| Objective: The cognitive impairment of brain caused by diabetes has been confirmed by many studies,and the pathological process involved is complicated.It mainly includes microangiopathy,increased inflammatory response,abnormal insulin signal pathway and glucose oxidative phosphorylation and increased oxidative stress.Among them,the increase of protein oxidative damage caused by oxidative stress may lead to the dysfunction of neuronal nutrition and substance transport,which is one of the mechanisms of neurological damage caused by diabetes.Proteasome metabolic pathway is an important pathway of oxidative damage protein degradation.It has been found that PA28 is a functional regulator of proteasome,which can bind to20 s proteasome and regulate(enhance)the activity of proteasome hydrolase activity.This study focused on the changes of PA28 protein in the cerebral cortex and hippocampus of diabetic rats,and its subsequent effects on proteasome composition,function and oxidative damage protein metabolism,aiming to explore whether PA28 protein plays a regulatory role in the increase of oxidative damage caused by diabetes.Methods: The diabetic rat models were induced by high fat and high sugar diet combined with low dose streptozotocin(Streptozotocin,STZ).The blood glucose,triglyceride,cholesterol,food intake,water intake,urine volume and body weight of the models were determined successfully;and the changes of PA28α(Psme1),PA28β(Psme2),proteasome composition subunits and protein oxidative damage in cerebral cortex and hippocampus of normal and diabetic rats were detected by Western blot,and the activity of proteasome was detected by specific kit;The AAV vectors inhibiting PA28α and PA28β were constructed respectively,and the effects on proteasome function and protein oxidative damage related indexes were detected after in vivo intervention of PA28 expression.Results:1.Establishment of diabetic rat models and detection of related indexesThe diabetic rat models were established by feeding with high-fat and high-sugar diet combined with low-dose STZ,and the related indexes of diabetes were detected.It was found that the diabetic rats showed polydipsia,polyphagia,polyuria,weight gain and then decrease,and the levels of blood glucose,triglyceride and cholesterol were higher than those of the control group,which successfully simulated the typical characteristics of diabetes.In the follow-up experiment,fasting blood glucose higher than 18mmol/L was selected as the inclusion criteria of diabetic animals.2.Detection of related indexes of oxidative damage in the brain of diabetic ratsDetection of lipid peroxidation and protein oxidation damage in rat brain: The content of MDA in DM group was significantly higher than that in Ctrl group,the total SOD activity in DM group was significantly lower than that in Ctrl group,and the content of DNP in cerebral cortex and hippocampus in DM group was significantly higher than that in Ctrl group.The content of 3-NT in cortex of DM group was significantly higher than that of Ctrl group,and the content of 3-NT in hippocampus of DM group was also higher than that of Ctrl group,but there was no statistical difference(P > 0.05).3.Detection of proteasome composition and activity in the brain of diabetic rats(1)The contents of catalytic subunit β1,β2 and β5 of 20 s core proteasome in rat brain: in the cortex and hippocampus,the contents of β1,β2 and β5 subunits in DM group were no statistical difference from those in Ctrl group(P>0.05).(2)20s proteasome activity in the brain: the 20 s proteasome activity in the cortex was significantly lower than that in the Ctrl group,and the 20 s proteasome activity in the hippocampus was also lower than that in the Ctrl group,but there was no statistical difference(P > 0.05).(3)In cerebral cortex and hippocampus,the content of Ubiquitin in DM group was significantly higher than that in Ctrl group(P< 0.05).4.Changes of PA28 regulatory proteins in the brain of diabetic rats.The contents of PA28α and PA28β in the cortex and hippocampus of rats: The contents of PA28α and PA28β in the DM group were significantly lower than those in the Ctrl group.5.Study on the effect of PA28 protein on oxidative damage protein in the brain of diabetic rats.5.1 PA28 inhibition experiment and effect verification:After in vivo intervention,the contents of PA28α and PA28β in the cortex and hippocampus of rats in the DM group and Ctrl group were significantly lower than those in the unintervened rats,and the proteasome activity in the cortex and hippocampus in the DM group and Ctrl group interfered with PA28 inhibition were significantly lower than those in the unintervened rats(P < 0.05).5.2 Effects of in vivo intervention of PA28 expression on the regulation of protein metabolism in brain oxidative damage:(1)The detection of proteasome function after in vivo intervention of PA28expression: Except that the Ubiquitin content of Ctrl group intervened by PA28αinhibition was higher than that of unintervened Ctrl group,but there was no statistical significance(P > 0.05),and the contents of Ubiquitin in cortex and hippocampus of DM group and Ctrl group after PA28 intervention in vivo were significantly higher than those of unintervened rats(P < 0.05).(2)Detection of protein oxidative damage related indexes after in vivo intervention to inhibit PA28 expression: After in vivo intervention to inhibit PA28 expression,the contents of DNP and 3-NT in cortex and hippocampus of rats in DM group and Ctrl group were significantly higher than those in unintervened rats(P <0.05),and protein oxidative damage increased.6.A preliminary study on the expression of PA28 protein in the brain of diabetic rats at different stagesThe expression of PA28α and PA28β proteins in the brain of short-term diabetic rats showed an increasing trend,but there were no statistical significance(P>0.05).However,the protein expression of PA28α and PA28β in the brain of long-term diabetic rats showed a downward trend,showing a bi-directional trend of increase at first and then decrease,which were statistically significant(P < 0.05).Conclusions:The diabetic rat models were successfully induced by high-fat and high-sugar diet combined with low-dose STZ.The model showed typical clinical symptoms of diabetes,such as increased blood glucose and blood lipid,polydipsia,polyphagia,polyuria,weight loss and so on.Oxidative modifications such as lipid peroxidation and protein oxidative damage increased in the brain of diabetic rats,and there was no significant change in the composition of β1,β2 and β5 subunits in cognitive related brain areas(cortex and hippocampus)of diabetic rats.But proteasome degradation activity decreased.The content of PA28,a proteasomal regulatory protein,in the diabetic brain was significantly decreased,and the proteasome function was inhibited and oxidative damage protein increased significantly after PA28 intervention in vivo.The duration of diabetes may affect the expression of PA28.In summary,Our results suggest that diabetes-related factors can reduce the expression of proteasome regulatory protein PA28,which affect the proteasome degradation function,and then lead to the weakening of protein oxidative damage degradation pathway and the increase of oxidative damage protein. |
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