ABSTRACT
PURPOSE: The objective of the study was to investigate efficacy and mechanisms of mouse adipose-derived mesenchymal stem cell-derived exosomes (mADSC-Exos) in the benzalkonium chloride (BAC)-induced mouse dry eye model. METHODS: Exosomes in the mADSC culture supernatant were isolated by ultracentrifugation. Western blotting, nanoparticle tracking analysis, and transmission electron microscopy were used to characterize mADSC-Exos. An experimental mouse model of dry eye was established by instillation of 0.2% BAC. mADSC-Exos were administered following BAC treatment. The positive control group was treated with commercial eye drops (0.1% pranoprofen). Corneal fluorescein staining, tear secretion, and tear film break-up time (BUT) were evaluated, and histologic analysis of the cornea and conjunctiva was performed by hematoxylin and eosin and periodic acid-Schiff staining. Apoptosis in the corneal epithelium was detected with the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and by Western blotting. Levels of pro-inflammatory cytokines in the cornea and conjunctiva were evaluated by flow cytometry, and mRNA and protein levels of NLR family pyrin domain-containing 3 (NLRP3) pathway components were assessed by quantitative real-time PCR and Western blotting, respectively. RESULTS: mADSC-Exos were characterized as vesicles with a bilayer membrane. The particle size distribution peak was at 134 nm. mADSC-Exos specifically expressed cluster of differentiation (CD)9, CD63, and CD81. mADSC-Exos treatment repaired ocular surface damage. Additionally, mADSC-Exos inhibited cell apoptosis, decreased the levels of interleukin (IL)-1ß, IL-6, IL-1α, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, and increased levels of the anti-inflammatory cytokine IL-10. Meanwhile, NLRP3 inflammasome activation and upregulation of caspase-1, IL-1ß, and IL-18 were reversed by mADSC-Exos. CONCLUSIONS: mADSC-Exos alleviate ocular surface inflammation, suggesting that it is a promising treatment for dry eye.
Subject(s)
Dry Eye Syndromes , Exosomes , Mesenchymal Stem Cells , Animals , Benzalkonium Compounds/toxicity , Dry Eye Syndromes/metabolism , Exosomes/metabolism , Inflammasomes/adverse effects , Inflammasomes/metabolism , Mesenchymal Stem Cells/metabolism , Mice , NLR Family, Pyrin Domain-Containing 3 ProteinABSTRACT
Menin, encoded by the Men1 gene, is responsible for ß-cell tumor formation in patients with multiple endocrine neoplasia type 1. Recently, menin has been proven to negatively regulate ß-cell proliferation during pregnancy. However, it is unclear whether menin is involved in pancreatic ß-cell proliferation in response to other physiological replication stimuli, such as glucose. In this study, we found that the menin level was significantly reduced in high glucose-treated INS1 cells and primary rat islets, both with increased proliferation. A similar observation was found in islets isolated from rats subjected to 72-h continuous glucose infusion. The glucose-induced proliferation was inhibited by menin overexpression. Further molecular studies showed that glucose-induced menin suppression was blocked by PI3K/Akt pathway inhibitors. A major PI3K/Akt substrate, Foxo1, was shown to enhance menin transcription levels by binding the promoter region of the Men1 gene. Therefore, we conclude that glucose inhibits menin expression via the PI3K/Akt/Foxo1 pathway and hence promotes pancreatic ß-cell proliferation. Our study suggests that menin might serve as an important intracellular target of glucose to mediate the mitogenic effect that glucose exerts in pancreatic ß-cells.
Subject(s)
Gene Expression Regulation/drug effects , Glucose/pharmacology , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/pathology , Transcription Factors/metabolism , Animals , Cell Line , Cell Proliferation , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Silencing , Glucose/administration & dosage , Insulin-Secreting Cells/metabolism , Male , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction , Transcription Factors/antagonists & inhibitors , Transcription Factors/geneticsABSTRACT
The Men1 gene has been identified as the gene responsible for MEN-1, a hereditary syndrome transmitted as an autosomal dominant trait. Disruption of the Men1 gene results in defects in the development of multiple organs, including pancreatic islets. Homozygous disruption of Men1 in mice causes embryonic lethality, making it difficult to determine the genes involved in defects of pancreatic islets during embryonic development. In this study, embryoid bodies formed from null mutant (Men1-/-) and wild-type (Men1+/+) embryonic stem cells were used as a model system to investigate the effects of the Men1 gene on pancreatic islet development. Using RT-PCR, SOX17, FOXA2 and NKX2.2 were found to be differentially expressed between the two embryoid bodies. Additionally, the gene expression profile of these Men1-/- embryoid bodies was characterized in detail by DNA microarray techniques, and a series of putative menin-targeted genes was identified. Our study suggests a critical role for Men1 in pancreatic islet development, and indicates that genes such as SOX17, FOXA2, NKX2.2 and SOX4 are potential targets of Men1.
