| PURPOSEDiabetic corneal neuropathy belongs to diabetic peripheral neuropathy, is oneof corneal disease risk factor. It provides new content to fully articulatethe mechanism of diabetes corneal neuropathy for a comprehensive elucidate the pathogenesis of diabetic neuropathy. In this study, we researched silencing signal regulators factor 1(Sirt1) acting on the micro-RNA(mi RNA), and targeting NOX4 gene to achieve the protective effect against diabetic peripheral neuropathy. METHODSIn this study, we chose type 2 diabetes model BKS.Cg-m + / + Leprdb / J mice(BKS-db/db) as an animal model, and first we explored the expression of SIRT1 in the TG tissue. Using adenovirus system to overexpress SIRT1 gene, by subconjunctival injection to make SIRT1 overexpression in TG tissue and then use mi RNA Array gene chip technology to detect m RNAs in TG tissue regulated by SIRT1.By bioinformatic analysis to explore possible mi R-182 target genes, and verified on cultured TG neurons.Primary trigeminal ganglion neurons were cultured in vitro,and were detected the occurrence of growth and survival under mi R-182 agomir vitro.Corneal damage-healing model was constructed on BKS-db/db mice, then mi R-182 mimics or inhibitors were injected through subconjunctival way. Corneal specimens were harvested after 72 hours, and it was detected of the expression of related genes and protein of epithelial cells and nerve degeneration, and corneal nerve was stained to demonstrate corneal nerve changes.Corneal epithelial healing was observed by sodium fluorescein staining, and corneal peripheral nerve degeneration was evaluated by corneal sensation, and β-tublin corneal nerve staining. RESULTSBy Westernblot and quantitative PCR technology, combined with immunofluorescence co-localization technology, we found a significant decrease in the expression of SIRT1 in TGof BKS-db/db mice.By micro Array gene chip technology, total of 29 mi RNAs were found by the difference expression regulated by SIRT1, including 19 genes upregulated, 11 downregulated genes. We selectedmi R-182 for further research, the most significant difference one in the expression of mi RNA.Applying two SIRT1 activator resveratrol(RSV) and SRT1720, treated TG primary cultured cells, we found mi R-182 significantly increased with the expression of SIRT1. To further validate the results, we constructed the SIRT1 overexpression adenoviral system and the SIRT1 interference adenoviral system, and found mi R-182 expression significantly changedwith the significantly changed expression of SIRT1 positively. It showed SIRT1 regulatedthe expression of mi R-182.Application of mi R-182 agomir treated primary cultured trigeminal ganglion cells, the results showed that compared with normal cultured trigeminal ganglion cells mi R-182 agomir significantly increased the length of the trigeminal ganglion cell synapses after treatment.By immunofluorescence, immunohistochemistry, q RT-PCR analysis showed that, compared to thedb/+control mice, SIRT1 expression in the cornea and TG of BKS-db / db mice was significantly reduced. After subconjunctival injection of mi R-182 agomir, it significantly promotedthe corneal epithelium healing of BKS-db / db mice, and significantly promoted corneal nerve repair and restored corneal sensitivity.By the same methods, we found NOX4 expression was up-regulated in TG cells and cornea of BKS-db/dbmice. By subconjunctival injection overexpression NOX4 adenovirus, NOX4 can significantly block the epithelium healing and neuroprotective effects of mi R-182, and by subconjunctival injection NOX4 interfering RNA NOX 4 can significantly promote the corneal epithelium healing of BKS-db/db mice and significantly promote corneal nerve restore. CONCLUSIONSThis study uncovered a role for Sirt1-responsive mi R-182 in protecting against diabetic peripheral nerve injury in trigeminal sensory neurons and keratopathy using the NOX4 pathway. |
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