Effects Of Recurrent Non Severe Hypoglycemia On Glutamate Metabolism Of Astrocytes In Diabetes And Its Mechanism

Background and purposes:Hypoglycemia is a common adverse side effects,in the process of diabetic hypoglycemic recurring of non severe hypoglycemia in the condition of chronic high sugar can lead to cognitive decline,but its mechanism is not clear,studies have shown that mitochondrial function to maintain the astrocyte glutamate metabolism and cognitive function plays an important role,based on this,this research mainly observe mice diabetes condition of severe hypoglycemia recurrently astrocyte glutamate metabolic ability changes,clear mitochondrial function in the role,and to explore by improving mitochondrial function and influence nerve protection mechanism of glutamic acid metabolism,This study provides a theoretical basis for the study on the mechanism of cognitive dysfunction caused by diabetes mellitus combined with recurrented non-severe hypoglycemia.Materials and Methods Firstly,an 8-week-old male C57/BL6 J mouse model of diabetes was established by intraperitoneal injection of streptozotocin,and then insulin glargine was used for hypoglycemia,and regular insulin was injected intraperitoneally for recurrented non-severe hypoglycemia intervention.The mice were divided into four groups.The expressions of GLAST,GLT-1,GS and GFAP in the hippocampus of mice induced by recurrented non-severe hypoglycemia in vivo were studied by Western Blot.Secondly,primary astrocytes of C57/BL6 J mice were extracted and cultured to establish the model of recurrented hypoglycemia under high glucose and divided into four groups.Cell activity was detected by CCK-8 assay.Hoechst staining was used to detect cell apoptosis.Glutamate detection kit tests the ability of cells to uptake glutamate;The expressions of Gl AST,GLT-1,GS and mitochondrial dynamics-related proteins in astrocytes were detected by Western Blot.DCFH-DA probe was used to detect intracellular reactive oxygen species.Mitosox Red fluorescent probe was used to detect cell mitochondrial superoxide.Mitotracker Red CMXROS was used to observe the morphology of mitochondria.Cell oxygen consumption rate(OCR)was measured by Seahorse XF24.Finally,SS31 mitochondrial protectant(100n M/ well)was administered during each repeated hypoglycemia period to further observe the changes of glutamate uptake and release capacity,protein expression of Gl AST,GLT-1,GS,mitochondrial function and other related indexes of astrocytes in each group.Results1.Recurrent non-severe hypoglycemia can reduce the expression of Gl AST,GLT-1 and GS in the hippocampus of diabetic mice,and activate astrocytes.2.Recurrent hypoglycemia under high glucose can reduce the protein expression of Gl AST,GLT-1 and GS in astrocytes,and reduce the ability of glutamate uptake and clearance rate.3.Recurrent hypoglycemia under high glucose can change the mitochondrial function of astrocytes.4.After SS31 intervention,the mitochondrial function of astrocytes was improved,and the expressions of Gl AST,GLT-1 and GS were enhanced,as well as the uptake and clearance rate of glutamate were enhanced.Conclusion Recurrent non-severe hypoglycemia induces excessive activation of astrocytes and mitochondrial dysfunction,leading to decreased glutamate uptake capacity of astrocytes and slower glutamate clearance rate,resulting in imbalance of extracellular glutamate homeostasis,which may be one of the reasons for cognitive decline of Recurrent non-severe hypoglycemia in diabetes.