RESUMO
Purpose: Pterygium is an ocular surface disease characterized by the invasion of fibrovascular tissue from the bulbar conjunctiva to the cornea and is associated with abnormal tear function caused by changes in tear composition and osmolarity. In this study, the effect of two different surgical techniques to remove primary pterygium: conjunctival autograft surgery (CAG) and amniotic membrane transplantation (AMT), on changes in MUC2 and MUC5AC tear mucins concentration were evaluated. Methods: Forty-four patients (>18 years old) with primary unilateral pterygium (> 1.0 mm long, measured from the limbus to the apex on the cornea) were randomly enrolled, and assigned to the AMT or CAG group by using the permuted block technique. Patients with systemic inflammatory diseases or other eye comorbidities were excluded from the study. Tear break-up time (TBUT) and best-corrected visual acuity (BCVA) assessments were performed before surgery and at 1, 3, and 6 months after surgery. Tears were collected concurrently with the clinical evaluations, and MUC2 and MUC5AC concentrations were subsequently measured by means of ELISA. Results: At 6 months after CAG or AMT, TBUT and BCVA were significantly lower (P < 0.05) in comparison with the baseline values in the study subjects. The tear mucin concentrations of both MUC2 and MUC5AC were significantly higher (P < 0.0001) in patients with pterygium before any surgical procedure than in healthy individuals. The concentration of MUC2 increased at 1 and 3 months after CAG surgery and decreased at 6 months; however, the MUC2 concentration decreased on the AMT group in all time point measurements. Interestingly, the MUC5AC concentration significantly increased at 1 month after AMT or CAG and then decreased at 3 and 6 months after surgery. Finally, an inverse correlation was found between both MUC2 and MUC5AC tear mucins concentration and the TBUT. Conclusions: These results suggest that pterygium excision via both CAG or AMT changes the concentrations of the tear mucins MUC2 and MUC5AC during the evaluated times, and these changes could affect tear film stability and clinical recovery after pterygium treatment. Translational Relevance: The tear film stability during pterygium excision was evaluated to determine adequate treatments.
Assuntos
Âmnio , Túnica Conjuntiva , Mucina-5AC , Mucina-2 , Pterígio , Lágrimas , Humanos , Masculino , Pterígio/cirurgia , Pterígio/metabolismo , Feminino , Pessoa de Meia-Idade , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/transplante , Mucina-2/metabolismo , Lágrimas/metabolismo , Âmnio/transplante , Âmnio/metabolismo , Seguimentos , Mucina-5AC/metabolismo , Idoso , Adulto , Autoenxertos , Acuidade Visual , Ensaio de Imunoadsorção Enzimática , Transplante Autólogo/métodos , Estudos ProspectivosRESUMO
The protein epidermal growth factor (EGF), which plays a crucial role in promoting cell proliferation and survival, has recently demonstrated potential in reducing inflammation. In this study, we examined the impact of EGF on the anti-inflammatory and anti-proliferative properties of pterygium, a prevalent hypervascular proliferative disease affecting the ocular surface. In surgically excised tissues, markers for fibrotic and inflammatory signals, including VIM, ACTA2, FAP, MMP2, VCAM1, ICAM1, CD86, IL6, and IL1B were upregulated in the pterygium body stroma compared to the normal conjunctival stroma. EGF exerted anti-inflammatory and anti-vasculogenic effects on pterygial fibroblasts when co-cultured with M1 macrophages. Moreover, exosomes derived from EGF-preconditioned M1 macrophages suppressed the heightened inflammatory and vasculogenic signals in pterygial fibroblasts induced by exosomes from M1 macrophages. Paradoxically, the proliferation of pterygial fibroblasts was inhibited by EGF in the in vitro microenvironment with M1 macrophages, despite EGF being known as a growth factor. EGF-preconditioning of M1 macrophages rescued the increased proliferation of pterygial fibroblasts induced by exosomes from M1 macrophages. In conclusion, our findings demonstrate that EGF effectively mitigates inflammation and proliferation in pterygial fibroblasts within a microenvironment containing M1 macrophages.
Assuntos
Proliferação de Células , Fator de Crescimento Epidérmico , Fibroblastos , Inflamação , Macrófagos , Pterígio , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Humanos , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Pterígio/metabolismo , Pterígio/patologia , Inflamação/metabolismo , Inflamação/patologia , Inflamação/tratamento farmacológico , Exossomos/metabolismo , Células Cultivadas , Masculino , Técnicas de Cocultura , Microambiente Celular/efeitos dos fármacos , Feminino , Pessoa de Meia-Idade , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Túnica Conjuntiva/efeitos dos fármacosRESUMO
ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca2+ concentration ([Ca2+]i) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca2+i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca2+]i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1) preincubation with the Ca2+ chelator EGTA, 2) preincubation with the SERCA inhibitor thapsigargin (10-6 M), which depletes ER Ca2+ stores, 3) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4) preincubation with the voltage-gated calcium channel antagonist nifedipine (10-5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10-5 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca2+]i was significantly lower after chelating Ca2+i with 2 mM EGTA in Ca2+-free buffer. The peak [Ca2+]i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10-5 M and BzATP at 10-4 M. Incubating CGCs with dantrolene alone decreased [Ca2+]i and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca2+i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca2+]i. This study contributes to the understanding of diseases arising from defective Ca2+ signaling in nonexcitable cells.NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca2+]i) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca2+]i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca2+]i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca2+]i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca2+i homeostasis.
Assuntos
Trifosfato de Adenosina , Cálcio , Células Caliciformes , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Cálcio/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Ratos , Células Cultivadas , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/efeitos dos fármacos , Agonistas Purinérgicos/farmacologia , Ratos Sprague-Dawley , Sinalização do Cálcio/efeitos dos fármacos , Humanos , Masculino , Fosfolipases Tipo C/metabolismoRESUMO
The conjunctiva has immune-responsive properties to protect the eye from infections. Its innate immune system reacts against external pathogens, such as fungi. The complement factor C5a is an important contributor to the initial immune response. It is known that activation of transient-receptor-potential-vanilloid 1 (TRPV1) and TRP-melastatin 8 (TRPM8) channels is involved in different immune reactions and inflammation in the human body. The aim of this study was to determine if C5a and mucor racemosus e voluminae cellulae (MR) modulate Ca2+-signaling through changes in TRPs activity in human conjunctival epithelial cells (HCjECs). Furthermore, crosstalk was examined between C5a and MR in mediating calcium regulation. Intracellular Ca2+-concentration ([Ca2+]i) was measured by fluorescence calcium imaging, and whole-cell currents were recorded using the planar-patch-clamp technique. MR was used as a purified extract. Application of C5a (0.05-50 ng/mL) increased both [Ca2+]i and whole-cell currents, which were suppressed by either the TRPV1-blocker AMG 9810 or the TRPM8-blocker AMTB (both 20 µM). The N-terminal peptide C5L2p (20-50 ng/mL) blocked rises in [Ca2+]i induced by C5a. Moreover, the MR-induced rise in Ca2+-influx was suppressed by AMG 9810 and AMTB, as well as 0.05 ng/mL C5a. In conclusion, crosstalk between C5a and MR controls human conjunctival cell function through modulating interactions between TRPV1 and TRPM8 channel activity.
Assuntos
Cálcio , Complemento C5a , Túnica Conjuntiva , Células Epiteliais , Humanos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/microbiologia , Cálcio/metabolismo , Complemento C5a/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Sinalização do Cálcio , Canais de Cátion TRPV/metabolismoRESUMO
Pterygium is often associated with chronic ultraviolet (UV) radiation exposure and characterized by the overgrowth of conjunctiva and extracellular matrix (ECM) remodeling. Notably, several studies in the skin have demonstrated that chronic UV radiation can upregulate Granzyme B (GrB) expression and increase ECM degradation. The aim of this study was to compare GrB expression between pterygium and healthy controls and to further link this GrB expression to mast cells. Post-mortem pterygium tissues and conjunctival tissues from age-matched controls were used to assess GrB expression via immunofluorescence and microscopy. We found a significantly higher density of GrB+ cells from pterygium specimens compared to healthy controls. Furthermore, many of the GrB+ cells in pterygium specimens co-expressed tryptase, a mast cell marker. These findings suggest a role for conjunctival mast cell-secreted GrB in the pathogenesis of pterygium and highlight GrB as a possible therapeutic target in delaying or halting pterygium progression.
Assuntos
Túnica Conjuntiva , Granzimas , Pterígio , Humanos , Pterígio/metabolismo , Pterígio/patologia , Granzimas/metabolismo , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Mastócitos/metabolismo , Adulto , Estudos de Casos e Controles , Idoso de 80 Anos ou mais , Triptases/metabolismoRESUMO
Purpose: The purpose of this study was to determine the association between corneal images provided by in vivo confocal microscopy (IVCM) with clinical parameters and conjunctival expression of HLA-DR antigen in patients with dry eye disease (DED). Methods: Two hundred fourteen eyes of 214 patients with DED were analyzed, consisting of 2 groups of patients - 63 with autoimmune dry eye disease (AIDED) and 151 with non-autoimmune dry eye disease (NAIDED). Patients underwent a full clinical examination, including symptom screening, using the Ocular Surface Disease Index (OSDI) questionnaire, and objective analysis of DED signs by Schirmer's testing, tear break-up time (TBUT), Oxford's test, and IVCM corneal imaging. The IVCM scoring criteria were based on corneal sub-basal nerve density (ND), nerve morphology (NM), and inflammatory cell (IC) density. Quantification of conjunctival HLA-DR antigen was performed by flow cytometry. Results: The total IVCM score (T-IVCM) as well as the IVCM-IC subscore (sc) were positively correlated with HLA-DR levels with r = 0.3, P < 0.001 and r = 0.3, P < 0.01, respectively in the total population of patients with DED. The IVCM-NDsc was negatively correlated with TBUT in patients with AIDED (r = -0.2, P < 0.05) and with the Schirmer's test in patients with NAIDED (r = -0.24, P < 0.05). However, the IVCM-NMsc was positively correlated with the Oxford score only in patients with AIDED (r = 0.3, P < 0.05). Conclusions: The proposed IVCM scoring system showed significant correlations with clinical parameters along with conjunctival HLA-DR quantification in patients with DED. Translational Relevance: The IVCM grading score represents an interesting point of commonality among clinical parameters, imaging, and molecular investigation of the ocular surface.
Assuntos
Túnica Conjuntiva , Córnea , Síndromes do Olho Seco , Antígenos HLA-DR , Microscopia Confocal , Humanos , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Síndromes do Olho Seco/diagnóstico , Masculino , Feminino , Antígenos HLA-DR/metabolismo , Pessoa de Meia-Idade , Túnica Conjuntiva/patologia , Túnica Conjuntiva/metabolismo , Córnea/patologia , Córnea/inervação , Córnea/metabolismo , Córnea/diagnóstico por imagem , Idoso , Adulto , Imagem Multimodal/métodos , Citometria de Fluxo/métodos , Lágrimas/metabolismoRESUMO
Pterygium is a frequent eye surface condition that is characterized by a high rate of proliferation, fibrovascular development, cellular migration, corneal infiltration, and angiogenesis. We investigated that ex vivo primary pterygium and conjunctival cell cultures were generated to analyze the effect of trehalose on cellular proliferation. After trehalose treatment, we performed microarray analysis to evaluate changes in the mRNA profile. We analyzed gene ontology (GO) and KEGG pathways to identify hub genes that changed expression levels after treatment and were associated with pterygium development. We selected three genes to verify their expression levels using qRT-PCR. The study also evaluated the impact of trehalose treatment on cell migration through a wound-healing assay. Our results suggested that pterygium cell proliferation was inhibited in a dose-dependent manner by trehalose. 2354 DEG were identified in pterygium and conjunctiva cells treated with trehalose compared to untreated groups. Functional enrichment analysis showed that differentially expressed mRNAs are involved in proliferation, vasculature development, and cell migration. We identified ten hub genes including upregulated (RANBP3L, SLC5A3, RERG, ANKRD1, DHCR7, RAB27B, GPRC5B, MSMO1, ASPN, DRAM1) and downregulated (TNC, PTGS2, GREM2, NPTX1, NR4A1, HMOX1, CXCL12, IL6, MYH2, TXNIP). Microarray analysis and functional investigations suggest that trehalose affects the pathogenesis of pterygium by modifying the expression of genes involved in crucial pathways related to cell function.
Assuntos
Movimento Celular , Proliferação de Células , Túnica Conjuntiva , Pterígio , Trealose , Pterígio/metabolismo , Pterígio/tratamento farmacológico , Pterígio/genética , Pterígio/patologia , Humanos , Trealose/farmacologia , Trealose/metabolismo , Proliferação de Células/efeitos dos fármacos , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/patologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Masculino , Pessoa de Meia-IdadeRESUMO
Linear polyethylenimine (L-PEI) has numerous applications, such as in pharmaceutical formulations, gene delivery, and water treatment. However, due to the presence of secondary amine groups, L-PEI shows a relatively high toxicity and low biocompatibility. Here, various organic anhydrides were used to modify L-PEI to reduce its toxicity and enhance its functionality. We selected methacrylic anhydride, crotonic anhydride, maleic anhydride, and succinic anhydride to modify L-PEI. The structure of the resulting derivatives was characterized using 1H NMR and FTIR spectroscopies, and their behavior in aqueous solutions was studied using turbidimetric and electrophoretic mobility measurements over a broad range of pHs. A fluorescence flow through method determined the mucoadhesive properties of the polymers to the bovine palpebral conjunctiva. Methacrylated L-PEI and crotonylated L-PEI showed strong mucoadhesive properties at pH 7.4, likely due to covalent bonding with mucin thiol groups. In contrast, maleylated and succinylated L-PEI were poorly mucoadhesive as the pH was above their isoelectric point, resulting in electrostatic repulsion between the polymers and mucin. The toxicity of these polymers was evaluated using in vivo assays with planaria and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assay in human alveolar epithelial cells. Moreover, the irritancy of polymers was assessed using a slug mucosa irritation assay. The results demonstrated that anhydride modification mitigated the adverse toxicity effects seen for parent L-PEI.
Assuntos
Anidridos , Polietilenoimina , Polietilenoimina/química , Animais , Humanos , Anidridos/química , Bovinos , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/metabolismoRESUMO
Purpose: To evaluate the therapeutic efficacy of topical application of a neurokinin-1 receptor (NK1R) antagonist in a rabbit model of nonallergic ocular redness. Methods: Nonallergic ocular redness was induced in rabbits by a single, topical application of dapiparzole hydrochloride eye drops (0.5%, 1%, 2%, or 5%). The NK1R antagonist L-703,606 was topically applied to the eye at the same time of induction or 20 min after induction, and phosphate buffered saline (PBS) treatment served as the control. Superior bulbar conjunctival images were taken every 30 s for the first 2 min, followed by every 4 min for 8 min, and then every 10 min until 1 h. The severity of ocular redness was evaluated on the images using ImageJ-based ocular redness index (ORI) calculations. Results: The ORI scores were significantly increased after the application of 0.5%, 1%, 2%, or 5% dapiparzole at each time point evaluated, with the most severe redness induced by the 5% dapiprazole that led to a maximal mean increase in ORI score of 14 at 20 min post-induction and thus used for subsequent evaluation of therapeutic efficacy of NK1R antagonism. Topical L-703,606, when applied at the same time as dapiprazole induction, significantly suppressed the increase of ORI scores at all time points (â¼40% decrease). Furthermore, when applied at 20 min after dapiprazole induction, L-703,606 rapidly and effectively suppressed the increase of ORI scores at 30, 40, 50, and 60 min (â¼30% decrease). Conclusions: Topical blockade of NK1R effectively prevents and alleviates nonallergic ocular redness in a novel animal model.
Assuntos
Modelos Animais de Doenças , Antagonistas dos Receptores de Neurocinina-1 , Animais , Coelhos , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Antagonistas dos Receptores de Neurocinina-1/administração & dosagem , Soluções Oftálmicas/administração & dosagem , Masculino , Receptores da Neurocinina-1/metabolismo , Relação Dose-Resposta a Droga , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/metabolismoRESUMO
Conjunctival fibrosis is a common postoperative complication of glaucoma filtration surgery, resulting in uncontrolled intraocular pressure and surgery failure. Therefore, there is an urgent need to understand the molecular mechanisms underlying conjunctival fibrosis and to explore novel pharmacologic anti-fibrosis therapies for glaucoma filtration surgery. Herein, the 4-dimensional data-independent acquisition (4D-DIA) quantitative proteomic results, coupled with experimental data, revealed the activation of the Wnt/ß-catenin pathway in transforming growth factor (TGF)-ß1-induced human conjunctival fibroblasts (HConFs). Treatment with ICG-001, a Wnt/ß-catenin inhibitor, effectively inhibited cell proliferation and migration in TGFß1-treated HConFs. ICG-001 treatment alleviated the increased generation of extracellular matrix proteins induced by TGFß1. In addition, ICG-001 reduced the expression level of α smooth muscle actin (α-SMA) and inhibited cell contractility in TGFß1-treated HConFs. Proteomics data further suggested that αB-crystallin (CRYAB) was a downstream target of Wnt/ß-catenin, which was up-regulated by TGFß1 and down-regulated by ICG-001. Immunoblotting assay also indicated that ICG-001 reduced the expressions of ubiquitin and ß-catenin in TGFß1-treated HConFs, implying that CRYAB stabilized ß-catenin by inhibiting its ubiquitination degradation. Exogenous CRYAB promoted cell viability, increased extracellular matrix protein levels, and up-regulated α-SMA expression of HConFs under TGFß1 stimulation. CRYAB rescued TGFß1-induced fibrotic responses that were suppressed by ICG-001. In conclusion, this study elucidates the regulatory mechanism of the Wnt/ß-catenin/CRYAB pathway in conjunctival fibrosis, offering promising therapeutic targets for mitigating bleb scarring after glaucoma filtration surgery.
Assuntos
Túnica Conjuntiva , Fibroblastos , Fibrose , Fator de Crescimento Transformador beta1 , Via de Sinalização Wnt , Humanos , beta Catenina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes , Proliferação de Células/efeitos dos fármacos , Túnica Conjuntiva/patologia , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos dos fármacos , Proteômica/métodos , Pirimidinonas/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Although glaucoma is a leading cause of irreversible blindness worldwide, its pathogenesis is incompletely understood, and intraocular pressure (IOP) is the only modifiable risk factor to target the disease. Several associations between the gut microbiome and glaucoma, including the IOP, have been suggested. There is growing evidence that interactions between microbes on the ocular surface, termed the ocular surface microbiome (OSM), and tear proteins, collectively called the tear proteome, may also play a role in ocular diseases such as glaucoma. This study aimed to find characteristic features of the OSM and tear proteins in patients with glaucoma. The whole-metagenome shotgun sequencing of 32 conjunctival swabs identified Actinobacteria, Firmicutes, and Proteobacteria as the dominant phyla in the cohort. The species Corynebacterium mastitidis was only found in healthy controls, and their conjunctival microbiomes may be enriched in genes of the phospholipase pathway compared to glaucoma patients. Despite these minor differences in the OSM, patients showed an enrichment of many tear proteins associated with the immune system compared to controls. In contrast to the OSM, this emphasizes the role of the proteome, with a potential involvement of immunological processes in glaucoma. These findings may contribute to the design of new therapeutic approaches targeting glaucoma and other associated diseases.
Assuntos
Glaucoma , Microbiota , Proteoma , Lágrimas , Humanos , Glaucoma/metabolismo , Glaucoma/microbiologia , Proteoma/metabolismo , Masculino , Feminino , Lágrimas/metabolismo , Pessoa de Meia-Idade , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Idoso , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/microbiologia , Metagenoma , AdultoRESUMO
Purpose: This study aimed to explore protective effects and potential mechanism of ectoine, a natural osmoprotectant, on ocular surface mucin production in dry eye disease. Methods: A dry eye model was established in C57BL/6 mice exposed to desiccating stress (DS) with untreated (UT) mice as controls. DS mice were topically treated with 2.0% ectoine or PBS vehicle. Corneal epithelial defects were assessed by Oregon Green Dextran (OGD) fluorescent staining. Conjunctival goblet cells, ocular mucins, and T help (Th) cytokines were evaluated by immunofluorescent staining or ELISA, and RT-qPCR. Results: Compared with UT mice, corneal epithelial defects were detected as strong punctate OGD fluorescent staining in DS mice with vehicle, whereas ectoine treatment largely reduced OGD staining to near-normal levels. Conjunctival goblet cell density and cell size decreased markedly in DS mice, but was significantly recovered by ectoine treatment. The protein production and mRNA expression of two gel-forming secreted MUC5AC and MUC2, and 4 transmembrane mucins, MUC1, MUC4, MUC16, and MUC15, largely decreased in DS mice, but was restored by ectoine. Furthermore, Th2 cytokine IL-13 was inhibited, whereas Th1 cytokine IFN-γ was stimulated at protein and mRNA levels in conjunctiva and draining cervical lymph nodes (CLNs) of DS mice, leading to decreased IL-13/IFN-γ ratio. Interestingly, 2.0% ectoine reversed their alternations and restored IL-13/IFN-γ balance. Conclusions: Our findings demonstrate that topical ectoine significantly reduces corneal damage, and enhances goblet cell density and mucin production through restoring imbalanced IL-13/IFN-γ signaling in murine dry eye model. This suggests therapeutic potential of natural osmoprotectant ectoine for dry eye disease.
Assuntos
Modelos Animais de Doenças , Síndromes do Olho Seco , Células Caliciformes , Interferon gama , Interleucina-13 , Camundongos Endogâmicos C57BL , Mucinas , Animais , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/tratamento farmacológico , Camundongos , Células Caliciformes/metabolismo , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/patologia , Interferon gama/metabolismo , Mucinas/metabolismo , Mucinas/biossíntese , Mucinas/genética , Interleucina-13/metabolismo , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Epitélio Corneano/metabolismo , Epitélio Corneano/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Diamino AminoácidosRESUMO
Objectives: To examine changes in tear oxidative stress levels and tear film functions in patients with blepharoptosis and dermatochalasis following conjunctiva-Müller muscle resection (CMMR) and blepharoplasty surgeries. Materials and Methods: This prospective study included 32 healthy controls and 62 patients with blepharoptosis or dermatochalasis. CMMR surgery was performed in 20 eyes and upper blepharoplasty was performed in 42 eyes. Tear oxidative stress markers (8-hydroxy-2'-deoxyguanosine [8-OHdG] and 4-hydroxy-2-nonenal [4-HNE]) were quantified by enzyme-linked immunosorbent assay and tear film functions were evaluated preoperatively and at 1 and 6 months postoperatively. The same assessments were performed in the control group at the same time points. Results: Preoperative tear 8-OHdG and 4-HNE levels were lower in healthy controls (52.8±13.5 ng/mL and 27.8±6.4 ng/mL, respectively) compared to patients with dermatochalasis (86.1±37.2 ng/mL and 29.8±11.1 ng/mL, respectively) and blepharoptosis (90.4±39.3 ng/mL and 43.1±4.2 ng/mL, respectively) (p<0.001). 8-OHdG levels were increased at 1 month after CMMR, while both markers were decreased 1 month postoperatively in the blepharoplasty group (p=0.034). Schirmer 1 and OSDI scores did not change throughout the visits in both patient groups, but a temporary decrease in tear break-up time (TBUT) was observed after CMMR (p=0.017). Conclusion: Dermatochalasis and blepharoptosis were associated with higher tear oxidative stress levels. CMMR surgery caused a temporary decrease in TBUT scores and an increase in oxidative stress in the first postoperative month.
Assuntos
8-Hidroxi-2'-Desoxiguanosina , Blefaroplastia , Blefaroptose , Túnica Conjuntiva , Músculos Oculomotores , Estresse Oxidativo , Lágrimas , Humanos , Estresse Oxidativo/fisiologia , Blefaroptose/cirurgia , Blefaroptose/metabolismo , Feminino , Masculino , Estudos Prospectivos , Lágrimas/metabolismo , Blefaroplastia/métodos , Pessoa de Meia-Idade , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/cirurgia , Músculos Oculomotores/cirurgia , Músculos Oculomotores/metabolismo , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Adulto , Biomarcadores/metabolismo , Ensaio de Imunoadsorção Enzimática , Idoso , Aldeídos/metabolismoRESUMO
Subconjunctival fibrosis is critical to the outcomes of several ophthalmic conditions or procedures, such as glaucoma filtering surgery. This study aimed to investigate the anti-fibrotic effect of celastrol on subconjunctival fibrosis and to further reveal the underlying mechanisms. We used celastrol-loaded nanomicelles hydrogel hybrid as a sustained-release drug. A rabbit model of subconjunctival fibrosis following silicone implantation was used for in vivo study, and TGF-ß1-induced human pterygium fibroblast (HPF) activation as an in vitro model. The effects of celastrol on inhibiting TGF-ß1-induced migration and proliferation of HPFs were evaluated by scratch wound assay and CCK-8, respectively. Immunofluorescence and western blotting were used to examine the effect of celastrol on the expression of α-SMA, collagen I, fibronectin, and the targets of the Hippo signaling pathway. We found that in vivo celastrol treatment reduced the expression of YAP and TAZ in subconjunctival tissue. Moreover, celastrol alleviated collagen deposition and subconjunctival fibrosis at 8 weeks. No obvious tissue toxicity was observed in the rabbit models. Mechanistically, celastrol significantly inhibited TGF-ß1-induced proliferation and migration of HPFs. Pretreatment of HPFs with celastrol also suppressed the TGF-ß1-induced protein expression of α-SMA, collagen I, fibronectin, TGF-ßRII, phosphorylated Smad2/3, YAP, TAZ, and TEAD1. In conclusion, celastrol effectively prevented subconjunctival fibrosis through inhibiting TGF-ß1/Smad2/3-YAP/TAZ pathway. Celastrol could serve as a promising therapy for subconjunctival fibrosis.
Assuntos
Fibrose , Glaucoma , Triterpenos Pentacíclicos , Animais , Coelhos , Fibrose/tratamento farmacológico , Triterpenos Pentacíclicos/farmacologia , Glaucoma/cirurgia , Glaucoma/tratamento farmacológico , Humanos , Silicones , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proliferação de Células/efeitos dos fármacos , Masculino , Hidrogéis , Triterpenos/farmacologia , Triterpenos/administração & dosagem , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Fator de Crescimento Transformador beta1/metabolismo , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/patologia , Túnica Conjuntiva/metabolismo , Próteses e Implantes/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Preparações de Ação Retardada , Doenças da Túnica Conjuntiva/prevenção & controleRESUMO
Tear matrix metalloproteinase (MMP)-9 is an inflammatory signal in patients with dry eye (DE). In the present study, to understand the action mechanism of probiotic LB101 (Lactobacillus plantarum NK151 and Bifidobacterium bifidum NK175 [4:1] mix) against DE, we investigated its effect on tear amount and inflammatory marker expression levels in mice with unilateral exorbital lacrimal gland excision/atropine-benzalkonium chloride application (EB) or fecal microbiota transplantation from mice with EB (eFMT). Oral gavage of LB101 increased EB-suppressed tear amount and decreased EB-induced blinking number. Furthermore, LB101 decreased EB-induced TNF-α, IL-1ß, and MMP-9 expression, TNF-α+ and NF-κB+CD11c+ cell populations, and edema in the conjunctiva, while EB-suppressed IL-10 and occludin expression increased. LB101 also decreased EB-induced TNF-α and IL-1ß expression and NF-κB+CD11c+ cell population in the colon. eFMT also decreased tear amount and increased blinking number in the transplanted mice. eFMT increased TNF-α, IL-1ß, and MMP-9 expression and TNF-α+ and NF-κB+CD11c+ cell populations in the conjunctiva and TNF-α and IL-1ß expression and NF-κB+CD11c+ cell populations in the colon. Oral gavage of LB101 increased eFMT-suppressed tear amount and decreased eFMT-induced blinking number. Furthermore, LB101 decreased TNF-α, IL-1ß, and MMP-9 expression, TNF-α+ and NF-κB+CD11c+ cell populations, and edema in the conjunctiva and TNF-α and IL-1ß expression and NF-κB+CD11c+ cell population in the colon, while eFMT-suppressed IL-10 and occludin expression decreased. Furthermore, LB101 increased eFMT-suppressed Muribaculaceae, Prevotellaceae, and Lactobacillaceae populations in the gut microbiota, while eFMT-induced Bacteroidaceae population decreased. These findings suggest that DE may cause gut dysbiosis, which may be a risk factor for DE, and LB101 may alleviate DE with gut inflammation by suppressing the expression of MMP-9 and proinflammatory cytokines TNF-α and IL-1ß with the regulation of gut microbiota-involved NF-κB signaling.
Assuntos
Síndromes do Olho Seco , Microbioma Gastrointestinal , Metaloproteinase 9 da Matriz , NF-kappa B , Probióticos , Transdução de Sinais , Animais , Metaloproteinase 9 da Matriz/metabolismo , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/tratamento farmacológico , Microbioma Gastrointestinal/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Probióticos/farmacologia , Probióticos/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Lágrimas/metabolismo , Transplante de Microbiota Fecal , Fator de Necrose Tumoral alfa/metabolismo , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/microbiologia , Túnica Conjuntiva/patologiaRESUMO
PURPOSE: The purpose of this study was to identify conjunctival transcriptome differences in patients with Acanthamoeba keratitis compared with keratitis with no known associated pathogen. METHODS: The host conjunctival transcriptome of 9 patients with Acanthamoeba keratitis (AK) is compared with the host conjunctival transcriptome of 13 patients with pathogen-free keratitis. Culture and/or confocal confirmed Acanthamoeba in 8 of 9 participants with AK who underwent metagenomic RNA sequencing as the likely pathogen. Cultures were negative in all 13 cases where metagenomic RNA sequencing did not identify a pathogen. RESULTS: Transcriptome analysis identified 36 genes differently expressed between patients with AK and patients with presumed sterile, or pathogen-free, keratitis. Gene enrichment analysis revealed that some of these genes participate in several biologic pathways important for cellular signaling, ion transport and homeostasis, glucose transport, and mitochondrial metabolism. Notable relatively differentially expressed genes with potential relevance to Acanthamoeba infection included CPS1 , SLC35B4 , STEAP2 , ATP2B2 , NMNAT3 , and AKAP12 . CONCLUSIONS: This research suggests that the local transcriptome in Acanthamoeba keratitis may be sufficiently robust to be detected in the conjunctiva and that corneas infected with Acanthamoeba may be distinguished from the inflamed cornea where no pathogen was identified. Given the low sensitivity for corneal cultures, identification of differentially expressed genes may serve as a suggestive transcriptional signature allowing for a complementary diagnostic technique to identify this blinding parasite. Knowledge of differentially expressed genes may also direct investigation of disease pathophysiology and suggest novel pathways for therapeutic targets.
Assuntos
Ceratite por Acanthamoeba , Túnica Conjuntiva , Transcriptoma , Ceratite por Acanthamoeba/parasitologia , Ceratite por Acanthamoeba/diagnóstico , Ceratite por Acanthamoeba/genética , Humanos , Túnica Conjuntiva/parasitologia , Túnica Conjuntiva/metabolismo , Masculino , Adulto , Feminino , Pessoa de Meia-Idade , Acanthamoeba/genética , Perfilação da Expressão Gênica , Adulto Jovem , Microscopia Confocal , Idoso , Análise de Sequência de RNARESUMO
PURPOSE: To establish and characterize a dry eye model in New Zealand rabbits by subcutaneous injections of scopolamine hydrobromide (SCOP). METHODS: Twenty New Zealand male rabbits were injected subcutaneously SCOP for 14 consecutive days; subcutaneous saline was used as a negative control. The correlated clinical parameters of ocular surface dryness were detected in vivo using tear secretion and corneal fluorescein staining. The expression of IL-1ß and TNF-α on the ocular surface and in lacrimal glands were analyzed by real-time PCR and western blot on the 14th day. The expression of Mucin-5 subtype AC (MUC5AC) was detected by Immunofluorescence staining in conjunctival tissue. RESULTS: The SCOP-treated rabbits exhibited significantly decreased aqueous tear secretion and increased corneal fluorescein staining scores over time. Both the mRNA expression levels and the protein expression levels of IL-1ß and TNF-α were significantly increased after SCOP treatment compared with those after saline treatment. The loss of conjunctival MUC5AC was found in the SCOP-injected rabbits. Some infiltrated inflammatory cells and atrophic acinar cells were observed in the lacrimal gland after SCOP treatment. The disordered structures of the ocular surface and lacrimal glands were also observed. CONCLUSIONS: This study showed that repeated subcutaneous SCOP injections successfully elicited some of the typical dry eye symptoms commonly seen in humans.
Assuntos
Túnica Conjuntiva , Modelos Animais de Doenças , Síndromes do Olho Seco , Interleucina-1beta , Aparelho Lacrimal , Escopolamina , Lágrimas , Fator de Necrose Tumoral alfa , Animais , Coelhos , Síndromes do Olho Seco/induzido quimicamente , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/tratamento farmacológico , Masculino , Escopolamina/toxicidade , Lágrimas/metabolismo , Injeções Subcutâneas , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Túnica Conjuntiva/efeitos dos fármacos , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/efeitos dos fármacos , Aparelho Lacrimal/patologia , Fator de Necrose Tumoral alfa/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Mucina-5AC/metabolismo , Mucina-5AC/genética , Western Blotting , RNA Mensageiro/genética , Córnea/metabolismo , Córnea/efeitos dos fármacos , Córnea/patologiaRESUMO
The purpose of this study is to evaluate loose suture-related inflammation and activation of conjunctiva-associated lymphoid tissue (CALT) in patients after keratoplasty. The patients who were treated with keratoplasty at the First Affiliated Hospital of Harbin Medical University between 2015 and 2022 were recruited into the study. We evaluated the time and location of loose suture development in patients after keratoplasty. In addition, in vivo confocal microscopy was used to evaluate the activation of CALT and the accumulation of inflammatory cells around loose sutures. Meso Scale Discovery assay detection kits were used to evaluate the inflammatory cytokines in the tears of patients before and after the loose suture was removed. In this study, we collected the information from 212 cases (212 eyes) who had PK (126 eyes) and DALK-treated (86 eyes) for corneal transplantation, including 124 males and 88 females, aged 14-84 years old. The average age was 50.65 ± 16.81 years old. Corneal sutures were more prone to loose at 3 months and 6 months after keratoplasty, and the frequent sites were at 5 and 6 o'clock. An increased number of inflammatory cells could be observed around the loose sutures than normal sutures (P < 0.001). In CALT, the density of diffuse lymphocytes (P < 0.001), follicles (P < 0.001), and parafollicular lymphocytes (P < 0.001) were higher and the central reflection of the follicles (P < 0.001) was stronger when suture loosening happened. The levels of inflammatory cytokines such as IL-1ß (P = 0.003), IL-8 (P = 0.012), and TNF-α (P < 0.001) were higher in the tears of the patients with loose sutures. The activation of CALT was partly settled after removing the loose sutures. In conclusion, loose sutures after corneal transplantation can lead to increased infiltration of inflammatory cells, activation of CALT, and increased secretion of inflammatory cytokines in the tears of patients. Regular follow-up to identify and solve the problem in time can avoid suture-related complications.
Assuntos
Túnica Conjuntiva , Transplante de Córnea , Tecido Linfoide , Suturas , Humanos , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Idoso , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Túnica Conjuntiva/cirurgia , Idoso de 80 Anos ou mais , Transplante de Córnea/efeitos adversos , Adolescente , Suturas/efeitos adversos , Adulto Jovem , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Citocinas/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Inflamação/etiologia , Lágrimas/metabolismoRESUMO
Background: Microbial keratitis is one of the leading causes of blindness globally. An overactive immune response during an infection can exacerbate damage, causing corneal opacities and vision loss. This study aimed to identify the differentially expressed genes between corneal infection patients and healthy volunteers within the cornea and conjunctiva and elucidate the contributing pathways to these conditions' pathogenesis. Moreover, it compared the corneal and conjunctival transcriptomes in corneal-infected patients to cytokine levels in tears. Methods: Corneal and conjunctival swabs were collected from seven corneal infection patients and three healthy controls under topical anesthesia. RNA from seven corneal infection patients and three healthy volunteers were analyzed by RNA sequencing (RNA-Seq). Tear proteins were extracted from Schirmer strips via acetone precipitation from 38 cases of corneal infection and 14 healthy controls. The cytokines and chemokines IL-1ß, IL-6, CXCL8 (IL-8), CX3CL1, IL-10, IL-12 (p70), IL-17A, and IL-23 were measured using an antibody bead assay. Results: A total of 512 genes were found to be differentially expressed in infected corneas compared to healthy corneas, with 508 being upregulated and four downregulated (fold-change (FC) <-2 or > 2 and adjusted p <0.01). For the conjunctiva, 477 were upregulated, and 3 were downregulated (FC <-3 or ≥ 3 and adjusted p <0.01). There was a significant overlap in cornea and conjunctiva gene expression in patients with corneal infections. The genes were predominantly associated with immune response, regulation of angiogenesis, and apoptotic signaling pathways. The most highly upregulated gene was CXCL8 (which codes for IL-8 protein). In patients with corneal infections, the concentration of IL-8 protein in tears was relatively higher in patients compared to healthy controls but did not show statistical significance. Conclusions: During corneal infection, many genes were upregulated, with most of them being associated with immune response, regulation of angiogenesis, and apoptotic signaling. The findings may facilitate the development of treatments for corneal infections that can dampen specific aspects of the immune response to reduce scarring and preserve sight.
Assuntos
Túnica Conjuntiva , Córnea , Citocinas , Ceratite , Lágrimas , Transcriptoma , Humanos , Lágrimas/metabolismo , Citocinas/metabolismo , Citocinas/genética , Córnea/metabolismo , Córnea/imunologia , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/imunologia , Ceratite/genética , Ceratite/imunologia , Ceratite/metabolismo , Idoso , Perfilação da Expressão GênicaRESUMO
A pterygium is a common conjunctival degeneration and inflammatory condition. It grows onto the corneal surface or limbus, causing blurred vision and cosmetic issues. Ultraviolet is a well-known risk factor for the development of a pterygium, although its pathogenesis remains unclear, with only limited understanding of its hereditary basis. In this study, we collected RNA-seq from both pterygial tissues and conjunctival tissues (as controls) from six patients (a total of twelve biological samples) and retrieved publicly available data, including eight pterygium samples and eight controls. We investigated the intrinsic gene regulatory mechanisms closely linked to the inflammatory reactions of pterygiums and compared Asian (Korea) and the European (Germany) pterygiums using multiple analysis approaches from different perspectives. The increased expression of antioxidant genes in response to oxidative stress and DNA damage implies an association between these factors and pterygium development. Also, our comparative analysis revealed both similarities and differences between Asian and European pterygiums. The decrease in gene expressions involved in the three primary inflammatory signaling pathways-JAK/STAT, MAPK, and NF-kappa B signaling-suggests a connection between pathway dysfunction and pterygium development. We also observed relatively higher activity of autophagy and antioxidants in the Asian group, while the European group exhibited more pronounced stress responses against oxidative stress. These differences could potentially be necessitated by energy-associated pathways, specifically oxidative phosphorylation.