Protective Effect On The Diabetic Corneal Nerve By Inhibiting The Expression Of MiR-181a In Mice Model

Purpose Diabetic keratopathy is one of the blindness factors that seriously threaten the vision of diabetic patients,and there is still no effective treatment.Diabetic corneal neuropathy can cause corneal sensory disturbances and abnormal nutrient metabolism,which is the most important cause of diabetic corneal lesions.In this study,we investigated the protective effect of microRNA on diabetic corneal nerve and its molecular mechanism by inhibiting microRNA 181a(miR-181a),acting on autophagy-related gene Atg5 and anti-apoptotic gene Bcl-2,in order to prevent and treat diabetic corneal neuropathy provides new ideas.Methods In this study,8 weeks old wild-type C57BL/6 male mice were selected,and a diabetic model was established by continuous intraperitoneal injection of streptozotocin(STZ).Healthy mice were used as normal controls.We chose miR-181 a,which has significant differences in the expression of trigeminal ganglion(TG)in normal and diabetic mice.The target genes of miR-181 a were predicted by bioinformatics software Targetscan et al.The target genes of Atg5 and Bcl-2 were screened for miR-181 a and verified on primary cultured mouse trigeminal neurons.Mouse TG cells were cultured in vitro to detect synaptic growth length and cell survival of primary cultured TG cells in vitro under miR-181 a antagomir and negative control.The corneal epithelial damage repair model of diabetic mice was established by epithelial scraping method.The healing of corneal epithelium was observed by subconjunctival injection of miR-181 a antagomir and negative control.After 48 hours,corneal specimens were collected and corneal nerve staining was performed to observe the degree of repair of diabetic corneal nerve.The expression of autophagy-related proteins Atg5,LC3-II and anti-apoptotic protein Bcl-2 were detected by Western blot and immunofluorescence assay.Results Continuous injection of STZ to establish a mouse model of type I diabetes,which has classic diabetes signs such as elevated blood sugar,weight loss,polydipsia and polydipsia.miR-181 a,which was significantly up-regulated in the trigeminal ganglion of type I diabetic mice,was screened by real-time PCR.TG cells were isolated in vitro and added to miR-181 a antagomir and negative control(miRNA antagomir NTC)for primary culture in vitro.Immunofluorescence showed TG cell synaptic length after miR-181 a antagomir treatment 48 hours later.A significant increase.Western blot analysis showed that the expression of autophagy-related proteins Atg5 and LC3-II was down-regulated and the expression of anti-apoptotic protein Bcl-2 was down-regulated in TG cells cultured in high glucose environment compared with normal TG cells.miR-181 a antagomir treatment After the negative control group,the expression of Atg5,LC3-II and Bcl-2 was significantly increased.The corneal epithelium was scraped off and the epithelial injury repair model was constructed.The corneal epithelial healing of diabetic mice was significantly delayed compared with that of normal mice.The diabetic mice were treated with subcutaneous submucosal injection of miR-181 a antagomir and the corneal epithelial healing rate was significantly higher than that of the negative control group.Accelerated,corneal nerve staining showed that it significantly promoted the repair of corneal nerve endings.The results of Western blot and immunofluorescence showed that the expression of autophagy-related proteins Atg5 and LC3-II was weaker and the expression of anti-apoptotic protein Bcl-2 was weaker than that of normal mice.The expression of Atg5,LC3-II and Bcl-2 in TG tissues of diabetic mice after subconjunctival injection of 181 a antagomir was significantly enhanced compared with the NTC control group.Conclusion This study demonstrates that by inhibiting the expression of miR-181 a in corneal epithelium of diabetic mice,Atg5 can be targeted to activate autophagy and Bcl-2 inhibits apoptosis,thereby promoting the regeneration of trigeminal ganglion neurons and corneal nerve fibers in diabetic mice,and protecting the wound healing of diabetic corneal epithelium.