Reactive Oxygen Species Are Essential Mediators In Epidermal Growth Factor-Stimulated Corneal Epithelial Cell Growth And Wound Healing

Reactive oxygen species(ROS),including superoxide anion(O₂),hydroxyl radical ((?)OH) and hydrogen peroxide(H₂O₂),are well known as byproducts of normal aerobic metabolism,or generated by profession phagocytic cells.They can also be derived from exogenous sources,either being taken up directly by cells from the extracellular milieu, or produced as a consequence of the cell exposure to some environmental insult.In general,ROS are considered to be harmful to cells and tissues.In the past,the studies showed that ROS are harmful for corneal epithelial cells, mitochondria produces high level of ROS under pathological condition,Unusual stimulation or wound such as UV-light exposure,refraction surgery,chemical burn can cause oxidative stress,leading to cell function damage or cell death.In other studies of ocular tissues,ROS-induced damage is associated with cataract and macular degeneration.The possibility of ROS as a beneficial element in corneal tissue and mitogenic signaling pathways has never been explored,even though the cornea expresses wide spectrum of growth factor receptors,and those receptors are known to respond to their respective growth factors to induce cell growth or cell differentiation as well as other cellular functions.Our study used human corneal epithelial cell line and primary cultured rabbit corneal epithelial cells as study models,we found epidermal growth factor(EGF) can stimulate corneal epithelial cells generate ROS shortly,which were involved in EGF-stimulated cell proliferation,adhesion,migration and wound healing.We also found that,short period of low concentration extracellular H₂O₂ improved corneal epithelial cell growth and wound healing.Part one:Detection of intracellular ROS and the effect on cell proliferation,adhesion,migration and wound healingTo verify if EGF-stimulated cells can indeed generate intracellular ROS,HCE and RCE cells were gradually deprived serum overnight.EGF(5 ng/ml)-stimulated ROS production in cells was captured as DCF-DA fluorescence and detected by confocal microscopy.The results showed EGF(5 ng/ml)-stimulated cells generate a short period of ROS production in 90 min.Inhibition of ROS fluorescence was done in cells pretreated with anitoxidant N-acetylcysteine(NAC at 40 mM).In order to clarify that. the observed EGF-induced ROS generation in HCE cells is not a artifact due to the transformed cell line,we conducted a similar experiment using primary epithelial cell culture from rabbit comea.The confocal study result shown that primary RCE cells also respond to 5 ng/ml EGF by generating transiently during 60 min.This EGF-stimulated ROS generation could be eradicated by preloading the cells with antioxidant NAC(40 mM),or free radical scavenger,mannitol(100 mM),confirming the results observed in HCE cells.We next determined the potential source of the EGF-stimulated ROS in the corneal epithelial cells,the enzymatic systems of NADPH oxidase,the lipoxygenase (LOX),and mitochondrial ROS production activations were inhibit by special inhibitors, RCE cells preloaded with NADPH oxidase or LOX inhibitor showed the effect that blocked EGF-induced ROS production.However,cells preloaded with mitochondrial inhibitor showed little change under the same conditions.We found that EGF can stimulate corneal epithelial cells generate intracellular ROS,the ROS observed is likely produced from NADPH oxidase and LOX.5 ng/ml EGF induced cell proliferation effect was quantified using BrdU assay,as shown in results,5 ng/ml EGF-stimulated primary RCE cells showed an increase of 28%in newly synthesized DNA and 30%for HCE cells over the untreated control cells.The mitogenic effect of EGF was totally eliminated when free radical scavenger mannitol(100 mM),or antioxidant NAC(40 mM) was present in the cells.These results suggested that the present of intracellular ROS is essential for EGF-induced cell proliferation.The phosphorylation of the MAPK,MEK,AKT which were related with cell proliferation and survival signalings were test by western blotting.ROS production during EGF stimulation correlated with cell growth and activations of the MAPK signaling pathway,while elimination of ROS by antioxidant or inhibitors suppressed cell growth and inhibited MAPK signaling.For cell adhesion assay,cells were trypsinized and seeded onto fibronectin-coated culture slides.Cells were fixed and photographed at 30,60,120,180,and 300 min.This experiment was repeated and the data showed that the physiological function of EGF was eradicated without ROS,suggested that ROS are required for EGF cellular function on cell adhesion.The beneficial effect of EGF with or without NAC on migration and wound healing were studied using the scrape-wound cell model and pig cornea organ culture model.HCE cells were grown to confluence and then the plate was gently scraped across the monolayer of cells to generate an even gap(the scrape wound).5 ng/ml EGF alone induced fast cell migration and closed completely by 16 hrs.In contrast,cells treated with NAC + EGF were slow in cell migration and the wound closure is not improved at 16 hrs.We used cornea organ culture model by fresh pig eyeballs.A center cornea epithelial detriment wound of 9 mm in diameter was made,and the injured area was stained by Richardson's dye.Wound healing process was photographed for 48 hrs. The corneas treated with 40 mM NAC,the wounding region showed very slow improvement compared with EGF group.These results proved strong evidence that ROS induced endogenously by EGF are nessential for the wound healing process of corneal epithelium.Part two:The effect of low concentration of extracellular ROS (H₂O₂) on cell proliferation,adhesion,migration and wound healing To study if extracellular ROS have the similar effect on cell physiological function as intracellular ROS,we used different concentration of extracellular ROS(hydrogen peroxide,H₂O₂) to stimulate HCE cells for 4 hrs.BrdU assay showed that 10-30 uM H₂O₂ can stimulate corneal epithelial cell proliferation,20 uM H2O2 showed the highest effect on cell proliferation,.However,lower(<10μM ) or higher(> 40μM) H₂O₂ didn't improve,or even depressed cell proliferation.According to these results,we chose 20μM H₂O₂ to treat the cells seeded on fibronetin coated slides.Cells were fixed and photographed after 5 hrs.The results showed that cells adhere in higher numbers and faster in the presence of 20μM H₂O₂ comparing to control group and antioxidant group,low concentration of H₂O₂ has the similar effect as EGF in cell adhesion.The wounded pig cornea organ culture model was made as described above,the cornea received control,20μM H₂O₂,NAC+ H₂O₂ three different treatments.After 48 hrs culture,the corneas received 20μM H₂O₂ treatment showed a completed healing like EGF group.However,30 min pretreatment of 40 mM NAC depressed the wound healing.The folloing novel findings were drawn based on the data:1.This study provides the first evidence that epidermal growth factor can stimulate corneal epithelial cells generate intracellular ROS shortly.2.These intracellular ROS were likely related generated from NADPH oxidase and LOX,and not from mitochondria.3.The presence of ROS in corneal epithelial cells appeared to be essential for mitogenic signaling pathways MER,INK,ERK1/2 and survival pathway AKT activation.4.Intracellular ROS has a physiological function in cell proliferation,adhesion, migration and cornea epithelium wound healing.]5.Low concentration of H₂O₂ can mimic the effect of EGF on cell proliferation, adhesion,migration and cornea epithelium wound healing.