The Neuroprotective Effect Of Interleukin-12 On Diabetic Retinopathy And Its Mechanism

Background and objectives:Diabetic retinopathy(DR)is one of the common complications of diabetes mellitus,and the retinal neuropathy occurs earlier and more significantly than microangiopathopathy.Studies have found that interleukin-12(IL-12)has a repairing effect on ganglion cell injury,but the protective effect of IL-12 on DR remains unclear.In this study,diabetic mouse model and high glucose cultured primary M iller cells were used to explore the neuroprotective effect of Interleukin-12(IL-12)on diabetic retinopathy and the mechanism of JAK2/STAT4 signaling pathway,providing a new theoretical basis for the prevention and treatment of DR.Methods:Animal experiments:C57BL/6J mice were intraperitoneally injected with 1%Streptozocin(STZ)solution at the dose of 50mg/kg for 5 days to establish the diabetic mouse model.The diabetic mice were randomly divided into diabetic group,IL-12 30ng group,IL-12 60ng group and IL-12 90ng group,and normal mice as control group.After 2w,the IL-12 treatment group was given a single intravitreal injection of IL-12 with a dose of lul/eye at a concentration of 30ng/ul,60ng/ul and 90ng/ul,respectively.The basic conditions of mice were measured before modeling,after 1w and after 2w.The thickness and cell quantity of retinal ganglion cell layer(GCL),inner nuclear layer(INL)and outer nuclear layer(ONL)were measured by HE staining.The expression of glial fibrillary acidic protein(GFAP),caspase-3 and B-cell lymphoma-2(Bcl-2)were detected by immunohistochemical staining.The diabetic mice were randomly divided into diabetic group,IL-12 60ng group,and normal mice as control group.Intravitreal injection was performed.The relative mRNA expression of glutamate/aspartate transporter(GLAST),glutamine synthetase(GS)JAK2 and STAT4 were detected by RT-qPCR.The protein expression of GFAP,GLAST,GS,p-JAK2,p-STAT4,Caspase-3 and Bcl-2 were detected by immunohistochemistry staining,immunofluorescence staining and western blot.Cell experiment:The primary mouse retinal Müller cells were cultrued with tissue block adhesion method and purifed in vitro.The cells were identified by GFAP+GS immunofluorescence double staining.The cells were divided into control group,high glucose group(30mmol/L glucose)and IL-12 treatment group(30mmol/L glucose+5?g/mL IL-12).The growth status of the primary Miller cell in each group was observed.The expression of GLAST,GS and GFAP in the primary Müller cells in each group were detected by immunofluorescence staining.Results:1.The diabetic mice showed signs of diabetes such as weight loss,hyperglycemia,increased food intake,water intake and urine volume.Compared with the control group,the thickness and cell quantity of retinal GCL,INL and ONL were decreased and the structure of retinas was disordered in the diabetic group.The expression of GLAST,GS,p-JAK2,p-STAT4 and Bcl-2 were decreased,while the expression of GFAP and Caspase-3 increased in the diabetic group.Compared with the diabetic group,the expression of GLAST,GS,p-JAK2,p-STAT4 and Bcl-2 were increased,while the expression of GFAP and Caspase-3 was decreased in IL-12 60ng group.2.GS+GFAP immunofluorescence double staining identified the primary Müller cells.Compared with the control group,Müller cells in high glucose group grew at a slower rate and had an abnormal cell morphology,and the expression of GLAST and GS decreased,while the expression of GFAP increased.After IL-12 treatment,the growth status of Müller cells was improved,the expression of GLAST and GS increased,and the expression of GFAP decreased.Conclusion:IL-12 can up-regulate the expression of GLAST and GS and down-regulate the expression of GFAP,leading to changes in the expression of Bcl-2/Caspase-3 in the diabetic mice retinas,thus improving the structure of retinas and the growth state of Müller cells.It may be related to the activation of JAK2/STAT4 signaling pathway.