, encapsulating the essence of the adage: "no bitterness, no intelligence" within the realm of phytochemistry.
Assuntos
Alpinia , Frutas , Sesquiterpenos Policíclicos , Sementes , Paladar , Alpinia/química , Sementes/química , Sesquiterpenos Policíclicos/metabolismo , Frutas/química , Metabolômica , Metaboloma , Análise Espaço-Temporal , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/metabolismoRESUMO
Primordial germ cells (PGCs) in Xenopus are specified through the inheritance of germ plasm. During gastrulation, PGCs remain totipotent while surrounding cells in the vegetal mass become committed to endoderm through the action of the vegetal localized maternal transcription factor VegT. We find that although PGCs contain maternal VegT RNA, they do not express its downstream targets at the mid-blastula transition (MBT). Transcriptional repression in PGCs correlates with the failure to phosphorylate serine 2 in the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAPII). As serine 5 is phosphorylated, these results are consistent with a block after the initiation step but before the elongation step of RNAPII-based transcription. Repression of PGC gene expression occurs despite an apparently permissive chromatin environment. Phosphorylation of CTD-serine 2 and expression of zygotic mRNAs in PGCs are first detected at neurula, some 10 hours after MBT, indicating that transcription is significantly delayed in the germ cell lineage. Significantly, Oct-91, a POU subclass V transcription factor related to mammalian Oct3/4, is among the earliest zygotic transcripts detected in PGCs and is a likely mediator of pluripotency. Our findings suggest that PGCs are unable to respond to maternally inherited endoderm determinants because RNAPII activity is transiently blocked while these determinants are present. Our results in a vertebrate system further support the concept that one strategy used repeatedly during evolution for preserving the germline is RNAPII repression.
Assuntos
Xenopus/embriologia , Xenopus/genética , Zigoto/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Masculino , Modelos Biológicos , Fatores de Transcrição de Octâmero/metabolismo , Oogênese/genética , Células-Tronco Pluripotentes/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Transcrição Gênica , Xenopus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
Temporomandibular joint (TMJ) growth requires orchestrated interactions between various cell types. Recent studies revealed that fibrocartilage stem cells (FCSCs) in the TMJ cartilage play critical roles as cell resources for joint development and repair. However, the detailed molecular network that influences FCSC fate during TMJ cartilage development remains to be elucidated. Here, we investigate the functional role of Igf1 in FCSCs for TMJ cartilage growth and homeostasis by lineage tracing using Gli1-CreER+ ; Tmflfl mice and conditional Igf1 deletion using Gli1-/Col2-CreER+ ; Igf1fl/fl mice. In Gli1-CreER+ ; Tmflfl mice, red fluorescence+ (RFP+ ) FCSCs show a favorable proliferative capacity. Igf1 deletion in Gli1+ /Col2+ cell lineages leads to distinct pathological changes in TMJ cartilage. More serious cartilage thickness and cell density reductions are found in the superficial layers in Gli1-CreER+ ; Igf1fl/fl mice. After long-term Igf1 deletion, a severe disordered cell arrangement is found in both groups. When Igf1 is conditionally deleted in vivo, the red fluorescent protein-labeled Gli1+ FCSC shows a significant disruption of chondrogenic differentiation, cell proliferation, and apoptosis leading to TMJ cartilage disarrangement and subchondral bone loss. Immunostaining shows that pAkt signaling is blocked in all cartilage layers after the Gli1+ -specific deletion of Igf1. In vitro, Igf1 deletion disrupts FCSC capacities, including proliferation and chondrogenesis. Moreover, the deletion of Igf1 in FCSCs significantly aggravates the joint osteoarthritis phenotype in the unilateral anterior crossbite mouse model, characterized by decreased cartilage thickness and cell numbers as well as a loss of extracellular matrix secretions. These findings uncover Igf1 as a regulator of TMJ cartilage growth and repair. The deletion of Igf1 disrupts the progenitor capacity of FCSCs, leading to a disordered cell distribution and exaggerating TMJ cartilage dysfunction. © 2023 American Society for Bone and Mineral Research (ASBMR).
Assuntos
Cartilagem Articular , Fator de Crescimento Insulin-Like I , Articulação Temporomandibular , Animais , Camundongos , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Fibrocartilagem , Homeostase , Células-Tronco , Articulação Temporomandibular/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Fator de Crescimento Insulin-Like I/metabolismoRESUMO
Due to the worldwide ecological and environmental issues induced by heavy metal pollution, including zinc and manganese, the ratio-metric discrimination of Zn2+ and Mn2+ based on CDs is urgently required. In this work, reduced CDs (re-CDs) with the intrinsic dual emissive peaks are obtained, and specific discrimination of Zn2+ and Mn2+ is realized by re-CDs with ratio-metric mode. With the addition of Zn2+, the fluorescent (FL) intensity at 650 nm increases obviously, while that at 680 nm progressively decreases. However, the presence of Mn2+ would induce the quenching of FL intensity at 680 nm while that at 650 nm remains constant. Then the Zn2+ and Mn2+ can be separately determined with the ratio of FL intensity at 650 nm to that at 680 mm (F650/F680). Under optimal conditions, the limit of detection (LOD) of Zn2+ is determined to be 9.09 nmol/L, and that for Mn2+ is estimated to be 0.93 nmol/L, which is much lower than previously reported work and standard level of Zn2+ and Mn2+ permitted in drinking water by WHO. Moreover, the specific recognition of Mn2+ and Zn2+ can be realized via the addition of different masking agents (ethylenediamine for Zn2+ and triethanolamine for Mn2+). Furthermore, our results reveal that the structural changes from -NH-CO to -NC-OH induced by Zn2+ contribute to the shift of FL peak from 680 to 650 nm while both static and dynamic quenching processes are involved in the detection of Mn2+. The ratio-metric probe was successfully applied to Zn2+ and Mn2+ determination in human serum samples and Sandy Lake water.
RESUMO
INTRODUCTION: Optic nerve injury is a leading cause of irreversible blindness worldwide. The retinal ganglion cells (RGCs) and their axons cannot be regenerated once damaged. Therefore, reducing RGC damage is crucial to prevent blindness. Accordingly, we aimed to investigate the potential influence of the gut microbiota on RGC survival, as well as the associated action mechanisms. METHODS: We evaluated the effects of microbiota, specifically Bifidobacterium, on RGC. Optic nerve crush (ONC) was used as a model of optic nerve injury. Vancomycin and Bifidobacterium were orally administered to specific pathogen-free (SPF) mice. RESULTS: Bifidobacterium promoted RGC survival and optic nerve regeneration. The administration of Bifidobacterium inhibited microglia activation but promoted Müller cell activation, which was accompanied by the downregulation of inflammatory cytokines and upregulation of neurotrophic factors and retinal ERK/Fos signaling pathway activation. CONCLUSIONS: Our study demonstrates that Bifidobacterium-induced changes in intestinal flora promote RGC survival. The protective effect of Bifidobacterium on RGC can be attributed to the inhibition of microglia activation and promotion of Müller cell activation and the secondary regulation of inflammatory and neurotrophic factors.
Assuntos
Traumatismos do Nervo Óptico , Células Ganglionares da Retina , Camundongos , Animais , Células Ganglionares da Retina/metabolismo , Traumatismos do Nervo Óptico/terapia , Traumatismos do Nervo Óptico/metabolismo , Neuroglia/metabolismo , Axônios/metabolismo , Fatores de Crescimento Neural/metabolismo , Cegueira/metabolismo , Sobrevivência Celular/fisiologia , Modelos Animais de DoençasRESUMO
Introduction: Ainaxiang (Blumea balsamifera (Linn.) DC.) is cultivated for the extraction of (-)-borneol and other pharmaceutical raw materials due to its abundant volatile oil. However, there is limited knowledge regarding the structural basis and composition of volatile oil accumulation in fresh B. balsamifera leaves. Methods: To address this problem, we compare the fresh leaves' morphology, microstructure, and volatile metabonomic at different development stages, orderly defined from the recently unfolded young stage (S1) to the senescent stage (S4). Results and discussion: Distinct differences were observed in the macro-appearance and microstructure at each stage, particularly in the B. balsamifera glandular trichomes (BbGTs) distribution. This specialized structure may be responsible for the accumulation of volatile matter. 213 metabolites were identified through metabolomic analysis, which exhibited spatiotemporal accumulation patterns among different stages. Notably, (-)-borneol was enriched at S1, while 10 key odor metabolites associated with the characteristic balsamic, borneol, fresh, and camphor aromas of B. balsamifera were enriched in S1 and S2. Ultra-microstructural examination revealed the involvement of chloroplasts, mitochondria, endoplasmic reticulum, and vacuoles in the synthesizing, transporting, and storing essential oils. These findings confirm that BbGTs serve as the secretory structures in B. balsamifera, with the population and morphology of BbGTs potentially serving as biomarkers for (-)-borneol accumulation. Overall, young B. balsamifera leaves with dense BbGTs represent a rich (-)-borneol source, while mesophyll cells contribute to volatile oil accumulation. These findings reveal the essential oil accumulation characteristics in B. balsamifera, providing a foundation for further understanding.