The Mechanisms Of Corneal Alkali Burn And Rapamycin-induced Damage Repair Using A Mouse Model

Cornea is the part of the eye exposed to the outer environment and thus most likely to receive damage due to various insults.Corneal chemical injuries account for 11.5%to 22.1%of ocular emergencies.Among them,corneal alkali burns are usually more severe than other types of injuries because alkali can rapidly penetrate tissues due to their lipophilic properties.Most injuries still result in loss of vision even with medical and surgical management due to corneal scarring and neovascularization.Therefore,the repair of corneal alkali burn is not only focused by fundamental science research,but also an important medical problem to be solved urgently.However,a large number of studies have focused on the effects of drugs on corneal alkali burn,and few studies on the mechanism of systemic response caused by injury,leading to ineffective screening of target drugs.Corneal alkali burn-induced response undergoes a highly complex cascade mechanism,which involves cytokine-mediated interactions among corneal epithelial cells,stromal cells,and immune cells.However,what is more important is the corneal neovascularization caused by injury,the corneal fibrosis caused by stromal cell differentiation and severe inflammation.Recently,rapamycin（Rapa）has been shown to induce anti-corneal fibrosis and anti-angiogenesis upon corneal injury,but the mechanisms that Rapa-mediated mammalian target of rapamycin（m TOR）regulate the corneal neovascularization were less studied.The aim of this thesis is to determine underlying DNA methylation-regulated mechanism,transcriptional factor-regulated mechanism and the MAPK/ERK1/2 and PI3K/AKT signaling pathways regulating mechanism,as well as corneal alkali burn-induced immune response and the drug repair mechanism on corneal alkali burn-induced angiogenesis and damage repair using Rapa.We use Hematoxylin&Eosin（H&E）staining,immunofluorescence staining（IF）,bisulfite sequencing PCR（BSP）,electrophoretic mobility shift assay（EMSA）,Western blot（WB）,Enzyme-linked immunosorbent assay（ELISA）and flow cytometry（FCM）,sequentially from the morphological,histological,and molecular levels in mouse model,drawing several conclusions as follows.Ⅰ.The alkali burn-induced corneal opacity,as well as regulations on corneal angiogenesis from DNA methylation,transcriptional factor and MAPK/ERK1/2 and PI3K/AKT signaling pathways under the Rapa treatment in mice:1.Alkali burn causes corneal neovascularization mediated by vascular endothelial growth factor（VEGF）,and induces quiescent keratinocytes to differentiate into fibroblasts which express alpha-smooth muscle actin（α-SMA）and those are transformed into myofibroblasts,leading to corneal opacity.Rapa protects the corneas from alkali burn injury through reducing VEGF-mediated angiogenesis andα-SMA-mediated corneal fibrosis.2.The cell proliferation induced by alkali burn is mainly from the corneal stromal cells,whereas the Rapa-induced cell proliferation from the corneal epithelial cells.These results suggest a feasibility of Rapa as a treatment candidate,namely it can effectively promote the proliferation of epithelial cells to achieve the purpose of repairing the wound surface as soon as possible,and it possibly inhibit the proliferation of corneal stromal cells to ensure the stromal uniformity and corneal good refraction.3.Alkali burn induces the increase of DNA methyltransferases 3b（Dnmt3b）expression and mediates the de novo DNA methylation of m TOR gene promoter,while Rapa mediates the reduction or removal of methylation induced by alkali burn through inhibiting the expression of Dnmt3b.Such regulatory mechanism mainly relies on some key Cp Gs sites in promoter to achieve its effect on transcription level in corneal alkali burn.4.Transcription factor ETS-variant 5（ETV5）binds to m TOR demethylation sites induced by Rapa and negatively regulates gene expression,which plays an important role in the process of damage repair.5.In a mouse model of corneal alkali burn,one of the mechanisms of angiogenesis is the injury-induced activation of the PI3K/Akt/m TOR signaling pathway and high expression of hypoxia-inducible factor-1alpha（HIF-1α）,promoting the high expression of VEGF.Therefore,we can draw a regulation axis of angiogenesis induced by alkali burn:PI3K/Akt/m TOR/HIF-1/VEGF.6.Both MAPK/ERK1/2 and PI3K/Akt signaling pathways regulate the VEGF expression through m TOR molecule.Therefore,combined inhibition of the both pathways contribute to the treatment of angiogenesis diseases.Ⅱ.The immune response and inflammation-related angiogenesis to corneal alkali burn and Rapa treatment after alkali burn:1.Alkali burn causes that the leukocytes infiltrate into damaged corneal stroma from the limbus,including macrophages and neutrophils,releasing interleukin-1α（IL-1α）and tumor necrosis factor-alpha（TNF-α）,activating Toll-like receptor 4（TLR4）and Nucleotide binding oligomerization domain-like receptors 3（NLRP3）pathway and recruiting matrix metalloproteinases-2（MMP-2）to develop the acute inflammation response and inflammation-related angiogenesis of alkali burn corneas.Rapa decreases the inflammatory response and inflammation-related angiogenesis by inhibiting leukocytes infiltration and cytokines expression.2.Rapa promotes the alkali burn-indued transformation of CD4+T cells to CD4+CD25^highTreg cells in corneas.