The Effect Of GSK-3 Beta Inhibitor On The Expression Of Autophagy Related Protein In Diabetic Retinopathy

Objective:Diabetic retinopathy?DR?is a common complication of diabetes,a kind of main microvascular disease,and it is also one of the main reason of blindness,itis of great significance to study the pathogenesis of the prevention and treatment of diabetic retinopathy.The aim of this research is to studyThe effect of GSK-3 beta inhibitor on the expression of autophagy related protein in diabetic retinopathy.In this study,the expression of GSK-3 beta inhibitor on the expression of autophagy related proteins in diabetic retinopathy was detected and its regulatory mechanism was explored to provide a theoretical basis for the treatment of diabetic retinopathy.Methods:1.The animal experiment part1.1.Establishment of model 1 diabetic rat modelAfter 1 weeks of adaptive feeding,Eighty male healthy SD rats with weight180-200g were randomly divided into normal control group?Control group,20 rats?and model group?DM group,60 only?according to the principle of weight balance.The model group was given intraperitoneal injection of 1%streptozotocin?STZ,60 mg/kg,pH=4.4 citric acid citric acid three sodium buffer solution?for injection,and fasting 16h before injection.Rats in the control group were injected with the same volume citric acid citric acid three sodium buffer.After 72 h,the caudal vein was collected,and the blood glucose test paper was used to detect the fasting blood glucose?fasting blood glucose,FBG?,and FBG more than 11.1mmol/L,and the model of type 1 diabetic rats was considered to be successful.1.2.Changes of retinopathy and autophagy related protein expression in diabetic ratsThe rats were randomly divided into 4 wk model group,8 wk model group,4 wk methisoindigo group and 8 wk methisoindigo group,with 15 rats in each group.The control group was divided into 4 wk control group and 8 wk control group,10 rats in each group.The group continued to feed with regular chow,and gavage of Methisoindigo to the drug group every day.After the group was finished,they were fed with normal diet,and the drug group was administrated with methisoindigo daily.The rats were administered 1 mg per 100g.The rats were anesthetized on weeks 4 and8,respectively.the abdominal aorta was taken from the aorta,and the serum was centrifuged.The vascular endothelial growth factor?VEGF?and the hypoxia inducible factor-1 alpha?HIF-1-alpha?of rats were detected by ELISA.Take out the eyeball,clean up the muscle tissue around the eyeball,and separate the retina.The expression of GSK-3 beta,VEGF,LC3A/B and ATG5 in the retina of each group was detected by immunohistochemical?IHC?.The changes of GSK-3?,LC3A/B,ATG5were detected using WB method.2.The cell experiment part2.1.Cell culture and treatmentThe rat retinal vascular endothelial cells are provided by the pharmacology room of the Pharmacy College of Shandong University.The cells were culturedin Dulbecco's modified Eagle's medium?DMEM?supplemented with 10%?v/v?fetalbovine serum and maintained in a humidified incubator of 95%air/5%CO₂ at37?.The myocardial cells were randomly divided into following groups:?1?Control group?normal glucose concentration,5.5 mM?;?2?DMSO group?0.1%?;?3?High glucose group?HG,33 mM?;?4?HG+Methisoindigo group;Methisoindigo was freshly dissolved in DMSO as a stock solution and diluted with DMEM when used.Cells were incubated for a period of 48 hours and processed further for downstream assays and experiments.2.2.Research processMTT assay was used to determine the in vitro effect of Methisoindigo on RMEC cells viability under high glucose.To examine possible relationship between GSK-3?,LC3A/B,ATG5were investigated by Western blot analysis.Results:1.The general characteristics of DM rats and the pathological changesCompared with the control group,the rats in group DM gradually developed mental malaise,and the symptoms of stench and stench aggravated the symptoms of diabetes,such as polydipsia,polydipsia,polyuria and weight loss.60 rats were successfully divided into DM group and methisoindigo group.The FBG levels in group DM and methisoindigo group increased significantly.In the 4wk DM group,the retina of the retina occurred,and the retinal neovascularization and visual acuity decreased.With the progression of diabetes,the retinal vessels in the 8 wk DM rats were obviously increased,the cornea was blurred,and the diopter blurred appeared abnormal.Lighten up.In DM group,the levels of VEGF and HIF-1?in serum increased significantly.Compared with the rats in model group,the levels of VEGF and HIF-1?in the treatment group decreased.2.The changes of GSK-3 beta protein and autophagy related protein in the rat retinaWB and IHC results showed that at 4 weeks after STZ injection,the expression of GSK-3?was significantly increased,and compared with the control group,the expression of GSK-3?was still markedly increased in 8wk DM group.Compared with Control group,the expressions of LC3A/B?ATG5 in 4 weeks and 8weeks were significantly increased,but compared with DM group,the expressions of LC3A/B?ATG5 were significantly reduced.3.The changes of GSK-3 beta protein and autophagy related protein in RMEC cellsMTT results showed that the proliferation rate of RMEC cells treated with DMSO showed no obvious change compared with control group?P>0.05?.When the cells were exposed to high glucose at a dose of 33 mM for 48 h,the proliferation rate of H9C2 was increased significantly?P<0.05?.Compared with HG group,the proliferation rate of RMEC cells treated with Methisoindigo showed no obvious change?P>0.05?.WB results showed that the expressions of GSK-3?and the autophagy related protein in HG group were significantly increased compared with control group.Compared with HG group,Methisoindigo treatment significantly lowered the expression of GSK-3?and autophagy related protein.Conclusion:1.The rats were induced by STZ,the newborn blood vessels in the retina of rats increased significantly,and the visual acuity of rats decreased.2.In the retina of 4wkDM and 8wkDM rats,the expression of GSK-3?was elevated,and the content of VEGF,ATG5 and LC3A/B increased significantly,and the level of autophagy increased.In the 4wk and the 8wk administration group,the expression of VEGF,ATG5 and LC3A/B decreased and the level of autophagy decreased while the expression of GSK-3?was reduced.3.In high glucose cultured RMEC cells,Inhibition ofthe expression of GSK-3?can reduce the expression of autophagy related proteins and decrease the level of autophagy.It indicates that inhibition of GSK-3?can regulate the change of autophagy and participate in the occurrence and development of diabetic retinopathy.It can be further studied as a target for the treatment of diabetic retinopathy.