Purpose: We used ultraperformance liquid chromatography coupled with quadrupole/time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS/MS) to analyze the metabolic profile of reflex tears obtained from patients with dry eye disorders. Methods: We performed a cross-sectional study involving 113 subjects: 85 patients diagnosed with dry eye syndrome (dry eye group) and 28 healthy volunteers (control group). Reflex tears (20–30 μl) were collected from the tear meniscus of both eyes of each subject using a Schirmer I test strip. MS data were acquired with a standard workflow by UPLC-Q/TOF-MS/MS. Metabolites were quantitatively analyzed and matched with entries in the Metlin, Massbank, and HMDB databases. Least absolute shrinkage and selection operator (LASSO) regression was conducted to detect important metabolites. Multiple logistic regression was used to identify the significant metabolic biomarker candidates for dry eye syndrome. Open database sources, including the Kyoto Encyclopedia of Genes and Genomes and MetaboAnalyst, were used to identify metabolic pathways. Results: After the LASSO regression and multiple logistic regression analysis, 4 of 20 metabolic biomarker candidates were significantly correlated with Ocular Surface Disease Index score, 42 of 57 with fluorescein breakup time, and 26 of 57 with fluorescein staining. By focusing on the overlap of these three sets, 48 of 51 metabolites contributed to the incidence of dry eye and there were obvious changes in different age groups. Metabolic pathway analysis revealed that the main pathways were glucose metabolism, amino acid metabolism, and glutathione metabolism. Conclusion: Dry eye syndrome induces changes in the metabolic profile of tears, and the trend differs with age. This evidence reveals the relationship between changes in metabolites, symptoms of dry eye syndrome, and age.

Retinopathy of prematurity (ROP), which is characterized by retinal neovascularization (RNV), is a major cause of neonatal blindness. The primary treatment for ROP is anti-vascular endothelial growth factor (VEGF) therapy, which is costly and can rapidly lead to desensitization. Celastrol, a bioactive compound extracted from Tripterygium wilfordii Hook F. (“Thunder of God Vine”), has been shown to exert anticancer and anti-inflammatory effects. However, whether celastrol has antiangiogenic activity and can suppress inflammation to inhibit ROP progression is unclear. This was investigated in the present study in vitro as well as in vivo using a mouse model of oxygen-induced retinopathy (OIR). Our results showed that celastrol treatment reduced neovascular and avascular areas in the retina and inhibited microglia activation and inflammation in OIR mice. Celastrol also inhibited proliferation, migration, and tube formation in cultured human retinal microvascular endothelial cells, and reversed the activation of the microRNA (miR)-17-5p/hypoxia-inducible factor (HIF)-1α/VEGF pathway in the retina of OIR mice. These results indicate that celastrol alleviates pathologic RNV in the retina by protecting neuroglia and suppressing inflammation via inhibition of miR-17-5p/HIF-1α/VEGF signaling, and thus has therapeutic potential for the prevention and treatment of ROP. Abbreviations: BSA, bovine serum albumin; COX2, cyclooxygenase 2; ECM, endothelial cell medium; FBS, fetal bovine serum; HDAC, histone deacetylase; HIF-1, hypoxia-inducible factor 1; HRMEC, human retinal microvascular endothelial cell; Hsp70, heat shock protein; IB4, isolectin B4; ICAM-1, intercellular adhesion molecule 1; IL-1β/6, interleukin 1 beta/6; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein 1; miRNA, microRNA; MMP, matrix metalloproteinase; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor-kappa B; OIR, oxygen-induced retinopathy; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; PI3K, phosphatidylinositol-3-kinase; qRT-PCR, quantitative real-time PCR; RNV, retinal neovascularization; ROP, retinopathy of prematurity; RTCA, real-time cell analyzer; RVO, retinal vaso-obliteration; TNF-α, tumor necrosis factor alpha; VCAM-1, vascular cell adhesion molecule 1; VEGF, vascular endothelial growth factor.

Background: Hyperglycaemia-induced angiogenesis plays an important role in diabetic retinopathy (DR). This study aimed to investigate the role of sirtuin1 (Sirt1)/forkhead box O3 (FOXO3) pathway in the effects of high-glucose on human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were divided into normal control group (5 mM glucose), high glucose group (30 mM), 30 mM glucose + shsirt1 group, 30 mM glucose + Sirt1 over-expression group (30 mM + Sirt1), 30 mM glucose + Sirt1 agonist SRT group, 30 mM glucose + SRT + FOXO3 silencing group (30 mM + SRT + siFOXO3). Cell proliferation, migration, invasion and apoptosis were determined. Results: High glucose treatment reduced the expression of Sirt1 and FOXO3 in HUVECs. However, Sirt1 over-expression or SRT attenuated the high-glucose-induced inhibition of HUVEC proliferation and migration as well as reduced their apoptosis. In contrast, Sirt1 silencing deteriorated the high-glucose induced inhibition of HUVEC proliferation and migration and further increased HUVEC apoptosis. FOXO3 expression increased with the increase in Sirt1 expression, which was accompanied by enhanced cellular functions. These were abolished after FOXO3 silencing. In addition, Sirt1/FOXO3 regulated HUVEC activities via peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Conclusions: Sirt1/FOXO3 pathway is essential for the survival of endothelial cells under high-glucose and plays an important role in the development of diabetes-induced retinal vascular endothelial injury.

Background: The progression and recurrence of pterygium mainly occur due to the abnormal proliferation and migration of stromal pterygium fibroblasts. This research explores the aberrant expression of small nucleolar RNA U3 (U3 snoRNA) in pterygium and elucidates the molecular mechanisms of U3 snoRNA in pterygium development. Methods: Primary human conjunctival fibroblasts (HCFs) and human pterygium fibroblasts (HPFs) were separated and cultured from fresh conjunctiva grafts and pterygium tissues. The PLKO.1 lentiviral system and CRISPR/Cas9 recombinant construct were, respectively, used to overexpress and silence U3 snoRNA in HPFs and HCFs for further specific phenotype analysis. RNA-seq and TMT-labeled quantitative protein mass spectrometry were utilized to evaluate the effect of U3 snoRNA on mRNA transcripts and protein synthesis. Results: Reduced U3 snoRNA in pterygium promotes HCF or HPF cells’ proliferation, migration, and cell cycle but has no significant effect on apoptosis. U3 snoRNA modulates 18S rRNA synthesis through shearing precursor ribosomal RNA 47S rRNA at the 5′ external transcribed spacer (5′ ETS). Moreover, the altered U3 snoRNA causes mRNA and protein differential expression in HCF or HPF cells. Conclusions: The atypical U3 snoRNA regulates the translation of specific proteins to exert a suppressive function in pterygium through modulating the 18S rRNA synthesis. Here, we uncover a novel insight into U3 snoRNA biology in the development of pterygium.

Aim: Pterygium is a common ocular surface disease, which is affected by a variety of factors. Invasion of the cornea can cause severe vision loss. N6-methyladenosine (m6A) is a common post-transcriptional modification of eukaryotic mRNA, which can regulate mRNA splicing, stability, nuclear transport, and translation. To our best knowledge, there is no current research on the mechanism of m6A in pterygium.Methods: We obtained 24 pterygium tissues and 24 conjunctival tissues from each of 24 pterygium patients recruited from Shanghai Yangpu Hospital, and the level of m6A modification was detected using an m6A RNA Methylation Quantification Kit. Expression and location of METTL3, a key m6A methyltransferase, were identified by immunostaining. Then we used m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq), RNA sequencing (RNA-seq), and bioinformatics analyses to compare the differential expression of m6A methylation in pterygium and normal conjunctival tissue.Results: We identified 2,949 dysregulated m6A peaks in pterygium tissue, of which 2,145 were significantly upregulated and 804 were significantly downregulated. The altered m6A peak of genes were found to play a key role in the Hippo signaling pathway and endocytosis. Joint analyses of MeRIP-seq and RNA-seq data identified 72 hypermethylated m6A peaks and 15 hypomethylated m6A peaks in mRNA. After analyzing the differentially methylated m6A peaks and synchronously differentially expressed genes, we searched the Gene Expression Omnibus database and identified five genes related to the development of pterygium (DSP, MXRA5, ARHGAP35, TMEM43, and OLFML2A).Conclusion: Our research shows that m6A modification plays an important role in the development of pterygium and can be used as a potential new target for the treatment of pterygium in the future.

Dry eye disease is the most common ocular surface disease globally, requiring a more effective treatment. We observed that a high-fat diet induced macrophage polarization to M1 and further induced inflammation in the meibomian and lacrimal glands. A four-week treatment with melatonin (MLT) eye drops can regulate macrophage polarization and alleviate dry eye signs. To investigate the therapeutic effects and mechanisms of action of MLT on high-fat-diet-induced dry eye disease in mice, RAW 264.7 cells pretreated with LPS and/or MLT underwent digital RNA with the perturbation of genes sequencing (DRUG-seq). Results showed that IFT27 was up-regulated, and MAPK pathways were suppressed after MLT pre-treatment. ERK/JNK phosphorylation was reduced in meibomian glands of MLT-treated dry eye mice and increased in IFT27 knockdown RAW 264.7 cells. In summary, MLT regulated macrophage polarization via IFT27 and reduced ERK/JNK phosphorylation. These results support that MLT is a promising medication for dry eye disease.