| Purpose As a common ophthalmological emergency,corneal alkali burn often results in corneal opacity and neovascularization,sometimes even leads to loss of vision.The existing therapy for corneal alkali burns has the disadvantages of being invasive,limited in curative effect,and having the side effects which are related to serious ocular complications.Therefore,exploring safe and effective treatments for corneal alkali burns has become a focus of ophthalmology research.Previous studies suggested that alkali burns can increase the reactive oxygen species(ROS),which could promote inflammatory response and corneal neovascularization.In addition,newly formed vessels predispose to lipid exudation and deposition in the cornea.Therefore,we speculate that the lipid peroxidation induced cell death,ferroptosis,may play an important role in corneal alkali burns,and ferroptosis-targeted treatments may be a potential therapy for repairing corneal alkali burns.Ferroptosis is a novel,lipid peroxidation and labile iron dependent programmed cell death,which is totally different from apoptosis and necrosis.Both inceased ROS and increased free iron may induce accumulation of lipid peroxidants which attacts cellular memebrane and mitochondria,leading to ferroptosis.Ferrostatin-1(Fer-1)is a specific small molecular inhibitor of ferroptosis,which can rescue ferroptosis by trapping intracellular toxic free radicals.A number of studies have suggested that Fer-1 can effectively suppress the occurrence and development of a variety of ferroptosis-related diseases,indicating its potential for clinical translational treatment of related diseases.However,the role of ferroptosis in corneal alkali burn and the therapeutic effect of Fer-1are still unclear.In this study,the alkali burn model was used to detect whether ferroptosis was trigged in corneal injury,and to investigate whether the administration of Fer-1 could ameliorate the corneal damage caused by alkali burn.Topical drug delivery with eye drop is a preferred way of administration with the advantages of simplicity and minor side effects.However,due to tear flow,blink reflex and the existence of ocular surface barrier,the bioavailability of traditional eye drops is often very low.On the other hand,the poor water solubility and rapid hydrolysis of Fer-1 into inactive form in vivo limit its widely application in clinics.Liposomes contain a cell membrane-like phospholipid bilayer structure,which can enhance the penetration and solubility of the drug by encapsulating the drug in the liposome vesicle.At the same time,liposomes have the advantages of non-toxicity,enhanced drug stability,and biodegradability.Thus,liposome is one of the most potential drug delivery systems widely used in ophthalmology clinics.Based on the above-mentioned background,we designed a Fer-1 liposome eye drop.By using alkali burn mice as an animal model,we aimed to study whether Fer-1 liposomes can exert therapeutic effects in protecting corneal from alkali burn injury,as well as the underlying mechanisms.In addition,the safety of Fer-1 liposomes was evaluated through in vivo and in vitro experiments.Methods This study is divided into three parts.The first part aims to explore whether ferroptosis exists in the alkali burned cornea.Eighteen C57BL/6 mice were divided into3 groups.The first group was the healthy control group.The second and third groups were alkali burned in the right eye and treated with saline and 200 μM Fer-1 twice a day respectively.The eye drops were treated for 14 days.Before administration and on the3 rd,7th,and 14 th day after administration,slit lamp photographs of the mice were taken to evaluate the length,area of their neovascularization and clinical injury score.After 14 days of treatment,the mice were sacrificed.In each group,some treated eyeballs were dissected to isolate the cornea for photographing.Of these corneas,some were fixed in glutaraldehyde for observation of mitochondria by transmission electron microscope.The remaining corneas were used for extracting m RNA and running real-time fluorescent quantitative polymerase chain reaction.The other treated eyeballs were fixed and sectioned for H&E stained and immunohistochemistry.The second part is to study the therapeutic effect of Fer-1 liposomes on alkali burned cornea and its mechanism.First,the Fer-1 liposomes were prepared by the thin film rotary evaporation method.The dynamic light scattering measurement and transmission electron microscopy were then performed.For in vivo study,60 C57BL/6mice were divided into 6 groups.Except for the first group,the other 5 groups were alkali-burned in the right eye and treated with eye drops twice a day for 14 days.Among them,the second group was treated with saline.The third,fourth,fifth,and sixth groups were treated with 200 μM Fer-1,200 μM Fer-1 liposome,200 μM dexamethasone and200 μM dexamethasone liposome,respectively.The methods of in vivo study were same as that in the first part.For in vitro study,we evaluated the inhibitory effects of Fer-1and Fer-1 liposomes on the migration ability of human umbilical vein endothelial cells(HUVEC)induced by vascular endothelial growth factor(VEGF)in scratch wounding migration assays.The third part aims to verify the safety of Fer-1 liposomes.For in vitro study,human corneal epithelial cells(HCECs)treated with different concentrations of Fer-1 liposomes were subjected to CCK-8 assay and live/dead assay to detect cell viability.For in vivo study,the body weight of mice in groups 1-4 in the second part of study was monitored every 3 days during the administration.After the mice were sacrificed,routine blood test,liver and kidney function index tests were taken.The heart,liver,spleen,lung and kidney were also dissected for fixation,and sectioned for H&E staining.In addition,12 C57BL/6 mice without alkali burn were divided into 4 group and administered as groups 1-4 in the second part of study.On the 14 th day,ocular surface fluorescein staining was performed.The mice were also observed by slit lamp biomicroscopy and photographed.Results Compared with healthy controls,alkali burned corneas showed obvious corneal opacity,edema,inflammatory cell infiltration and neovascularization.Administration of Fer-1 attenuated the injuries,suggesting that alkali burn may cause ferroptosis in cornea.Following experiments demonstrated that alkali burn induced the upregulation of ROS related genes(Gclm,Gpx1,Gsr,Prdx1,Sod1)and ferroptosis marker Ptgs2,and accumulation of lipid peroxidants 4-HNE.Fer-1 treatment rescued these disturbance,corfirming that alkali burn triggered ferroptosis in cornea.In addition,after alkali burn induction,the expression of the ferroptosis inducer Acsl4 was upregulated,whereas the ferroptosis-suppressor Gpx4 was inhibited,which was accompanied by mitocondrial destruction.These mechanistic studies revealed that,corneal alkali burn resulted in activation of ferroptosis pathaway and inhibition of ferroptosis-suppressor.Meanwhile,accumulated ROS might attact mitochondria.Both of them accelerated the activation of ferroptosis.Fer-1 administration rescued the corneal injury from alkali burns,indicating that ferroptosis may serve as the therapeutic target for treating corneal alkali burn.Based on this,we further constructed studied Fer-1 lipisome to improve the bioavailability and therapeutic effect for ferroptosis induced corneal injury.The results showed that,after treatment,in addition to the ferroptosis markers,the expressions of the inflammation indicators including IL-1β and IL-6,and the neovascularization-related regulators including Vegfa,Vegfc,Vegfr3,Mmp2,α-Sma and CD31 were all restored.Both Fer-1 and Fer-1 liposomes could rescue the corneal injury induced by alkali burns,and the curative effects of Fer-1 liposomes were more significant.Scratch wound migration assays also showed that both Fer-1 and Fer-1liposome could inhibit the migration of HUVEC cells induced by VEGF,and the inhibitory effect of Fer-1 liposomes was more obvious.The data above indicated that the therapeutic effect of Fer-1 liposome were better than Fer-1 in the repair of alkali burned cornea.Moreover,we further accessed the safty of Fer-1 liposome in treating corneal alkali burns by detecting the biocompatibility of Fer-1 liposome.In vitro and in vivo results indicated that Fer-1 liposomes had no obvious toxic and side effects.Conclusions This study provides the first evidence that ferroptosis plays an important role in the progress of corneal injury induced by alkali burn.Fer-1,a specific ferroptosis inhibitor,could effectively alleviate the inflammatory response and neovascularization caused by alkali burns.In addition,Fer-1 liposome had a more desirable and beneficial therapeutic effect than Fer-1,as well as had no obvious toxic and side effects.Therefore,targeting ferroptosis by Fer-1 liposome could be supposed as a potential novel therapy for alkali burned cornea. |
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