| ObjectiveCorneal chemical injury,represented by alkali burn,was a common ophthalmic emergency,which could cause a series of ocular complications,such as corneal perforation and complicated cataract.Moreover,there was still a lack of effective clinical intervention measures.It was of great clinical significance to study new therapeutic targets and effective prevention and treatment measures for corneal chemical injury.The aim of this study was to investigate the effects of potassium glycyrrhizinate nano-micelles eye drops on corneal epithelial healing and corneal neovascularization,and the expression of high mobility group protein B1(HMGB1)and related signal pathway factors,such as receptor of advanced glycation endproducts(RAGE)and toll like receptor(TLR).Methods1.First,the formulation of dipotassium glycyrrhizinate-rebamipide micelle eye drops was optimized and its encapsulation rate and particle size were determined.Then the storage stability was evaluated.The rabbit eye irritation test and the chicken embryo allantoic membrane test were used to investigate the safety of potassium glycyrrhizinate nanomicelles eye drops.Coumarin 6 was used as the model drug to investigate the absorption characteristics in vivo.2.MTT assay was used to evaluate the effects of dipotassium glycyrrhizinate nano-micelles on cell viability.The cell scratch test was used to evaluate the effect of dipotassium glycyrrhizinate nanomicelles on cell proliferation.Hydrogen peroxide stimulated human corneal epithelial cells to construct an oxidative stress model.ROS,SOD and MDA were detected,and the protective effect of dipotassium glycyrrhizinate nano-micelles on human corneal epithelial cells was evaluated.3.The corneal alkali burn model of mice was established,and the mice in each group were treated according to the established experimental scheme.On the 1st,3rd,5th,7th and 14 th day after administration,the staining of corneal fluorescein sodium and tiger red were observed by slit lamp,and the repair speed of corneal epithelium in each group was detected;the growth of corneal neovascularization was observed;the density of corneal neovascularization in different groups was observed after perfusion with FITC dextran;the corneal sensitivity was measured by corneal sensitivity meter;the growth of corneal neovascularization was observed H&E staining was used to observe the pathological changes of corneal structure;TUNEL method was used to evaluate the apoptosis of corneal epithelial cells;ELISA was used to detect the content of VEGF,IL-6,IL-1 β and other inflammatory factors;Western blot was used to detect the expression of HMGB1,RAGE and TLR4.Results1.The average particle size,polydispersity index and zeta potential of the nano-micelle eye drops were 16.56 ± 0.91 nm,0.377 ± 0.12 and-(7.27 ± 0.591)MV,respectively.The entrapment efficiency of micellar eye drops was 99.97 ± 0.86%.The micellar eye drops were safe and had no irritation to the ocular surface.There was no bleeding,hemolysis and coagulation after contact with chick embryo allantoic membrane.In addition,dipotassium glycyrrhizinate can also promote the ocular surface absorption of coumarin 6.2.In a certain concentration range(less than 500 μg/mL),dipotassium glycyrrhizate nano-micelle had good cell tolerance and could promote human corneal epithelial cell migration.Compared with the model group,dipotassium glycyrrhizinate rebamipide nano-micelles could effectively reduce the level of oxidative stress and regulate the production of SOD and MDA(compared with model group,P<0.05).3.After alkali burn,corneal epithelium was damaged,healing was slow,epithelial cell apoptosis was increased and the corneal sensitivity was decreased.After 14 days of treatment with dipotassium glycyrrhizate nano-micelle eye drops,corneal epithelial healing rate was significantly accelerated(compared with PBS group,P<0.05),corneal sensitivity was improved,the neovascularization was significantly inhibited,and the levels of inflammatory factors in the cornea were decreased.In addition,Western Blot results revealed the expression of HMGB1/RAGE/TLR4 proteins was decreased in dipotassium glycyrrhizate nano-micelle eye drops(compared with PBS group,P<0.05).But the effect of 0.1%sodium hyaluronate as a positive control group was not as good as that of micelle group.ConclusionDipotassium glycyrrhizinate was a specific inhibitor of HMGB1,and the rebamipide micelles constructed by it could treat corneal alkali burns by regulating the HMGB1 signaling pathway,and effectively inhibit corneal neovascularization. |
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