Intervention And Mechanism Of "cocktail" Nano-drops On Corneal HMGB1 Signal Pathway

Objective:Corneal injury has a high incidence among ocular diseases,which seriously damages patients’vision and quality of life.It is an urgent clinical and scientific problem to study the development mechanism and explore effective pharmacotherapy.High mobility group box 1（HMGB1）can promote the release of NF-кB and other inflammatory molecules,resulting in delayed repair of corneal epithelial cells after injury,decreased sensitivity of corneal nerve and corneal tissue lesions.Inhibition of HMGB1 signaling pathway is an effective intervention after corneal epithelial injury.Vasoactive intestinal peptide（VIP）,a corneal nerve factor,promotes the differentiation and repair of neurons during nervous system development,also inhibits excessive inflammation and immune response,which plays an important role in maintaining and regulating the immune homeostasis of corneal tissues.The purpose of this study as to fabricate"cocktail type"nano-drops.Two small molecular active excipients,dipotassium glycyrrhizinate（DG）and a--glucosyl hesperidia（GH）,were used to construct stable nano-micellar eye drops.To solubilize and synergistically strengthen bisdemethoxycurcumin（BDMC）.To explore the regulatory effect of the eye drops on HMGB1 signaling pathway,the influence of VIP protein,a key molecule in the VIP signaling pathway,on the expression changes of cornea,was also explored to reveal the possible relationship between the multi-target regulation related molecules in this signaling pathway.Methods:1.Fabricating"cocktail"nano-drops.,namely dipotassium glycyrrhizin-a--glucosyl hesperidin-bisdemethoxycurcumin nano-micelle-drops（VIP）.The encapsulation rate,particle size,polydispersity index（PDI）,potential and solubility of the eye drops were determined,as well as the eye safety profiles and ocular absorption characteristics.2.After the corneal epithelial injury model in healthy mice was established successfully,the following indexes were detected at the specified time after drug administration in each group.The repair degree of corneal epithelium was examined by fluorescein sodium staining.Corneal nerve sensitivity was measured.Corneal histopathology was detected.Corneal nerve staining was performed to judge the recovery of nerve fibers.The expressions of NF-κB,IL-6,HMGB1,TLR2,TLR4,VIP and SHH were determined.3.After the successful establishment of corneal denervation mice model and corneal epithelial injury model,the following indicators were detected at the specified time after drug administration in each group.The repair degree of corneal epithelium was examined by fluorescein sodium staining.Corneal nerve sensitivity was measured.Corneal histopathology was detected.Corneal nerve staining was performed to judge the recovery of nerve fibers.The expressions of NF-κB,IL-6,HMGB1,TLR2,TLR4,TLR9,VIP and SHH were determined.4.After AQP1^-/-mice model and corneal epithelial injury model were successfully established,the following indexes were detected at the specified time after drug administration in each group.The repair degree of corneal epithelium was examined by fluorescein sodium staining.Rose-bengal staining was used to check the degree of dry eye.Corneal nerve sensitivity was measured.Corneal histopathology was detected.Tear secretion was measured.Results:1.The encapsulation efficiency of dipotassium glycyrrhizin-a--glucosyl hesperidin-bisdemethoxycurcumin nano-eye-drops was 98.37±2.26%,the average particle size was 4.06±0.22nm,PDI was 0.26±0.04,and the zeta potential was-（21.23±1.86）m V.The eyedrops had good eye tolerance and eye absorption characteristics.2.In the healthy mice model of corneal epithelial injury,0.5mg/m L dipotassium glycyrrhizin-a-glucosyl hesperidin-didemethoxycurcumin nano-eye-drops group significantly promoted the repair of corneal epithelial injury,the recovery of corneal nerve sensitivity,and the healing of corneal tissue and structure.The expression of NF-κB and IL-6,HMGB1,TLR4 and TLR2 were significantly decreased,while the expression of VIP and SHH were significantly increased.3.In the corneal denervation mice model of corneal epithelial injury,0.5mg/m L dipotassium glycyrrhizin-a--glucosyl hesperidin-bisdemethoxycurcumin nano-eye-drops group significantly promoted corneal epithelium damage repair,promoted corneal nerve recovery and promoted the regeneration of the corneal epithelium nerve fibers,as well as promoted the healing of corneal tissue structure.The expression of NF-κB and IL-6,HMGB1,TLR4,TLR2 and TLR9 were significantly decreased,while the expression of VIP and SHH were significantly decreased.4.In the AQP1^-/-mice model of corneal epithelial injury,0.5mg/m L dipotassium glycyrrhizin-a--glucosyl hesperidin-bisdemethoxycurcumin nano-eye-drops group significantly promoted the repair of corneal epithelial injury,the repair of corneal nerve sensitivity.It also increased the tear secretion,and ameliorated the symptoms of dry eye.Conclusion:This nano-eye-drops were safe and reliable,and had significant advantages in promoting the repair of corneal epithelial injury in normal mice,promoting the recovery and regeneration of corneal epithelial nerve fibers in corneal denervation mice,increasing the amount of tear secretion and improving the symptoms of dry eye in the eyes of AQP1^-/-mice.The mechanism was related to inhibiting HMGB1 signaling pathway to inhibit anti-inflammatory and enhancing VIP signaling pathway to improve corneal nerve regeneration role.