Mechanism Of DLK/JNK/c-JUN Signaling Pathway In Chronic Glaucoma-Induced Optic Nerve Damage

Purpose: Glaucoma is an extremely complex multifactorial disease that is one of the most irreversible causes of blindness worldwide due to its characteristic visual field defects and optic nerve(ON)atrophy.Glaucoma is a chronic disease that requires longterm treatment.In most cases,the disease is asymptomatic in its early stages.Early diagnosis through eye exams and additional treatment can slow the progression of the disease.Glaucoma is a serious disease of the visual organ that manifests primarily as optic neuropathy,in which progressive apoptosis of retinal ganglion cells(RGCs)is the primary cause of visual loss.However,the pathological and molecular mechanisms of RGC apoptosis are not well understood,and multiple factors may be involved in the optic nerve damage leading to RGC apoptosis.Current treatments provide good control of intraocular pressure,but cannot reverse the visual damage caused by glaucoma.The molecular mechanisms of optic nerve damage caused by chronic glaucoma remain to be investigated.In this study,the DLK/JNK/c-JUN pathway was used as a starting point to find an effective therapeutic target.Methods: Healthy male SD rats were randomly divided into a control group,a high IOP model group and a high IOP model group using sunitinib(DLK inhibitor).A rat chronic glaucoma model was established by ligating the suprascleral vein,and the DLK inhibitor sunitinib was injected intraperitoneally from 1 day before plastination until the end of the performance,and the retinal tissues were harvested1 day,1 week,2weeks and 4 weeks after plastination injury.The number of surviving p-c-JUN(+)RGCs in the whole retina was determined by Western blot for DLK,JNK,p-JNK,c-JUN,p-cJUN,MKK4,p-MKK4,MKK7 and p-MKK7,and by immunofluorescent staining for p-c-JUN markers.Thus,the role of the DLK/JNK/c-JUN pathway for RGC damage in chronic glaucoma was confirmed.We performed statistical analysis of all data using SPSSl3.0 software and reported the results as mean ± standard deviation(x±s).One-way ANOVA and Tukey’s post hoc test allowed comparison of multiple datasets,while independent samples t-test allowed a more accurate assessment of the similarity between the two datasets.Results: 1.One day after surgery,the experimental eyes of all groups of rats showed weak ocular reactions that disappeared after three days.No abnormal reactions were observed in the eyes of the control group.2.The IOP of rats in each group was not statistically significant before modeling.After 1d,1w,2w and 4w after modeling,the IOP in the model group and model+sunitinib group was higher than that in the blank control group,and the difference was statistically significant and could be maintained for 4 weeks.3.Western blot showed that the expression levels of DLK,p-JNK/JNK,p-cJUN/c-JUN,p-MKK4/MKK4 and p-MKK7/MKK7 proteins were significantly higher in the simulated group compared with the control group after simulation,with statistical differences(P<0.01).At 1day,1,2 and 4 weeks after simulation,DLK and p-JNK/JNK protein expression levels were significantly lower in the model+unitinib group compared with the model group,with statistical differences(P<0.01);The expression of p-c-JUN/cJUN,p-MKK4/MKK4,and p-MKK7/MKK7 proteins was lower,with statistical differences(P<0.05).The expression levels of certain proteins in the model + sunitinib group were statistically lower than in the control group(P<0.01).Trend in expression of individual proteins: model > model + sunitinib > control group.4.Immunofluorescence staining showed that the number of p-c-JUN(+)RGCs was significantly increased in the model group compared with the control group.After administration of the DLK inhibitor sunitinib for1 day,1,2 and 4 weeks,the number of p-c-JUN(+)RGCs decreased significantly compared to the model group and the difference between the two groups was statistically significant,P<0.05.Conclusion:1.In this study,we established a model of moderately high intraocular pressure by branding three suprascleral veins and were able to maintain a stable pressure for four hours,which proved to be relatively successful.Using this model,we simulated the progression of retinal disease in patients with chronic glaucoma in a clinical setting.2.We investigated the pathological and molecular biological mechanisms of the DLK/JNK/c-JUN signaling pathway in chronic glaucoma leading to optic nerve atrophy.The results showed that this pathway is involved in optic nerve injury and that the use of DLK pathway inhibitors reduced the apoptosis of some RGCs,which may have a protective effect on RGCs.3.The results suggest that this signaling pathway is involved in optic nerve injury.