Topical Calcitriol Application Promotes Diabetic Corneal Wound Healing And Reinnervation

Purpose:In the streptozotocin(STZ)-induced type 1 diabetic mice,vitamin D deficiency can delay corneal epithelial healing,which can not be rescued with supplemental diet.In this study,we used topical application of calcitriol and evaluated its efficiency on diabetic corneal epithelial healing and nerve regeneration.Methods:The peripheral bloods of mice were collected,and the blood glucose levels and 25(OH)D levels of normal mice and diabetic mice were measured.To study the role of calcitriol on diabetic corneal epithelial damage repair,the experimental groups were set up:normal mice group,diabetic mice group,diabetic mice + calcitriol group,and epithelial injury mice models were established.To evaluate the calcitriol efficiency on the diabetic corneal epithelial damage repair,we stained and evaluated the corneal epithelium defect area with sodium fluorescein at 0h,24 h,30h and 48 h after injury.Ki67 immunofluorescent staining was used to evaluate the proliferation of the corneal epithelium.In order to detect the effect of calcitriol on diabetic corneal nerve regeneration,the corneal sensitivity was measured at 3,7,14 days after injury,and corneal nerve regeneration was detected by corneal staining 14 days.The corneas of normal mice,diabetic mice and diabetic mice +calcitriol group were collected.Immunofluorescence staining,ELISA and RT-q PCR were used to detect the calcitriol effects on diabetic corneal neutrophil infiltration and macrophage transformation.The expression of NLRP3 signal pathway and related factors were detected by Western blot,ELISA and RT-q PCR.To further study the mechanism of calcitriol on diabetic corneal injury healing,we investigated the effect of blocking NLRP3 and IL-1β on diabetic epithelium wound healing.The removal rates were evaluated by fluorescein sodium photographing,epithelial defect area statistics,and epithelial proliferations were evaluated by Ki67 immunofluorescence staining.Corneal sensitivity and whole-mounted corneal staining were used to evaluate the effect of NLRP3 blocker and IL-1β blocking antibody on epithelial nerve regeneration in diabetic cornea.In addition,immunofluorescence staining and quantitative PCR were used to evaluate the effect of local application of calcitriol on the expression of desmosomes and hemi-desmosomes Results:Compared with normal mice,the blood glucose of diabetic mice increased and the level of 25(OH)D in serum decreased significantly(p<0.05).The results of corneal epithelial injury experiment showed that local application of calcitriol significantly accelerated the healing of diabetic corneal epithelium,and ki67 immunofluorescence staining showed a significant increase in corneal epithelial proliferation(p<0.05).The follow-up corneal sensitivity and corneal nerve staining showed that the local application of calcitriol promoted the nerve regeneration of diabetic cornea after injury,and the corneal sensation was significantly improved(p<0.05).The results of immunofluorescence staining,ELISA and RT-q PCR showed that,compared with the untreated diabetic cornea,the local application of calcitriol reduced the infiltration of neutrophils in the diabetic cornea and promoted the transformation of M1 macrophages to M2 macrophages(p<0.05).Subsequently,ELISA,RT-q PCR and Western blot results showed that the expression of key targets of NLRP3 signaling pathway(IL-1β,Pro-IL-1β,Pro-caspase-1,caspase-1,ASC)in diabetic cornea were significantly decreased after local application of calcitriol(p<0.05).NLRP3 antagonist MCC950 and IL-1 β blocking antibody significantly increased the healing rate,epithelial proliferation,corneal nerve density and sensitivity of diabetic corneal epithelium.Calcitriol can promote epithelial and nerve repair by inhibiting the excessive activation of NLRP3 inflammasome in diabetic cornea.In addition,calcitriol upregulated the expression of desmosome and semi-desmosome related genes in diabetic cornea(p< 0.05).Conclusion:The level of 25(OH)D in diabetic mice was significantly decreased,and the local application of calcitriol promoted the healing of corneal epithelial injury,nerve regeneration and sensory recovery in diabetic mice.These improvements may be related to the overactivated NLRP3 signaling and reduced inflammatory response in diabetic mice.