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Background: Dry eye disease has a high prevalence and exerts a significant negative effect on quality of life. In China, there are currently no available nasal sprays to promote natural tear production in patients with dry eye disease. We therefore evaluated the efficacy and safety of OC-01 (varenicline solution) nasal spray versus vehicle in Chinese patients with dry eye disease. Methods: This was a randomized, multicenter, double-masked, vehicle-controlled, phase 3 clinical trial conducted at ophthalmology departments in 20 hospitals across China (NCT05378945). Eligible patients had a diagnosis of dry eye disease based on patient symptoms, Eye Dryness Score (EDS), Schirmer's Test (with topical anesthesia) Score (STS), and corneal fluorescein staining (CFS) score. Participants were randomly assigned 1:1 using an Interactive Web Response System (IWRS) to receive OC-01 0.6 mg/mL twice daily (BID) or vehicle nasal spray. Participants, investigators, and sponsor were all masked to treatment assignment. The primary endpoint was the percentage of subjects in the intention-to-treat population achieving ≥10 mm improvement in STS from baseline at week 4. Findings: In total, 340 patients were randomized from 21 July 2022 to 04 April 2023, 78.8% were female. Patients in the OC-01 group (n = 176) had significantly higher achievement of ≥10 mm improvement in STS (35.8% [n = 63] versus 17.7% [n = 29], stratified odds ratio: 2.67, 95% CI: 1.570-4.533, p = 0.0002) and a significantly greater increase from baseline STS (least-squares mean difference [SE]: 3.87 [0.794], p < 0.0001) at week 4 versus the vehicle group (n = 164). In addition, OC-01 led to a numerically greater reduction in mean EDS from baseline at week 4 compared to the vehicle group (LS mean [SE] difference: -1.3 [2.20]; 95% CI: -5.64 to 2.99, p = 0.5467). The most common adverse event was mild, transient sneezing (78% of OC-01 administrations). No serious adverse events related to nasal administration occurred. Interpretation: OC-01 (varenicline solution) nasal spray BID has clinically meaningful efficacy for reducing the signs (as measured by STS) and may improve the symptoms (as measured by EDS) of dry eye disease, with an excellent safety and tolerability profile, in the Chinese population. Funding: Jixing Pharmaceutical Co. Ltd.
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BACKGROUND: Safely inhibiting the formation of scar in the glaucoma filtration surgery (GFS) has always been an issue for clinical glaucoma doctors. Anti-vascular endothelial growth factor (VEGF) agents can reduce angiogenesis, and anti-placental growth factor (PIGF) agents can affect reactive gliosis. However, the effect of conbercept, which can bind to both VEGF and PIGF, on human Tenon's fibroblasts (HTFs) is unknown. METHODS: HTFs were cultured in vitro and treated with conbercept or bevacizumab (BVZ). No drug was added to the control group. The effects of drugs on cell proliferation were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the collagen type I alpha1(Col1A1) mRNA expression level was measured using quantitative polymerase chain reaction (qPCR). HTF cell migration after drug interventions was evaluated using the scratch wound assay along with the measurement of the expression levels of VEGF and PIGF in human umbilical vein endothelial cells (HUVECs) using enzyme-linked immunosorbent assay, as well as the detection of the VEGF(R) mRNA expression level in HTFs using qPCR. RESULTS: After the addition of conbercept (0.01, 0.1, and 1 mg/mL) to the cultured HTFs or HUVECs, no significant cytotoxicity was observed compared with the control group, while the cytotoxicity of 2.5 mg/mL BVZ on HTFs was obvious. Conbercept significantly inhibited HTF cell migration and Col1A1 mRNA expression level in HTFs. It was superior to BVZ in inhibiting HTF migration. After the intervention with conbercept, the expression level of PIGF and VEGF in HUVECs significantly decreased; and the inhibitory effect of conbercept on the expression level of VEGF in HUVECs was weaker than that of BVZ. Conbercept was more advantageous than BVZ in inhibiting the expression level of VEGFR-1 mRNA in HTFs. However, its effect in terms of inhibiting the expression level of VEGFR-2 mRNA in HTFs was weaker than that of BVZ. CONCLUSION: The results suggested the low cytotoxicity and significant anti-scarring effect of conbercept in HTF with significant anti-PIGF and inferior anti-VEGF effects compared with BVZ, thus providing a better understanding of the role of conbercept in the GFS wound healing process.
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Antineoplásicos Inmunológicos , Bevacizumab , Cicatriz , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Humanos , Fibroblastos , Bevacizumab/efectos adversos , Bevacizumab/farmacología , Bevacizumab/uso terapéutico , Cicatriz/prevención & control , Glaucoma/cirugía , Células Endoteliales de la Vena Umbilical Humana , Colágeno Tipo I , Antineoplásicos Inmunológicos/efectos adversos , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéuticoRESUMEN
The accumulation of DNA damage induced by oxidative stress is a crucial pathogenic factor of endothelial loss in diabetic vascular complications, but it is still unknown whether aberrant glucose metabolism leads to defective DNA repair and accounts for hyperglycemia-induced endothelial oxidative stress injury. Here, we showed that Foxo1 knockdown alleviated diabetes-associated retinal DNA damage and vascular dysfunction. Mechanistically, FOXO1 knockdown avoided persistent DNA damage and cellular senescence under high glucose in endothelial cells by promoting DNA repair mediated by the MRN (MRE11-RAD50-NBS1 complex)-ATM pathway in response to oxidative stress injury. Moreover, FOXO1 knockdown mediated robust DNA repair by restoring glycolysis capacity under high glucose. During this process, the key glycolytic enzyme PFKFB3 was stimulated and, in addition to its promoting effect on glycolysis, directly participated in DNA repair. Under genotoxic stress, PFKFB3 relocated into oxidative stress-induced DNA damage sites and promoted DNA repair by interaction with the MRN-ATM pathway. Our study proposed that defective glycolysis-dependent DNA repair is present in diabetic endothelial cells and contributes to hyperglycemia-induced vascular dysfunction, which could provide novel therapeutic targets for diabetic vascular complications.
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Angiopatías Diabéticas , Hiperglucemia , Humanos , Proteínas de Ciclo Celular/metabolismo , Células Endoteliales/metabolismo , Reparación del ADN , Glucólisis , Daño del ADN , Estrés Oxidativo , Hiperglucemia/genética , Hiperglucemia/metabolismo , Glucosa/metabolismo , Angiopatías Diabéticas/metabolismo , Proteína Forkhead Box O1/metabolismo , Fosfofructoquinasa-2/genética , Fosfofructoquinasa-2/metabolismoRESUMEN
Diabetic retinopathy (DR) and other diabetic vascular complications are the leading cause of death and disability in patients with suboptimum glycemic control. In the pathogenesis of diabetic vascular diseases, hyperglycemia-induced oxidative stress, DNA damage, and poly-ADP-ribose-polymerase (PARP) hyperactivation play important roles in endothelial cell impairment. Adipose differentiation-related protein FBXW7 was reported to regulate PGC-1α stability and mitochondrial homeostasis. Here, we investigated the role and mechanism of FBXW7 in repairing endothelial oxidative stress injuries under hyperglycemic conditions. FBXW7 promoted the hampered activity of homologous recombination and non-homologues end joining pathway for repairing DNA double-strand breaks damage, an initiating factor for PARP hyperactivation and diabetic vascular complications. The abundant mobilization of DNA damage repair mediated by FBXW7 suppressed PARP activation, leading to downregulation of PARP expression and activity in both human endothelial cells and diabetic rat retinas. This provided a new method for PARP inhibition, superior to PARP inhibitors for treating diabetic vascular complication. Furthermore, FBXW7 rescued downregulated NAD+ levels and ameliorated mitochondrial dysfunction, thereby reducing superoxide production under hyperglycemic conditions. These effects reversed oxidative injury and vascular leakage in diabetic rat retina, providing a potential future treatment strategy.
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Angiopatías Diabéticas , Hiperglucemia , Animales , Humanos , Ratas , Células Endoteliales , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Estrés Oxidativo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Especies Reactivas de Oxígeno/farmacologíaRESUMEN
Background: Neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) are major causes of blindness in aged people. 30% of the patients show unsatisfactory response to anti-vascular endothelial growth factor (anti-VEGF) drugs. This study aims to investigate the relationship between serum metabolome and treatment response to anti-VEGF therapy. Methods: A prospective longitudinal study was conducted between March 2017 and April 2019 in 13 clinical sites in China. The discovery group were enrolled from Shanghai General Hospital. The validation group consisted of patients from the other 12 sites. Participants received at least one intravitreal injection of 0.5 mg anti-VEGF drug, conbercept, and were divided into two groups - responders and non-responders. Serum samples of both groups were processed for UHPLC-MS/MS analysis. We constructed principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) models to investigate the metabolic differences between two groups using SIMCA-P. Area under curve (AUC) was calculated to screen the biomarkers to predict treatment response. Metabolites sub-classes and enriched pathways were obtained using MetaboAnalyst5.0. Results: 219 eyes from 219 patients (nAMD = 126; PCV = 93) were enrolled. A total of 248 metabolites were detected. PCA and PLS-DA models of the discovery group demonstrated that the metabolic profiles of responders and non-responders clearly differed. Eighty-five differential metabolites were identified, including sub-classes of diacylglycerophosphocholines, lysophosphatidylcholine (LPC), fatty acids, phosphocholine, etc. Responders and non-responders differed most significantly in metabolism of LPC (p = 7.16 × 10^-19) and diacylglycerophosphocholine (p = 6.96 × 10^-17). LPC 18:0 exhibited the highest AUC, which is 0.896 with 95% confidence internal between 0.833 and 0.949, to discriminate responders. The predictive accuracy of LPC 18:0 was 72.4% in the validation group. Conclusions: This study suggests that differential metabolites may be useful for guiding treatment options for nAMD and PCV. Metabolism of LPC and diacylglycerophosphocholine were found to affect response to conbercept treatment. LPC 18:0 was a potential biomarker to discriminate responders from non-responders.
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Lymphoma is a serious malignant tumor that contains more than 70 different types and seriously endangers the body's lymphatic system. The lymphatic system is the regulatory center of the immune system and is important in the immune response to foreign antigens and tumors. Studies showed that multiple genetic variants are associated with lymphoma but determining the pathogenic mechanisms remains a challenge. In the present study, we first applied the Gene Ontology (GO) and KEGG pathway enrichment analyses of lymphoma-associated and lymphoma-nonassociated genes. Next, the Boruta and max-relevance and min-redundancy feature selection methods were performed to filter and rank features. Then, features preselected and ranked using the incremental feature selection method were applied for the decision tree model to identify the best GO terms and KEGG pathways and extract classification rules. Results indicate that our predicted features, such as B-cell activation, negative regulation of protein processing, negative regulation of mast cell cytokine production, and natural killer cell-mediated cytotoxicity, are associated with the biological process of lymphoma, consistent with those of recent publications. This study provides a new perspective for future research on the molecular mechanisms of lymphoma.
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Linfoma , Apoptosis , Ontología de Genes , Humanos , Internacionalidad , Linfoma/genética , ProbabilidadRESUMEN
Diabetic retinopathy (DR) is the leading cause of acquired blindness in middle-aged people. The complex pathology of DR is difficult to dissect, given the convoluted cytoarchitecture of the retina. Here, we performed single-cell RNA sequencing (scRNA-seq) of retina from a model of type 2 diabetes, induced in leptin receptor-deficient (db/db) and control db/m mice, with the aim of elucidating the factors mediating the pathogenesis of DR. We identified 11 cell types and determined cell-type-specific expression of DR-associated loci via genome-wide association study (GWAS)-based enrichment analysis. DR also impacted cell-type-specific genes and altered cell-cell communication. Based on the scRNA-seq results, retinaldehyde-binding protein 1 (RLBP1) was investigated as a promising therapeutic target for DR. Retinal RLBP1 expression was decreased in diabetes, and its overexpression in Müller glia mitigated DR-associated neurovascular degeneration. These data provide a detailed analysis of the retina under diabetic and normal conditions, revealing new insights into pathogenic factors that may be targeted to treat DR and related dysfunctions.
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Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Estudio de Asociación del Genoma Completo/métodos , Ratones , Retina/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula IndividualRESUMEN
BACKGROUND: Age-related macular degeneration (AMD) represents the leading cause of visual impairment in the aging population. The goal of this study was to identify aberrantly-methylated, differentially-expressed genes (MDEGs) in AMD and explore the involved pathways via integrated bioinformatics analysis. METHODS: Data from expression profile GSE29801 and methylation profile GSE102952 were obtained from the Gene Expression Omnibus database. We analyzed differentially-methylated genes and differentially-expressed genes using R software. Functional enrichment and protein-protein interaction (PPI) network analysis were performed using the R package and Search Tool for the Retrieval of Interacting Genes online database. Hub genes were identified using Cytoscape. RESULTS: In total, 827 and 592 genes showed high and low expression, respectively, in GSE29801; 4117 hyper-methylated genes and 511 hypo-methylated genes were detected in GSE102952. Based on overlap, we categorized 153 genes as hyper-methylated, low-expression genes (Hyper-LGs) and 24 genes as hypo-methylated, high-expression genes (Hypo-HGs). Four Hyper-LGs (CKB, PPP3CA, TGFB2, SOCS2) overlapped with AMD risk genes in the Public Health Genomics and Precision Health Knowledge Base. KEGG pathway enrichment analysis indicated that Hypo-HGs were enriched in the calcium signaling pathway, whereas Hyper-LGs were enriched in sphingolipid metabolism. In GO analysis, Hypo-HGs were enriched in fibroblast migration, membrane raft, and coenzyme binding, among others. Hyper-LGs were enriched in mRNA transport, nuclear speck, and DNA binding, among others. In PPI network analysis, 23 nodes and two edges were established from Hypo-HGs, and 151 nodes and 73 edges were established from Hyper-LGs. Hub genes (DHX9, MAPT, PAX6) showed the greatest overlap. CONCLUSION: This study revealed potentially aberrantly MDEGs and pathways in AMD, which might improve the understanding of this disease.
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Metilación de ADN/genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/genética , Degeneración Macular/genética , Biología Computacional , ARN Helicasas DEAD-box/genética , Marcadores Genéticos/genética , Humanos , Proteínas de Neoplasias/genética , Factor de Transcripción PAX6/genética , Mapas de Interacción de Proteínas , Proteínas tau/genéticaRESUMEN
The macular neovascular disease is a group disorder with complex pathogenesis of neovascularization for vision impairment and irreversible blindness, posing great challenges to precise diagnosis and management. We prospectively recruited participants with age-related macular degeneration (AMD), polypoidal choroidal vasculopathy (PCV), and pathological myopia (PM) and compared with cataract patients without fundus diseases as a control group. The serum metabolome was profiled by gas chromatography coupled with time-of-flight mass spectrometry (GC-TOFMS) analysis. Multivariate statistical methods as well as data mining were performed for interpretation of macular neovascularization. A total of 446 participants with macular neovascularization and 138 cataract subjects as the control group were enrolled in this study. By employing GC-TOFMS, 131 metabolites were identified and 33 differentiating metabolites were highlighted in patients with macular neovascularization. For differential diagnosis, three panels of specific metabolomics-based biomarkers provided areas under the curve of 0.967, 0.938, and 0.877 in the discovery phase (n = 328) and predictive values of 87.3%, 79%, and 85.7% in the test phase (n = 256). Personalized pathway dysregulation scores measurement using Lilikoi package in R language revealed the pentose phosphate pathway and mitochondrial electron transport chain as the most important pathways in AMD; purine metabolism and glycolysis were identified as the major disturbed pathways in PCV, while the altered thiamine metabolism and purine metabolism may contribute to PM phenotypes. Serum metabolomics are powerful for characterizing metabolic disturbances of the macular neovascular disease. Differences in metabolic pathways may reflect an underlying macular neovascular disease and serve as therapeutic targets for macular neovascular treatment.
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Coroides , Degeneración Macular , Angiografía con Fluoresceína , Fondo de Ojo , Humanos , Degeneración Macular/diagnóstico , MetabolómicaRESUMEN
Purpose: Adiponectin has been shown to exert potent anti-inflammatory activities in a range of systemic inflammatory diseases. This study aimed to investigate the potential therapeutic effects of KS23, a globular adiponectin-derived peptide, on endotoxin-induced uveitis (EIU) in rats and lipopolysaccharide (LPS)-stimulated mouse macrophage-like RAW 264.7 cells. Methods: EIU was induced in Lewis rats by subcutaneous injection of LPS into a single footpad. KS23 or phosphate-buffered saline (PBS) was administered immediately after LPS induction via intravitreal injection. Twenty-four hours later, clinical and histopathological scores were evaluated, and the aqueous humor (AqH) was collected to determine the infiltrating cells, protein concentration, and levels of inflammatory cytokines. In vitro, cultured RAW 264.7 cells were stimulated with LPS in the presence or absence of KS23, inflammatory cytokine levels in the supernatant, nuclear translocation of nuclear factor kappa B (NF-κB) subunit p65, and the expression of NF-kB signaling pathway components were analyzed. Results: KS23 treatment significantly ameliorated the clinical and histopathological scores of EIU rats and reduced the levels of infiltration cells, protein, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the aqueous humor. Consistently, KS23 decreased the expression of TNF-α and IL-6 in the supernatant of LPS-stimulated RAW 264.7 cells and inhibited the LPS-induced nuclear translocation of NF-κB p65 and the phosphorylation of IKKα/ß/IκBα/NF-κB. Conclusion: The in vivo and in vitro results demonstrated the anti-inflammatory effects of the peptide KS23 and suggested that KS23 is a compelling, novel therapeutic candidate for the treatment of ocular inflammation.
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BACKGROUND: Uveitis is a potentially sight-threatening form of ocular inflammation that affects the uvea in the wall of the eye. Currently available treatments for uveitis have exhibited profound adverse side effects. However, KS23 is a novel 23-amino-acid anti-inflammatory peptide derived from adiponectin that may have the capability to function as a safe alternative to these existing treatment options. We, therefore, evaluated the preventive effect of KS23 in experimental autoimmune uveitis (EAU). METHODS: EAU was induced in mice via immunization with the peptide interphotoreceptor retinoid binding protein 161-180 (IRBP161-180). KS23 was then administered every 2 days via intraperitoneal injection to induce protection against EAU. Clinical and histopathological scores were employed to evaluate the disease progression. Inflammatory cytokines were also quantified using ELISA, and the expression levels of specific chemokines and chemokine receptors were assessed via qRT-PCR. In addition, the proportions of Th1 and Th17 cells were detected via flow cytometry, and the expression levels of specific proteins were quantified from the retina of mice using western blot analysis, to elucidate the specific mechanism of action employed by KS23 to suppress the inflammation associated with EAU. RESULTS: KS23 was found to significantly improve EAU-associated histopathological scores, while decreasing the expression of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-6, and IL-17A), chemokines (LARC, RANTES, MIG, IP-10), and chemokine receptors (CCR6 and CXCR3). The proportions of Th1 and Th17 cells were also suppressed following intraperitoneal injection with KS23. The anti-inflammatory mechanism employed by KS23 was determined to be associated with the activation of AMPK and subsequent inhibition of NF-κB. CONCLUSIONS: KS23 decreased the proportions of Th1 and Th17 cells to effectively ameliorate the progression of EAU. It may, therefore, serve as a promising potential therapeutic agent for uveitis.
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Adiponectina/análogos & derivados , Adiponectina/farmacología , Antiinflamatorios/farmacología , Células TH1/efectos de los fármacos , Células Th17/efectos de los fármacos , Uveítis/inmunología , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Inflamación/inmunología , Inflamación/patología , Ratones , Péptidos/farmacología , Retina/inmunología , Retina/patología , Células TH1/inmunología , Células Th17/inmunología , Uveítis/patologíaRESUMEN
This is an in vitro study conducted to observe the safety and antiscarring effects of combined application of bevacizumab (BVZ) and 5-fluorouracil (5-Fu) or BVZ and mitomycin C (MMC) during glaucoma filtration surgery (GFS). The cytotoxicity of drug combinations in human Tenon's fibroblasts (HTFs) and human umbilical vein endothelial cells (HUVECs) was evaluated. Their effects on the levels of vascular endothelial growth factor (VEGF) in HUVECs, cell proliferation and migration in HTFs, and the expression of collagen type I alpha 1 (Col1A1) gene in HTFs were evaluated. In addition, the effects of combined drugs on VEGF(R) mRNA in HTFs were detected to explore the possible underlying drug mechanisms. The results showed that BVZ combined with 5-Fu demonstrated more significant antiscarring effects than BVZ or 5-Fu alone. However, the inhibitory effects of BVZ combined with MMC were similar to those of MMC alone. The cytotoxicity of the drug combinations was significantly greater than that of single drug, suggesting that combined application of BVZ and antimetabolites after GFS was safer when applied at different sites (such as subconjunctival injection at bilateral sides of the filtering bleb) or at varied time points.
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Purpose: To determine the relationship between choroidal thickness (ChT) at the posterior pole and refractive error and to explore the difference between the macular and peripapillary regions in children with myopia. Methods: A total of 340 healthy Chinese children underwent a series of comprehensive ocular examinations including cycloplegic refraction. Swept-source optical coherence tomography was used to measure the ChT in the macular and peripapillary regions. The Early Treatment of Diabetic Retinopathy Study grid was applied to define the sectors. Results: The mean spherical equivalent (SE) of the participants was -1.71 ± 2.22 diopter (D; range from -7.63 to 4.25 D). The mean ChT in the central foveal, parafoveal, and perifoveal regions were 229 ± 65 µm, 227 ± 60 µm, and 215 ± 50 µm, respectively, and the mean global peripapillary choroidal thickness (PPCT) was 136 ± 33 µm. The choroid in the macular region and the global PPCT was thinner in myopes compared to hyperopes. The area between the central fovea and the optic disc underwent the largest change as myopia worsened. SE, uncorrected visual acuity, cornea curvature radius (CR), retinal thickness (RT), and retinal nerve fiber layer thickness (RNFLT, except for the central fovea) were the independent factors of ChT in the macular region. SE, CR, RT, and RNFLT were the independent factors of PPCT temporally, inferiorly, and globally, while only CR, RT, and RNFLT were independently associated with PPCT superiorly and nasally. Conclusions: Choroidal thinning might be uneven during the development of myopia. SE only influenced the macular area and sectors temporal and inferior to the optic disc.
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Coroides/patología , Miopía/patología , Adolescente , Pueblo Asiatico , Niño , China , Estudios Transversales , Femenino , Humanos , Mácula Lútea/patología , Masculino , Miopía/fisiopatología , Errores de Refracción/patología , Tomografía de Coherencia Óptica/métodosRESUMEN
PURPOSE: To assess the anti-inflammatory effect and mechanism of a novel peptide, PAPep, in poly(I:C)-stimulated corneal fibroblasts. METHODS: Corneal fibroblasts were treated with poly(I:C) to elicit inflammation. Real-time polymerase chain reaction (PCR) and ELISA were used to measure the mRNA and protein levels of interleukin (IL)-6, monocyte chemotactic factor (MCP)-1, and interferon gamma (IFN-γ). Real-time PCR, immunofluorescence, and immunoblot were performed to determine ICAM-1 expression. Translocation of NF-κB p65 was observed by immunofluorescence. Phosphorylation of IκBα, NF-κB, and mitogen-activated protein kinase (MAPK) (p38, JNK and ERK) were detected by western blot. RESULTS: The results showed that PAPep effectively decreased mRNA and protein expression of IL-6, MCP-1, and IFN-γ in corneal fibroblasts exposed to poly(I:C). In addition, PAPep reduced mRNA and protein levels of ICAM-1. The NF-κB and MAPK(p38) pathway were inhibited by PAPep treatment, as indicated by suppression of p65 nuclear translocation, and IκBα, NF-κB, and p38 activation. PAPep showed no effect on JNK or ERK activity. CONCLUSIONS: PAPep attenuates the expression of inflammatory cytokines and ICAM-1 in corneal fibroblasts induced by poly(I:C) through blocking the NF-κB and MAPK(p38) pathway. PAPep may be considered a promising therapeutic agent for treating viral keratitis.
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Córnea/citología , Citocinas/metabolismo , Fibroblastos/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Péptidos/farmacología , Poli I-C/inmunología , Células Cultivadas , Córnea/efectos de los fármacos , Córnea/metabolismo , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , FN-kappa B/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
In keratitis, keratocytes play a vital role by releasing inflammatory cytokines and expressing intercellular cell adhesion molecule-1(ICAM-1). GC31 is a peptide derived from thrombomodulin, an endogenous protein with potential anti-inflammation properties. We evaluated the protective effect of GC31 in LPS- or poly(I:C)-induced corneal fibroblasts. Cultured keratocytes were treated with either LPS or poly(I:C); The mRNA and protein expressions of IL-6, IL-8, MCP-1, and IFN-γ were determined by real-time RT-PCR and ELISA. The expression level of ICAM-1 was estimated by real-time RT-PCR, immunofluorescence, and western blot. The underlying pathways were investigated by detecting NF-κB p65 translocation and phosphorylation of IκBα, p65, p38, JNK, and ERK. The MTS assay was used to measure cell viability of keratocytes after GC31 incubation. The elevation of IL-6, IL-8, MCP-1, and IFN-γ expression induced by LPS or poly(I:C) was significantly inhibited by GC31 in a dose-dependent manner at both mRNA and protein levels. GC31 also reduced the expression of ICAM-1 in keratocytes after LPS or poly(I:C) stimulation. LPS or poly(I:C) induced p65 translocation and phosphorylation of IκBα, p65, p38, and JNK were suppressed by GC31.GC31 is not only an effective inhibitor of LPS-induced inflammatory response, but it also inhibits poly(I:C)-induced release of inflammatory cytokines and ICAM-1 expression by blocking the NF-κB and MAPK (p38 and JNK) pathways. This suggested that GC31 may exert a protective effect in attenuating corneal inflammation by suppressing the immune response of the fibroblasts.
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Antiinflamatorios/farmacología , Quimiocinas/metabolismo , Córnea/metabolismo , Fibroblastos/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , FN-kappa B/antagonistas & inhibidores , Péptidos/farmacología , Trombomodulina/química , Células Cultivadas , Quimiocinas/antagonistas & inhibidores , Edema Corneal/tratamiento farmacológico , Humanos , Queratinocitos/efectos de los fármacos , Lectinas Tipo C/fisiología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Poli I-C , Polinucleótidos/farmacología , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Keratitis is a common cause of blindness. Current anti-inflammatory drugs used in keratitis have profound side effects. Small peptides derived from endogenous proteins potentially display both desired efficiency and safety. We identified an 11-amino-acid peptide, H-RN, from hepatocyte growth factor (HGF), an endogenous protein with anti-inflammatory properties. We evaluated the effects of H-RN in keratitis in vitro and in vivo. METHODS: In vitro, corneal fibroblasts were stimulated with LPS or poly(I:C), surrogates for bacteria and viruses. Inflammatory cytokines, intercellular cell adhesion molecule-1 (ICAM-1), translocation of NF-κB p65, activation of IκBα, NF-κB, and MAPKs were detected. In vivo, keratitis in rats was induced by LPS. Clinical, histological observation, and quantification of cytokines in the cornea were conducted. H-RN safety was measured by cell viability, clinical, histological, and microstructural observations. RESULTS: H-RN inhibited IL-6, monocyte chemotactic protein-1(MCP-1), Interferon- γ(IFN-γ), and ICAM-1 expression triggered by LPS or poly(I:C), alleviated the clinical manifestation and reduced the clinical score in keratitis in vivo. The histological disorder and proinflammatory cytokines of the cornea were also reduced. The translocation of NF-κB and phosphorylation of IκBα, NF-κB, p38, JNK, and ERK were significantly inhibited by H-RN. No sign of toxicity was observed. CONCLUSIONS: H-RN effectively attenuated keratitis in vivo and in vitro induced by LPS or poly(I:C) through blocking NF-κB and MAPK signaling pathways. It may be a promising and safe agent in treating keratitis.
Asunto(s)
Factor de Crecimiento de Hepatocito/uso terapéutico , Queratitis/tratamiento farmacológico , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fragmentos de Péptidos/uso terapéutico , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Córnea/patología , Citocinas/metabolismo , Activación Enzimática/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Mediadores de Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Queratitis/patología , Lipopolisacáridos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Fragmentos de Péptidos/farmacología , Fosforilación/efectos de los fármacos , Poli I-C , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas WistarRESUMEN
Keratitis is a worldwide sight-threatening disease. Current drugs generate various adverse effects. Large molecules hardly penetrate ocular tissues. Small peptides derived from endogenous protein display certain advantages. Previously we indentified a novel peptide (PAPep) from human pancreatitis-associated protein (PAP), a protein with protective effect against inflammatory diseases. To further examine the effect of PAPep on inflammatory disease and expand its scope of potential clinical application, especially in keratitis, we tested the effect of PAPep on various aspects of lipopolysaccharide (LPS)--induced corneal inflammation in vivo and in vitro. Dexamethasone (DXM) was used as a drug control. Our results suggested that PAPep suppressed the clinical manifestation, histological disorder and inflammatory cells infiltration and reduced the release of interleukin (IL)-6, IL-8 and monocyte chemotactic protein (MCP)-1 in the cornea. Moreover, PAPep inhibited LPS-induced mRNA and protein expression of the three cytokines in the corneal fibroblasts, prevented translocation of NF-κB and interrupted the phosphorylation of IKKα/ß/IκBα/NF-κB. Our study demonstrates that PAPep could effectively attenuate LPS-induced keratitis, more likely by virtue of inhibiting the activation of the IKKα/ß/IκBα/NF-κB pathway. PAPep may be considered to be a promising and safe drug for therapeutic application for ocular inflammation.