Plasma exosomal proteomic studies of corneal epithelial injury in diabetic and non-diabetic group.

OBJECTIVE

Diabetic Keratopathy (DK) is one of the significant complications of type II diabetes (T2DM) with pathogenesis not yet clarified. Since hyperglycemia is able to change the protein components contained in plasma exosomes, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as feasible to analyze the expression of plasma exosomal proteins in patients with T2DM and non-diabetic patients respectively, find critical biological markers, and explore the mechanism of DK as well as potential therapeutic targets.

METHOD

Blood and clinical information of corneal epithelial injury in a diabetic group (the study group) and a non-diabetic group (the control group), who were patients admitted to the Department of Ophthalmology, Yangpu Hospital, Tongji University School of Medicine from July 2020 to November 2020, were collected. The qEV size exclusion method was adopted to separate exosomes from plasma. The exosomes were then identified through transmission electron microscopy (TEM), nanoparticle tracking analyzer (NTA), and Western blot. The plasma exosomes of the study group and the control group were quantitatively analyzed by proteomics. A bioinformatics method is utilized to screen differential proteins and the expression of the differential proteins was verified by Western blot.

RESULT

TEM indicated that the exosomes had a double-concave disc-like appearance, with a size of about 100 nm, and Western blot expressed as CD63 and TSG101. The plasma exosomes of the study group and the control group were analyzed by quantitative proteomics with a total number of 952 proteins detected of which 245 proteins existed in the ExoCarta exosomal protein database. Through adoption of P-value to screen credible differential proteins, the heat map displayed 28 differential proteins, 7 upregulated proteins, and 21 downregulated proteins; the volcano map displayed 7 upregulated proteins and 22 downregulated proteins; the PPI interaction map displayed 12 upregulated proteins and 18 downregulated proteins. Through GO enrichment analysis, it was identified that the differential protein participated in the main biological processes and was involved in regulating the cell's stimulation response to insulin, the insulin receptor signaling pathway, and the activity of glycosylphosphatidylinositol phospholipase D as well as anti-oxidation. The enriched cell components include main components such as exosomes, blood particles, and cytoplasm. KEGG enrichment analysis indicated that the target protein FLOT2 was mainly concentrated in insulin-related signaling pathways. Western blot indicated that the expression of FLOT2 in the study group was lower compared with the control group while the expression of Exo70 was higher.

CONCLUSION

Proteomic analysis of the study group and the control group displayed a variety of proteins in plasma exosomes. The downregulated protein FLOT2 in the study group was closely related to the occurrence, development, and complication of DK in T2DM patients. The expression status of plasma FLOT2 protein in T2DM patients is expected to be a biomarker for diagnosing and monitoring of DK.