ABSTRACT
Background Limbal stem cells (LSCs) play an important role on the stability of corneal epithelium and corneal transparency.Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor can promote cell proliferation and reduce apoptosis,such as human embryonic stem cells and karatin epithelial cells.Objective This study was to investigate the improving effect of Y-27632,a ROCK inhibitor,on the activity of rabbit LSCs in corneal preservation medium.Methods Corneal preservation solution was prepared by adding 12.5％ chondroitin sulfate,10.0％ low molecular dextran,20.0 mg/L dexamethasone,100 mg/L tobramycin sulfate,9.5 g/L Hepes and 0.375 mg/L L-glutamine in MEM.The corneas of New Zealand white rabbits were collected and preserved in the corneal preservation solution with or without Y-27632 for 4,7,14 days,and the density and morphology of corneal endothelial cells were examined by using 0.25％ trypan blue staining and 0.2％ alizarin red staining.Isolated corneal epithelial cells were seeded on 3T3 feeder layer and cultured for 7-10 days until colonies formation.Colony shape of LSCs was observed under the light microscope,and colony-formation efficiency was analyzed after Giemsa staining by Image J software.Results The morphology and density of corneal endothelial cells were normal in the corneal preservation solution with and without Y-27632 for 4 days.In the seventh day after preservation,the cells remained the regular hexagon in shape in the preservation solution with Y-27632,however,the cellular membrane was slightly shrinking with the positive staining for alizarin red in the preservation solution without Y-27632.The density of corneal endothelial cells in the corneal preservation solution without Y-27632 was (2 262-± 75) cells /mm2,while in the preservation solution with Y-27632 was (2 425 ±95) cells/mm2(P＜0.001).The cloning spheres of LSCs were similar in preservation solution both with and without Y-27632 in the freshly isolated cornea or preserved corneas and exhibited more cells inside.But in 7 days and 14 days after preservation,the cloning spheres were much smaller in the preservation solution without Y-27632 group than those in the preservation with Y-27632 group.No significant differences were found in the cloning-formation rate and survival rate of corneal epithelial cells in corneas freshly isolated or preserved for 4 days in both groups (all at P＞0.05).In 7 days and 14 days after preservation,the active rates of corneal epitheli.al cells were (73.00±2.12)％ and (56.00±0.71)％ in the preservation solution with Y27632,which were significantly higher than (66.00 ± 4.00) ％ and (49.00 ± 0.71) ％ in the preservation solution without Y-27632,showing statistically significant differences between them (t =3.098,P =0.018;t =9.798,P =0.000).In addition,the cloning-formation rates of LSCs were (11.05±0.21)％ and (3.10±1.97)％ in the preservation solution with Y-27632 in 7 days and 14 days after preservation,revealing significantly elevation in comparison with (2.05 ± 1.20) ％ and (0.40 ±0.14) ％ in the preservation solution without Y-27632 (t =18.107,P =0.000;t=3.184,P=0.017).Conclusions Y-27632 promotes the vitality and cloning-formation ability of LSCs in corneal preservation medium,suggesting its potential use during storage of cornea.
ABSTRACT
Background Diclofenac sodium eye drops,pranoprofen eye drops and bromfenac sodium hydrate eye drops are three clinical commonly used nonsteroidal anti-inflammatory drugs(NSAIDs).The variation of cytoxicity among these drugs and whether the cytoxicity is related to the supplements are also unknown.Objective This study was to compare the cytotoxicity of three non-steroidal anti-inflammatory eye drops and their active components with cultured human corneal epithelial cells (HCECs) in vitro,and to discuss toxic origins of these drugs.Methods HCECs were cultured in different drugs with the final concentration of 0.10％,0.05％,0.02％ and 0.01％.Cell proliferation was evaluated by MTT assay.Then,0.002％ eye drops (1∶50) was added,and the migration and damage of the cells were deceted by transwell migration assay and lactate dehydrogenase (LDH) assay.Results The cytotoxicity of three nonsteroidal anti-inflammatory eye drops on HCECs was concentration-dependent (all at P=0.00).Diclofenac sodium eye drops showed the most dominant effects on the proliferation,migration and damage of HCECs among the three eye drops,while bromfenac sodium eye drops showed the least effect on the cell damage (proliferation:Fdrug =20.25,P=0.00;migration:F =103.43,P =0.00;damage:Fdrug =164.16,P =0.00).Compared with the eye drops,their active components showed less cytoxicity.Pranoprofen appeared the least effects on the proliferation,migration and damage of HCECs (proliferation:Fdrug =332.27,P =0.00;migration:F =332.27,P =0.00;damage:Fdrug=154.83,P=0.00).Conclusions The cytotoxicity ofdiclofenac sodium eye drops is more obvious than that of pranoprofen eye drops or bromfenac sodium hydrate eye drops.The cytotoxicity of the three eye drops originates from their supplements or the interaction between the supplements and active components.
ABSTRACT
Background Recent researches show that oxidative stress is involved in the progress of keratoconus.Nuclear factor-E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway plays a critical role in the defense against oxidative stress,but its function in keratoconus is unclear.Objective To investigate the differences of Nrf2-ARE signaling activation and matrix degenerating enzymes between keratoconus and normal corneal stromal cells.Methods Corneal stromal cells were isolated from keratoconus and normal cornea by using dispase and collagenase digestion.The cells were treated with hydrogen peroxide (H2O2) to mimic in vivo oxidative stress condition.Reactive oxygen species (ROS) production was measured by fluorescence substrate DCHF-DA incubation.Nrf2 level and the expression of Nrf2-ARE downstream antioxidant genes were analyzed by Western blot and real-time quantitative-PCR(RT-qPCR).The activity of matrix degenerating enzymes,including urokinase-type plasminogen activator (uPA)-uPA receptor (uPAR) system and matrix metalloproteinase-2 (MMP-2) were assessed by Western blot and gelatin zymography respectively.Results In normal culture,keratoconus corneal stromal cells assumed increased basal ROS and Nrf2 level when compared with normal cells(t =18.155,P＜0.01).However,after H2O2 treatment,the keratoconus corneal stromal cells showed increased ROS production,while decreased Nrf2 translocation and no significant difference in expression levels of Nrf2-ARE downstream antioxidant genes (Nrf2:t =62.123,P＜ 0.01 ; (nicotinamide adenine dinucleotide phosphate quinine oxidoreductase-1 [NQO-1]:t =2.209,P =0.092 ; hemo oxygenase-1 [HO-1]:t =0.293,P =0.784 ; superoxide dismutase [SOD2]:t =0.749,P =0.495).The contents of uPA-uPAR and the activity of MMP-2 also showed a higher level in keratoconus corneal stromal cells than normal cells,with significant differences between them (t =19.164,15.458,4.818,all at P＜0.01).Conclusions The defect of Nrf2-ARE signaling activation exists in the keratoconus corneal stromal cells,and correlats with the abnormal expression level of stromal degeneration enzymes,which suggests that the defect of Nrf2-ARE signaling activation may be involved in the progression of keratoconus.