The Anti-fibrosis Effects Of Peroxisome Proliferator-activated Receptor δ On Rat Corneal Wound Healing After Excimer Laser Keratectomy

ObjectivesCorneal opacity is a major cause of visual impairment worldwide.Corneal stromal fibrosis characterized by myofibroblasts and abnormal extracellular matrix(ECM)is usually the result of inappropriate wound healing.The commonly used steroids and mitomycin C(MMC)are associated with serious side effects.Therefore,it is necessary to explore a new treatment that is both effective and safe.Recently,it is reported that peroxisome proliferator-activated receptor(PPAR)8 plays important roles in the regulation of cell growth and differentiation as well as tissue wound healing.Extensive works by independent laboratories show that PPAR8 agonist inhibits cell proliferation and collagen synthesis in vascular smooth muscle cells(VSMC)and cardiac fibroblasts.In vivo,PPAR8 agonist treatment shows protection from liver fibrosis by reducing inflammation.However,whether activation of PPARδattenuates the corneal fibrosis is unknown.For the first time,we aimed to figure out whether the ligand activation of PPAR5 had an impact on corneal wound healing,especially on corneal fibrosis after laser ablation.Methods1.Transepithelial phototherapeutic keratectomy(PTK)4.5 mm in diameter and 50 or 70 pm in depth over central cornea were done in Sprague-Dawley rats.The corneas were evaluated by slit-lamp biomicroscopy,corneal confocal microscopy and HE staining to choose the suitable depth of laser ablation.2.We used GW501516,a highly selective PPAR8 agonist,to activate PPARδ and GSK3787,a selective and irreversible PPAR8 antagonist,to block PPAR8.PBS,GW501516,GSK3787 or GW501516 combined with GSK3787 were given subconjunctivally immediately after laser and twice a week until sacrificed.3.Deficiencies of corneal epithelium were examined by slit-lamp biomicroscopy at early time after laser.Corneal neovascularization(CNV)and corneal haze after laser were examined once a week by slit-lamp biomicroscopy until 4 weeks after laser.4.Cellular aspects of corneal wound healing and the changes of ECM were evaluated with in vivo confocal imaging 1,2 and 4 weeks after laser.The relative intensities of reflectivity for anterior stroma were analysed quantatively.5.Cellular aspects of corneal wound healing were examined by post-mortem HE staining and numbers of keratocytes in central cornea were counted 4 weeks after laser.6.The a-SMA,collagen type Ⅲ and fibronectin were examined by post-mortem immunofluorescent stainings 2 and 4 weeks after laser.7.The mRNA expression levels of Ki67 antigen,α-SMA,collagen type Ⅲ and collagen type Ⅰ were examined by RT-PCR 4 weeks after laser.Results1.Transepithelial PTK 4.5 mm in diameter and 70 μm in depth over central cornea produced suitable haze in Sprague-Dawley rats.2.Ligand activation of PPARδ inhibited corneal re-epithelialization:At 24 and 48 hours after surgery,the remaining corneal epithelial defect areas were largest in GW501516 group with significant differences.3.Ligand activation of PPARδ promoted corneal angiogenesis:The degrees of CNV in GW501516 group were higher than that in all other groups 1 week and 2 weeks after surgery with significant differences.4.Ligand activation of PPAR8 improved the corneal transparency:The hazes were lower in GW501516 group compared to GSK3787 group 4 weeks after laser with significant differences.5.Ligand activation of PPAR8 inhibited the activation and proliferation of keratocytes:At 4 weeks after surgery,corneal confocal microscopy showed that corneas in GW501516 group presented with quiet cells and low reflectivity of ECM while corneas in GSK3787 group presented with active cells and higher reflectivity of ECM.The average pixel intensities per micron of the central cornea for anterior stroma which indicated the relative intensity of reflectivity of the laser ablated stroma were lowest in GW501516 group with significant differences.Mean numbers of keratocytes in central cornea were lower in GW501516 group compared to PBS group and GSK3787 group with significant differences.The mRNA expression levels of Ki67 antigen in corneal stroma were lowest in GW501516 group with significant differences.6.Ligand activation of PPAR8 inhibited keratocytes transdifferentiation into myofibroblasts:At 2 weeks after surgery,immunofluorescent stainings for α-SMA were positive in the anterior and mid stroma without significant differences among all groups.At 4 weeks after surgery,the mRNA expression levels of α-SMA were lowest in GW501516 group with significant differences.7.Ligand activation of PPARδ inhibited ECM synthesis:At 4 weeks after surgery,immunofluorescent stainings for collagen type Ⅲ and fibronectin were lower in GW501516 group than in PBS group and GSK3787 group.The mRNA expression levels of fibronectin,collagen type Ⅲ and collagen type Ⅰ were lowest in GW501516 group with significant differences.ConclusionsGW501516 attenuated the activation and proliferation of keratocytes which could be reversed by GSK3787.Transdifferentiation from keratocytes into myofibroblasts and associated ECM synthesis as well as corneal haze were decreased by GW501516 at remodeling phase of wound healing.These results suggest the activation of PPAR8 has a potential anti-fibrosis effect on comeal wound healing,but the target should be carefully chosen because it delays corneal re-epithelialization and promotes corneal angiogenesis as well.