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BACKGROUND: We aimed to characterize the relationship between the serum 25-hydroxyvitamin D concentration and the circulating lipid concentrations of patients with NAFLD in the Hulunbuir region of China. METHODS: One hundred fifty-six patients, who were diagnosed with NAFLD in the Physical Examination Department of the Second Clinical College of Inner Mongolia University for the Nationalities between January 2021 and March 2023, were recruited as NAFLD group, and 160 healthy people were recruited as a control group during the same period. The serum 25(OH)VitD, TBIL, TG, TC, LDL-C, HDL-C, AST, ALT, GGT, and FPG activities of the participants were measured, and hepatic ultrasonography was performed. RESULTS: The BMI of the NAFLD group was higher than of the control group (p < 0.05). The serum 25(OH)VitD3 (p < 0.05) and the HDL-C concentrations of the NAFLD group were lower than those of the normal control group. However, the AST (p < 0.05), ALT (p < 0.05), and GGT (p < 0.05) activities, and the serum TG (p < 0.05), TC (p < 0.05), LDL-C (p < 0.05), and the fasting glucose (p < 0.05) concentrations of the NAFLD group were higher than those of the normal control group. The serum 25(OH)VitD3 concentrations of the NAFLD group significantly cor-related negatively with BMI (r = -0.302, p < 0.01), TG (r = -0.221, p < 0.05), and fasting glucose (r = -0.236, p < 0.05). The BMI, TG, and fasting glucose of vitamin D-deficient participants were higher than of the participants with adequate or insufficient levels of vitamin D (p < 0.05). Finally, the BMI of vitamin D-deficient participants was higher than of those with an adequate vitamin D status (p < 0.05). CONCLUSIONS: A deficiency of 25(OH)VitD is more common in people from the Hulunbuir region of China than elsewhere. In addition, the vitamin D status is significantly associated with NAFLD; as the serum vitamin D concentration decreases, patients with NAFLD show greater dyslipidemia and hyperglycemia and a higher BMI.
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Lipídeos , Hepatopatia Gordurosa não Alcoólica , Vitamina D , Humanos , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Feminino , Vitamina D/sangue , Vitamina D/análogos & derivados , Masculino , China/epidemiologia , Adulto , Lipídeos/sangue , Pessoa de Meia-Idade , Estudos de Casos e Controles , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/epidemiologia , Deficiência de Vitamina D/diagnóstico , Índice de Massa CorporalRESUMO
Glucocorticoid-induced cataract (GIC)-associated biomarkers were screened by ceRNA network construction. The GIC samples' GSE3040 were obtained from the NCBI-GEO database. R's Limma package was used to identify differentially expressed RNAs (DERs) between the normal and GIC samples group (4- and 16-h). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis for the mRNAs in the constructed GIC lncRNA-miRNA-mRNA ceRNA regulation network was implemented. A total of 1665 and 1443 DERs were obtained in the 4- and 16-h group, respectively. At two time points, 256 overlapping DERs were identified, of which 210 (17 lncRNAs and 203 mRNAs) had significant differential expression (4 down- and 206 up-regulated). A total of 534 co-expressed ligation pairs (all up-regulated) were obtained. A ceRNA regulation network was constructed. RPS6KA5, GAB1, CCR7, CCL2, COL4A4, and PPARG were obtained and significantly enriched in the 4 KEGG signaling pathways and were featured as GIC target molecules.
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Catarata , MicroRNAs , RNA Longo não Codificante , Catarata/induzido quimicamente , Catarata/genética , Redes Reguladoras de Genes , Glucocorticoides , Humanos , RNA Longo não Codificante/genéticaRESUMO
PURPOSE: The present study investigated a pathogenic mutation and its mechanism on membranous cataract in a congenital membranous cataract family. METHODS: An autosomal dominant four-generation Chinese congenital membranous cataract family was recruited and whole-exome sequencing was performed to screen for sequence variants. Candidate variants were validated using polymerase chain reaction and Sanger sequencing. Wild-type and mutant low-density lipoprotein receptor-related protein 5-like (LRP5L) plasmids were constructed and transfected into human lens epithelial cells (HLE B-3) and human anterior lens capsules. The cell lysates, nuclear and cytoplasmic proteins, and basement membrane components of HLE B-3 cells were harvested. LRP5L and laminin γ1 were knocked down in HLE B-3 cells using specific small-interfering RNA. The protein expression levels of LRP5L, laminin γ1, and c-MAF were detected using immunoblotting and immunofluorescence. RESULTS: We identified a novel suspected pathogenic mutation in LRP5L (c.107C > G, p.P36R) in the congenital membranous cataract family. This mutation was absent in 300 normal controls and 300 age-related cataract patients. Bioinformatics analysis with PolyPhen-2 and SIFT suggested that LRP5L-P36R was pathogenic. LRP5L upregulated laminin γ1 expression in the cytoplasmic proteins of HLE B-3 cells and human anterior lens capsules, and LRP5L-P36R inhibited the effects of LRP5L. LRP5L upregulated c-MAF expression in the nucleus and cytoplasm of HLE B-3 cells, and LRP5L-P36R inhibited c-MAF expression via inhibition of laminin γ1. CONCLUSION: Our study identified a novel gene, LRP5L, associated with congenital membranous cataract, and its mutant LRP5L-P36R contributed to membranous cataract development via inhibition of laminin γ1 and c-MAF.
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Catarata , Laminina , Povo Asiático , Catarata/genética , Humanos , Laminina/genética , Mutação , Mutação de Sentido Incorreto , Linhagem , Proteínas Proto-Oncogênicas c-maf/genéticaRESUMO
Fuchs endothelial dystrophy (FED) and late cornea allograft failure of cornea transplantation are associated with human corneal endothelial cells (HCEC) senescence. Kojic acid has various functions, however, its anti-senescence effect has never been identified. In this study, we investigated the anti-senescence effect of kojic acid on HCEC. Cell viability, migration ability and senescence were evaluated by MTT assay, migration assay, and senescence-associated beta-galactosidase (SA-ß-Gal) staining, respectively. Senescence-related protein expression was analyzed by western blotting and immunofluorescence assay. Angiogenesis of human umbilical vein endothelial cells (HUVEC) was examined by tube formation assay and spheroid sprouting assay. The results showed that kojic acid could inhibit HCEC senescence, characterized by enhancing migration, decreasing the levels of SA-ß-Gal staining, galectin 8, laminin α1, laminin α2, laminin γ1 and p21, and increasing that of p-NF-κB of senescent HCEC. The p-NF-κB inhibitor could reverse the anti-senescent effect of kojic acid, and p21 siRNA showed similar anti-senescence effect with kojic acid. In addition, kojic acid could alleviate HUVEC tube formation induced by senescent HCEC, which could be reversed by p-NF-κB inhibitor. The p21 siRNA could alleviate HUVEC spheroid sprouting induced by senescent HCEC. These results indicated that kojic acid might inhibit HCEC senescence and following resulted angiogenesis via NF-κB and p21 signaling pathways, possibly through downregulation of galectin 8 and laminins. Therefore, kojic acid is a promising drug for HCEC senescence-related diseases such as FED and late cornea allograft failure.
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Antioxidantes/farmacologia , Senescência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Endotélio Corneano/patologia , NF-kappa B/metabolismo , Pironas/farmacologia , Transdução de Sinais , Western Blotting , Linhagem Celular , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Endotélio Corneano/metabolismo , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , TransfecçãoRESUMO
PURPOSE: To investigate the roles of a selective MMP-2 and -9 inhibitor (SB-3CT) in corneal inflammatory lymphangiogenesis. METHODS: The expression of MMP-2 and -9 in the cornea after suture inplacement, treated with SB-3CT or negative control, was detected by real-time polymerase chain reaction (PCR). Inflammatory corneal neovascularization (NV) was induced by corneal suture placement. Mice were treated with SB-3CT eye drops (twice daily for 1 week, 5 µL per drop; 50, 100, or 200 µM). The outgrowth of blood and lymphatic vessels, and macrophage recruitment were analyzed by immunofluorescence assay. The expressions of vascular endothelial growth factor-C (VEGF-C) and its receptor VEGFR-3 were tested by real-time PCR. RESULTS: MMP-2 and -9 expression were suppressed significantly by treatment with SB-3CT. The data demonstrated, for the first time, that SB-3CT strongly reduced corneal lymphangiogenesis and macrophage infiltration during inflammation. Furthermore, expressions of VEGF-C and its receptor VEGFR-3 were significantly inhibited by SB-3CT during corneal lymphangiogenesis. CONCLUSIONS: These novel findings indicated that blockade of MMP-2 and -9 could inhibit lymphangiogenesis. Further investigation of this factor may provide novel therapies for transplant rejection and other lymphatic disorders.
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Neovascularização da Córnea/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 1 Anel/farmacologia , Linfangiogênese/efeitos dos fármacos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , RNA/genética , Sulfonas/farmacologia , Animais , Córnea/patologia , Neovascularização da Córnea/tratamento farmacológico , Neovascularização da Córnea/metabolismo , Modelos Animais de Doenças , Vasos Linfáticos/patologia , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: A birth population-based study was conducted in Danyang, Jiangsu Province, to evaluate major birth defects in emerging regions in China with similar maternal and neonatal care conditions. METHODS: We conducted a population-based study in a cohort of infants born in Danyang from 2014 to 2021, including 55 709 perinatal infants. Four categories of isolated birth defects were defined as cases: congenital heart defects (CHDs; n=2138), polydactyly (n=145), cleft lip with or without palate (CL/P; n=76) and accessory auricles (n=93). Infants with congenital malformations were identified by the Chinese Birth Defects Monitoring Network. RESULTS: Compared with autumn, conception in spring (OR=1.31 [1.16-1.48]) and winter (OR=1.39 [1.23-1.58]) was associated with an increased risk of CHD. Increased risk of CHD, CL/P and accessory auricles was significantly associated with non-local registered residence (OR=1.17 [1.07-1.28], OR=2.73 [1.52-4.88] and OR=2.11 [1.20-3.71], respectively). Individuals of Han nationality were less likely to have polydactyly (OR=0.23 [0.05-0.98]). CONCLUSIONS: The season of pregnancy was significantly associated with CHDs. Offspring of mothers with non-local registered hometown had greater risks of CHDs, CL/P and accessory auricles.
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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.
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BACKGROUND: Currently, no perfect treatment for neovascularization and lymphangiogenesis exist, and each treatment method has its complications and side effects. This study aimed to investigate the anti-angiogenic and anti-inflammatory effects of cannabidiol and its mechanism of action. METHOD: An in vivo corneal neovascularization (CNV) model was established using the suture method to investigate the inhibitory effects of CBD on suture-induced corneal inflammation, pathological blood vessel formation, and lymphangiogenesis. Additionally, the impact of CBD on immune cells was studied. In vitro methodologies, including cell sorting and co-culture, were employed to elucidate its mechanism of action. RESULTS: Compared with the CNV group, CBD can inhibit CNV, lymphangiogenesis, and inflammation induced via the suture method. In addition, CBD specifically induced CD45+CD11b+Gr-1+ cell upregulation, which significantly inhibited the proliferation of CD4+ T lymphocytes in vitro and exhibited a CD31+ phenotype, proving that they were myeloid-derived suppressor cells (MDSCs). We administered anti-Gr-1 to mice to eliminate MDSCs in vivo and found that anti-Gr-1 partially reversed the anti-inflammatory and angiogenic effects of CBD. Furthermore, we found that compared with MDSCs in the normal group, CBD-induced MDSCs overexpress peroxisome proliferator-activated receptor-gamma (PPAR-γ). Administering PPAR-γ inhibitor in mice almost reversed the induction of MDSCs by CBD, demonstrating the role of PPAR-γ in the function of CBD. CONCLUSION: This study indicates that CBD may induce MDSCs upregulation by activating the nuclear receptor PPAR-γ, exerting anti-inflammatory, antiangiogenic, and lymphangiogenic effects, and revealing potential therapeutic targets for corneal neovascularization and lymphangiogenesis.
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Anti-Inflamatórios , Canabidiol , Neovascularização da Córnea , Linfangiogênese , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides , Animais , Canabidiol/farmacologia , Canabidiol/uso terapêutico , Neovascularização da Córnea/tratamento farmacológico , Neovascularização da Córnea/patologia , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Camundongos , Linfangiogênese/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Modelos Animais de Doenças , Suturas , PPAR gama/metabolismo , Humanos , Inflamação/tratamento farmacológico , Masculino , Córnea/patologia , Córnea/efeitos dos fármacos , Células CultivadasRESUMO
PURPOSE: Accumulating evidence in animals suggests that leukocytes are involved in the pathogenesis of diabetic retinopathy. The present study was designed to investigate whether leukocytes from diabetic patients could kill retinal endothelial cells and whether that cytotoxicity could be inhibited in vivo by administration of berberine. METHODS: Human retinal endothelial cells (HRECs) were cocultured (24 h) with leukocytes freshly isolated from nondiabetic and diabetic patients, and leukocyte-mediated death of HRECs was analyzed with flow cytometry. HRECs or leukocytes were incubated with antibodies against intercellular adhesion molecule-1(ICAM-1) or integrin beta-2, or with various concentrations of berberine. The protein expression levels of inflammatory factors were investigated using western blots, and activities of antioxidant enzymes and malondialdehyde content were examined as markers of oxidative stress. In addition, leukocytes were isolated from 28 diabetic patients with retinopathy and nondiabetic patients before and after 1 month in vivo therapy with berberine. The effects of the berberine on leukocyte-mediated killing of endothelial cells was again assessed. RESULTS: Leukocytes from diabetic patients induced more apoptosis of HRECs in a coculture system than did cells from nondiabetic patients, and this killing occurred primarily via direct cell-cell contact. Berberine inhibited the leukocyte-mediated killing of HRECs in vitro, the decrease in antioxidant enzyme activities, the nuclear translocation of nuclear factor kappa B, and the increase in intercellular adhesion molecule-1 and inducible nitric oxide synthase expression and malondialdehyde content in HRECs cultured in high glucose. Berberine also decreased integrin beta-2 expression of leukocytes in vitro and in vivo. Oral consumption of berberine for 1 month likewise inhibited the diabetes-induced increase in leukocyte-mediated killing of HRECs. CONCLUSIONS: Our findings suggest that leukocytes from diabetic patients kill retinal endothelial cells, and that berberine can inhibit this leukocyte-mediated killing of vascular endothelium. Coculture of leukocytes with HRECs might serve as a biomarker to study the role of leukocytes in the development of diabetic retinopathy, and the data are consistent with berberine being a therapy against diabetic retinopathy.
Assuntos
Apoptose/efeitos dos fármacos , Berberina/farmacologia , Diabetes Mellitus Experimental/patologia , Células Endoteliais/patologia , Leucócitos/patologia , Retina/patologia , Animais , Berberina/administração & dosagem , Berberina/química , Antígenos CD18/metabolismo , Adesão Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Glucose/farmacologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
An Arg-Gly-Asp (RGD) motif in the fourth FAS1 domain of the human BIGH3 (transforming growth factor-ß1-inducible gene-h3) protein has been reported to play an important role in mediating tumor angiogenesis. The aim of this study was to investigate the inhibitory effect of a modified C-terminal fragment BIGH3 protein with an RGDRGD motif on corneal neovascularization in vitro and in vivo. Recombinant C-terminal fragment BIGH3 protein with wild-type sequence and modified C-terminal fragment BIGH3 protein containing an RGDRGD motif were successfully expressed and purified. We demonstrated that both proteins significantly inhibited vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) adhesion, migration, and tube formation and induced cell apoptosis but failed to inhibit HUVEC proliferation. We determined that the mechanism underlying this activity was an interaction between BIGH3 and αvß3 integrin, which blocked the phosphorylation of PI3K/Akt and ERK signaling pathways. The inhibitory effects of wild-type and modified C-terminal fragment BIGH3 proteins on angiogenesis were confirmed by a rabbit corneal neovascularization assay. More importantly, we provided evidence that the modified C-terminal fragment BIGH3 protein with an RGDRGD motif inhibited angiogenic activity far more effectively than did wild-type C-terminal fragment BIGH3. Collectively, our data show that a C-terminal fragment BIGH3 protein containing an RGDRGD motif might be promising as an effective drug in treating corneal neovascularization.
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Inibidores da Angiogênese/farmacologia , Neovascularização da Córnea/prevenção & controle , Proteínas da Matriz Extracelular/farmacologia , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Motivos de Aminoácidos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Animais , Apoptose/efeitos dos fármacos , Vasos Sanguíneos/efeitos dos fármacos , Western Blotting , Caspases/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neovascularização da Córnea/metabolismo , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana , Integrina alfaVbeta3/metabolismo , Mutagênese Sítio-Dirigida , Oligopeptídeos/química , Oligopeptídeos/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Plasmídeos , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Coelhos , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/genética , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
PURPOSE: To construct a scaffold using silk fibroin (SF) and chitosan (CS) that could replace the corneal stroma with the biological characteristics of the scaffold materials still intact. METHODS: To develop an organotypic corneal stroma, SF and CS were chosen to synthesise the tissue-engineered bioscaffold. We cultured primary rabbit corneal epithelial cells and corneal stromal cells in vitro. Keratocytes were used to assess cytotoxicity on SF-CS (SFCS) blends, which was determined by a Cell Counting Kit-8 assay. The corneal lamellar scaffolds were developed with sequential culture techniques to form cell-scaffold constructs. Implantation was tested in 15 New Zealand White rabbits. The corneal substitutes were analysed by light and electron microscopy. RESULTS: The reconstructed lamellar cornea was comparable to native tissue, with high levels of K3/12 expression in the corneal epithelial cells and vimentin in the stromal cells; moreover, the morphology and the position of the cells could be distinguished by histological methods. There was no sign of any immune reaction in or around the transplanted discs 12 weeks after implantation. CONCLUSION: A SFCS scaffold might be a suitable blend for corneal tissue engineering.
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Quitosana/química , Córnea/citologia , Substância Própria/citologia , Células Epiteliais/citologia , Fibroínas/química , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , CoelhosRESUMO
BACKGROUND: Cataracts are the main cause of reversible blindness worldwide. The ageing of the lens caused by ultraviolet B (UVB) radiation is mostly related to oxidative stress (OS). Little is known about whether OS induced by UVB enhances the sensitivity of lens epithelial cells to ferroptotic stress, which may be a new mechanism leading to age-related cataracts (ARCs). METHODS: Ferroptosis was detected by transmission electron microscopy (TEM), iron assay, lipid peroxidation (MDA) assay, real-time PCR, western blotting, and immunofluorescence. Genetic engineering technology was used to investigate the regulatory relationship among Sirtuin 6 (SIRT6), nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear receptor coactivator 4 (NCOA4), glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH1). Knockdown and overexpression of SIRT6 locally in vivo in rats were performed to probe the regulatory mechanism of SIRT6 in ferroptosis in ARCs. FINDINGS: Here, we observed that UVB can drastically induce ferroptosis in lens epithelial cells in vivo and in vitro. Surprisingly, inhibition of ferroptosis was the direct reason that melatonin rescued B-3, SRA01/04 and HEK-293 T cells survival; the pan-caspase inhibitor Z-Vad-FMK did not significantly reverse the death of UVB-irradiated cells compared with that in the UVB+DMSO group. SIRT6 was an upstream regulator of phosphorylated Nrf2 (p-Nrf2) and NCOA4 in B-3, SRA01/04 and HEK-293 T cells. Melatonin inhibited ferroptosis through the SIRT6/p-Nrf2/GPX4 and SIRT6/COA4/FTH1 pathways to neutralize lipid peroxidation toxicity, which protected cells against ferroptotic stress in vitro and delayed cataract formation caused by UVB exposure in rats. INTERPRETATION: Our findings reveal a novel causal role of melatonin in the pathogenesis of ARCs, which raises the possibility of selectively targeting the activation of SIRT6 and ferroptotic resistance as a latent antioxidative therapeutic strategy for ARCs.
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Catarata , Ferroptose , Melatonina , Sirtuínas , Animais , Humanos , Ratos , Catarata/prevenção & controle , Catarata/metabolismo , Ferritinas , Células HEK293 , Melatonina/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Oxirredutases/metabolismo , Sirtuínas/metabolismoRESUMO
Mutations in the coding region of the OPTN gene are associated with certain glaucomas. Although the function of the optineurin protein is yet to be elucidated, the most common mutation, E50K, is associated with a severe phenotype. Plasmids expressing wild-type Optineurin (WT) and mutant Optineurin(E50K) were transfected into RGC-5 and monitored by immunofluorescence staining and western blotting. The mutant Optineurin(E50K) induced the death of retinal ganglion cells by generation of reactive oxygen species accompanied disruption of mitochondrial transmembrane potential, down-regulation of bcl-2, and up-regulation of bax, which led to the release of cytochrome C from the mitochondria into the cytosol, which, in turn, resulted in the activation of caspase-9 and caspase-3, indicating that mutant Optineurin(E50K) acquired the ability to induce cell death through the mitochondrial caspase-dependent cell death pathway.
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Apoptose , Mitocôndrias/metabolismo , Proteínas Mutantes/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Transdução de Sinais , Fator de Transcrição TFIIIA/metabolismo , Substituição de Aminoácidos/genética , Animais , Western Blotting , Proteínas de Ciclo Celular , Fracionamento Celular , Linhagem Celular , Citometria de Fluxo , Humanos , Espaço Intracelular/metabolismo , Potencial da Membrana Mitocondrial , Proteínas de Membrana Transportadoras , Microscopia de Fluorescência , Propídio/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Coloração e Rotulagem , Frações Subcelulares/metabolismoRESUMO
PURPOSE: Allergic conjunctivitis is the most common cause leading to ocular redness (OR). Herein, using an animal model of allergic OR, we evaluated the therapeutic efficacy of topical blockade of substance P (SP) in treating red eye. METHODS: Allergic OR was induced in guinea pigs with topical histamine. Ocular SP was blocked using a specific SP receptor (neurokinin-1 receptor, NK1R) antagonist, L-703,606, via topical application 10 min before or 10 min after histamine instillation. Animal eyes were examined and a series of images were taken for up to 60 min post-OR induction. The severity of redness was analyzed using the quantitative ocular redness index (ORI). At the end of clinical examination, conjunctival tissues were collected for histological examination of conjunctival blood vessels and infiltrating eosinophils and neutrophils. In addition, SP concentration was quantified in the tear fluid and expression levels of inflammatory cytokines were assessed in the conjunctival tissues. RESULTS: Topical histamine application successfully induced red eye, evidenced by the significantly increased ORI during the observation period, with peak values at 10 min, along with significantly increased levels of SP in the tears. Topical treatment with L-703,606, either before histamine application or at the time of peak ORI, effectively reduced ORI and suppressed conjunctival blood vessel dilation, along with decreased eosinophil and neutrophil infiltration, and inflammatory cytokine expression in the conjunctiva, as well as reduced SP levels in the tears. CONCLUSIONS: Topical blockade of SP effectively prevents and treats allergy-related ocular redness by suppressing blood vessel dilation and allergic inflammation.
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Conjuntivite Alérgica , Substância P , Cobaias , Animais , Histamina/farmacologia , Histamina/uso terapêutico , Conjuntivite Alérgica/tratamento farmacológico , Conjuntivite Alérgica/etiologia , Túnica Conjuntiva/patologia , Eosinófilos/metabolismo , Eosinófilos/patologiaRESUMO
PURPOSE: To investigate the inhibitory effects of anti-mouse interleukin-17 (IL-17) monoclonal antibody (mAb) in high-responder corneal allograft rejection. METHODS: C57BL/6 or BALB/c mice corneal grafts were grafted onto BALB/c hosts. The neutralizing mouse IL-17 antibody and isotype control were injected intraperitoneally immediately after transplantation for experimental treatment. At appropriate times after treatment, recipient grafts were assessed clinically and histologically, and recipient corneal graft- infiltrating cells were detected by immunohistochemistry and quantified by real-time PCR. The cytokine spleen levels of T helper type 1 (Th1), Th2, and Th17 were analyzed by enzyme-linked immunosorbent assay. Flow cytometric analysis was used to evaluate the frequencies of IL-17-producing Th17 cells. RESULTS: Neutralization of IL-17 with anti-IL-17 mAb obviously prolonged allograft survival compared to the group that received isotype control. Neovascularizations and inflammatory immune cells in corneal stroma decreased in the allogeneic recipients treated with anti-IL-17 mAb. The mRNA (mRNA) level of graft-infiltrating cells, including neutrophiles, cluster of differentiation 4 (CD4) T cells, and CD8 T cells, decreased dramatically in the IL-17 neutralization group. At days 14 and 42, splenocytes from recipients treated with anti-IL-17 mAb produced significantly less of the pro-inflammatory cytokines interferon-gamma (IFN-γ), IL-12p40, and IL-17 compared to those from control Ig-treated recipients at day 14. However, Th2 cytokine IL-4 and IL-5 production increased, and IL-13 levels were not significantly different among the three groups. IL-6 production was elevated in recipients treated with anti-IL-17 mAb. Anti-IL-17 mAb reduced the percentage of Th17 in CD4+ T cells, but there was no statistical significance between anti-IL-17 mAb and the control group. CONCLUSIONS: Neutralization of mouse IL-17 bioactivity with anti-IL-17 mAb improves allogeneic corneal graft survival and inhibits corneal allograft rejection to a certain extent by inhibiting production of graft-infiltrating inflammatory cells and decreasing the secretion of pro-inflammatory cytokines.
Assuntos
Anticorpos Monoclonais/farmacologia , Transplante de Córnea , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Interleucina-17/antagonistas & inibidores , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Sobrevivência de Enxerto/imunologia , Interferon gama/biossíntese , Interferon gama/imunologia , Subunidade p40 da Interleucina-12/biossíntese , Subunidade p40 da Interleucina-12/imunologia , Interleucina-13/biossíntese , Interleucina-13/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-4/biossíntese , Interleucina-4/imunologia , Interleucina-5/biossíntese , Interleucina-5/imunologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Baço/citologia , Baço/imunologia , Linfócitos T/citologia , Transplante HomólogoRESUMO
PURPOSE: Endostatin plays an important role in inhibiting corneal neovascularization (CNV). The aim of this study was to evaluate the antiangiogenic activities of lipid-mediated subconjunctival injection of the modified RGDRGD (arginine- glycin- aspartic- arginine- glycin- aspartic- endostatin gene in a rabbit model of neovascularization in vivo. METHODS: A modified human endostatin gene containing an RGDRGD motif was obtained by rapid site-directed mutagenesis. Forty New Zealand white rabbits underwent alkaline burn and developed CNV, which were randomly divided into four groups: an experimental control group, a PCI empty vector group, a PCI-endostatin group, and a PCI-RGDRGD-endostatin group. The vector, endostatin, and RGDRGD-endostatin groups received injections into the superior bulbar conjunctiva after the burn. An injection of 5 µg was given twice at 1-week intervals. Four eyes of two rabbits received neither treatment nor alkaline burn and served as absolute normal controls. The areas of CNV were monitored after 7 and 14 days. Corneas were examined by histology, and VEGF (vascular endothelial growth factor) and CD31 (platelet endothelial cell adhesion molecule-1) expression was detected by immunohistochemistry after 7 and 14 days. Retina, liver, and kidney were examined by histology, and CD38 expression in the inflammatory cells was detected by immunohistochemistry at 90 days. RESULTS: Subconjunctival injection of both native endostatin and modified RGDRGD-endostatin genes resulted in a significant suppression of CNV in vivo, with modified RGDRGD-endostatin being more effective than native endostatin. The mean concentration of VEGF in the PCI-RGDRGD-endostatin group significantly decreased compared to the means in the other groups. Upon histological examination, the endostatin-treated and RGDRGD-endostatin-treated eyes showed significantly less neovascular area and fewer vessels than the control and vector-injected groups. Retinal, hepatic, and renal tissue sections were normal, and there was no inflammatory cell infiltration observed. CONCLUSIONS: Native and modified endostatin can significantly inhibit CNV by suppressing the expression of VEGF. However, modified endostatin with the RGDRGD motif is far more effective than the endostatin gene in antiangiogenic activity.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Córnea/efeitos dos fármacos , Neovascularização da Córnea/tratamento farmacológico , Endostatinas/administração & dosagem , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/uso terapêutico , Animais , Sequência de Bases , Córnea/metabolismo , Córnea/patologia , Neovascularização da Córnea/genética , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Neovascularização da Córnea/prevenção & controle , Modelos Animais de Doenças , Endostatinas/química , Endostatinas/genética , Endostatinas/uso terapêutico , Feminino , Vetores Genéticos/uso terapêutico , Humanos , Imuno-Histoquímica , Injeções Intraoculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Qingganjiuwei powder (QGJWS) is a well-known traditional drug containing nine kinds of medicinal materials. This drug is commonly used in the Inner Mongolia region and exerts remarkable clinical effects on hepatic protection. AIM OF THE STUDY: To investigate whether QGJWS inhibits liver fibrosis in rats and to reveal its potential mechanisms. METHODS: Liver fibrosis model was induced by CCl4 for 8 weeks in SD rats. Next, rats were intragastrically administered quantum satis doses of QGJWS (0.525, 1.575, 4.725 g/kg per day) or Silymarin (SIL; 120 mg/kg per day) for 8 weeks. Afterwards, the rats were sacrificed, and serum aminotransferase (ALT and AST) levels, histopathological changes as well as the mRNA and protein expression of matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitor of metalloproteinase1 (TIMP1), collagen type â (COL1), α-smooth muscle actin (α-SMA), combined with phosphorylation levels of extracellular signal-regulated kinase (ERK), C-Jun amino-terminal kinases (JNKs) and stress-activated protein kinase-2 (p38) protein in liver tissues were measured in each groups, respectively. RESULTS: The symptoms and signs of the model rats were consistent with the diagnostic criteria of liver fibrosis. By contrast, treatment with QGJWS clearly improved the general condition of rats. Also, the morphology and structure of liver can be ameliorated, there are fewer hepatocyte necrosis and lymphocytic infiltration and pseudolobuli in QGJWS treatment groups as demonstrated by histopathological analysis, thus helping bring about lower METAVIR scores. QGJWS administration also dramatically decreased serum ALT and AST levels. Further immunohistochemistry, western blotting and Real-Time PCR analysis revealed that QGJWS significantly enhanced the mRNA and protein expression of MMP2, MMP9, and downregulated the expression levels of COL1, TIMP1 and α-SMA. Furthermore, QGJWS reduced the activities of mitogen-activated protein kinases (MAPKs) pathway in liver by inhibited the phosphorylation of ERK, JNKs and p38 proteins. CONCLUSIONS: QGJWS offers notable protection against CCl4-induced liver fibrosis in rats, which may be due to its ability to inhibited the MAPKs signaling pathway.
Assuntos
Tetracloreto de Carbono/toxicidade , Medicamentos de Ervas Chinesas/uso terapêutico , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Medicina Tradicional da Mongólia/métodos , Animais , Medicamentos de Ervas Chinesas/isolamento & purificação , Cirrose Hepática/metabolismo , Masculino , Pós , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Resultado do TratamentoRESUMO
Objectives: China has implemented universal hepatitis B vaccination since 2002 and provided charge-free hepatitis B immunoglobulin (HBIG) to infants of HBV-infected mothers since July 2011. We aimed to compare mother-to-child transmission (MTCT) in children born before and since July 2011.Methods: In total, 5,149 children of HBV-infected mothers were tested for HBV markers. Group one contained 1,160 children born during August 2002-June 2011 and group two contained 3,989 children born during July 2011-June 2016.Results: In total, 92 (1.8%, 95% confidence interval [95%CI] 1.4-2.2) children were infected with HBV. None (0%, 95%CI 0.0-0.1) of 3,716 children of mothers with negative hepatitis B e antigen (HBeAg) was infected, whereas 92 (6.4%, 95%CI 5.2-7.8) of 1,433 children of HBeAg-positive mothers were infected (p < 0.0001). Among children of HBeAg-positive mothers, MTCT occurred in 10.3% (19/185) (95%CI 6.3-15.6) in group one and 5.8% (73/1,248) (95%CI 4.6-7.3) in group two (p = 0.02).Conclusions: Implementing charge-free active-passive immunoprophylaxis greatly reduces MTCT of HBV in children of HBeAg-positive mothers, highlighting the importance of timely administration of both hepatitis B vaccine and HBIG to prevent MTCT. The still remaining MTCT suggests that reducing maternal virus load before delivery is an additional important measure.
Assuntos
Hepatite B , Complicações Infecciosas na Gravidez , Estudos de Coortes , Feminino , Hepatite B/prevenção & controle , Antígenos de Superfície da Hepatite B , Vacinas contra Hepatite B , Humanos , Lactente , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Gravidez , Complicações Infecciosas na Gravidez/prevenção & controleRESUMO
PURPOSE: To determine if there is increased mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage with age in the lenses of rats. We also explored the immunolocalization of 8-oxoguanine DNA glycosylase 1 (OGG1) and AP endonuclease 1 (APE1) in the lens and studied three of the predominant base excision repair (BER) enzymes: OGG1, APE1, and DNA polymerase gamma (Polgamma). METHODS: The methods used by this study include the selection of twenty-six male Wistar rats in each group (2 months old and 26 months old) and fourteen male Wistar rats in the 16 months old group. The total DNA of lenses were isolated and the DNA genome was amplified by a long extension-polymerase chain reaction (LX-PCR). We examined mtDNA and nDNA damage with a quantitative polymerase chain reaction (QPCR) assay that was combined with EvaGreen. We also studied the gene expression of mRNA and protein in these key BER enzymes with real time-polymerase chain reaction (RT-PCR) and western blot analysis. RESULTS: There was an increase in oxidative DNA damage, which exists primarily in the mtDNA. The amount of 8-hydroxy-2'-deoxy-guanosine (8-OHdG) in DNA was significantly increased with age. Our experiments demonstrated that the gene expression of mRNA and protein in these key BER enzymes decreased with age. OGG1 and APE1 were localized by immunohistochemistry within lens epithelial cells (LECs) and superficial fiber cells. CONCLUSIONS: The gene expression of mRNA and protein in these key BER enzymes decreased with age, which caused a decrease in the repairing capability of the mtDNA and the accumulation of mtDNA damage. The increased mtDNA damage and decreased expression of BER enzymes may cause a "vicious cycle" of oxidative stress that contributes to the accumulation of mtDNA mutations and age-related cataract pathogenesis.
Assuntos
Envelhecimento/patologia , Dano ao DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Mitocondrial/genética , Cristalino/enzimologia , Cristalino/patologia , 8-Hidroxi-2'-Desoxiguanosina , Envelhecimento/genética , Animais , Núcleo Celular/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Ensaio de Imunoadsorção Enzimática , Regulação Enzimológica da Expressão Gênica , Imuno-Histoquímica , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de SequênciaRESUMO
OBJECTIVE: To construct the eukaryotic express vector of wild type and R555W mutated transforming growth factor beta induced (BIGH3) gene, and to determine the effects of overexpression of R555W mutated BIGH3 in human corneal epithelial (HCE) cells. METHODS: The full coding domain sequence of BIGH3 gene was cloned from human corneal tissue from operative spackman with RT-PCR. Mutagenesis of R555W-BIGH3 was performed in rapid site-directed mutagenesis technique in the expression vector pIRES2-EGFP. The two types of BIGH3 gene were transient transfected to HCE cells. Immunoblotting was used to detect the expression of BIGH3. The cell apoptosis rates were observed by flow cytometry. The morphological changes of the cells were examined by electron microscopy. The viability of caspase-3 was examined. RESULTS: Wild type and mutated BIGH3 gene were successfully amplified by PCR. After HCE cells transfected with two types eukaryotic expression plasmid, the BIGH3 were detected in the HCE cells. The cell apoptosis were observed by flow cytometry and electron microscopy. The viability of caspase-3 was increased in the R555W mutated type. CONCLUSIONS: The R555W mutated BIGH3 is successfully obtained by rapid site-directed mutagenesis technique. Overexpression of R555W mutated BIGH3 induces apoptosis in HCE cells through activation of caspase-3.