| ObjectiveDiabetic keratopathy(DK)is a common diabetic ocular complication,and its pathogenesis has not been fully clarified,and there are no effective surgical or medical interventions in clinical practice.Therefore,it is of great significance to research new therapeutic targets and effective prevention and treatment measures.In this study,we investigated the pathophysiology and epithelial damage of streptozocin(STZ)-induced type?diabetic mice and the occurrence of high mobility group B1(HMGB1)and receptor of advanced glycation endproducts(RAGE)in diabetic corneal lesion's developmental changes;the role of HMGB1 and its inhibitor dipotassium glycyrrhizinate in the repair of diabetic corneal epithelium/nerve damage was also investigated;the fabrication of dipotassium glycyrrhizinate nanomicelles,and its synergistically regulation of the HMGB1 signaling pathway to promote diabetic corneal epithelial/nerve damage recovering were also evaluated in this study.Methods1.C57BL/6J mice were intraperitoneally injected with STZ to establish type ?diabetes model.The weight,blood glucose,tear secretion,and intraocular pressure of normal and diabetic mice were measured on 4,8,12,and 16 weeks after the establishement of the diabetic animal model.The eye surface and coreal epithelium were observed using a slit lamp staining after the corneal fluorescein or tiger red staining in each group.Scanning electron microscope was used to observe the microscopic changes of the corneal epithelium/endothelium microstructure of mice in pre-determined time points.TUNEL method was used to evaluate the apoptosis of mouse corneal epithelial cells.Corneal sensitivity was measured with a corneal sensitivity meter,and corneal nerve staining was also performed to observe the cornealnerve density of mice in each group.Western Blot and immunohistochemical methods were used to evaluate the expression profiles of HMGB1/RAGE.2.After performing a disease model of corneal epithelial injury in diabetic mice,exogenous HMGB1 peptide solution and dipotassium glycyrrhizinate solution were administrated with conjunctival injection,and the repair profiles of corneal epithelial injury in each group were observed with cornea fluorescein staining.Corneal nerve density after epithelial repair was evaluated with nerve antibody staining.Western blot was performed to evaluate the expression profiles of HMGB1 and RAGE.3.anomicelle micelles eye drops with dipotassium glycyrrhizinate as nanocarriers with Gen1121(Gen)as the model drug were fabricated with simple thin-film method.Parameters of nanomicelles such as encapsulation efficiency,micelle size,and storage stability were evaluated.The antioxidant activity profiles of nanomicelle eye drops were evaluated with FRAP and ABTS methods;The in vitro artificial membrane permeation characters,in vitro release profiles,and in vivo ocular permeation were evaluated.Pharmacological activities were evaluated with corneal epithelial injury model in diabetic mice.The study was explored with the following five groups: PBS eye drops group,dipotassium glycyrrhizinate eye drops group,Gen eye drops group,dipotassium glycyrrhizate&Gen mixture eye drops group,nanomicelle eye drops group.Each group was treated according to the pre-determined experimental protocol.The corneal epithelial healing speed,corneal nerve density and corneal sensitivity of mice at pre-determined time points were evaluated.Western blot and immunohistochemical experiments were explored to evaluate the expression profiles of HMGB1/RAGE.Quantitative levels of IL-6 and IL-1? in cornea were determined with ELISA.Results1.Reduced tear secretion,damaged tear film and ocular surface,increased intraocular pressure were observed in diabetic mice.Increased corneal epithelial cell apoptosis,decreased corneal sensitivity,and decreased nerve density were also observed to diabetic mice.Results from western blot and immunohistochemistry evaluations showed that the expression of HMGB1/RAGE continued to increase with the duration of diabetes.The diabetic corneal epithelium/nerve healing were delayed.2.Nanomicelle eye drops based on dipotassium glycyrrhizinate as nanocarriers could significantly promote corneal/nerve healing,while the exogenous HMGB1 peptide delayed corneal/ nerve healing in diabetic mice.3.The average micelle size of the nanomicelle eye drops based on dipotassium glycyrrhizinate as nanocarriers was 29.5 ± 2.05 nm,and the encapsulation efficiency of Gen was 98.96 ± 0.95%.This nanomicelle eye drops showd well ocular tolerance.The antioxidant capacity of Gen in nanomicelles was highly improved,and the abilitites ofin vitro artificial membrane permeation and in vivo ocular permeation were also greatly improved.Nanomicelle eye drops helped heal of damaged corneal epithelium and corneal nerve when compared to other tested group(p<0.05).Western Blot and immunohistochemical results revealed that the expression levels of HMGB1/RAGE in nanomicelle eye drops group was much lower than other tested groups(p<0.05).The levels of inflammatory factors in corneas were also reduced in nanomicelle eye drops group(p<0.05).ConclusionThe HMGB1 played an important role in DK,and might be a potential target for the prevention and treatment of DK.The nanomicelle eye drops based on dipotassium glycyrrhizinate as nanocarriers could exert multi-target regulations of HMGB1 signaling pathway.This novel nanomicelle eye drops might be a promising nanomedicine for DK. |
PDF Full Text Request