The Study Of The Role In Glutamate-glutamine Cycle Between Neuron-astrocyte In Hypoglycemia-related Cognitive Dysfunction

Objective:Hypoglycemia is one of the most common complications during the treatment of diabetes,which seriously threatens the health and quality of life of patients with diabetes.Literature at home and abroad suggests that hypoglycemia events are closely related to the occurrence of cognitive dysfunction in patients with diabetes.Excitatory amino acid toxicity is one of the pathological mechanisms of hypoglycemia brain damage and then leads to cognitive dysfunction.The glutamate-glutamine cycle between the coupling of neuron-astrocyte maintains the balance of glutamate in the brain and plays an important role in protecting neuronal functions.Therefore,this study observed the effects of hypoglycemia on the glutamate-glutamine cycle in the neuronal-astrocyte coupling,and further explored the mechanisms of hypoglycemic neuron damage,as well as excitatory amino acid toxicity affecting learning and memory related signaling pathway NMDA receptor-ERK-CREB in hypoglycemia-related cognitive dysfunction.Aim to provide theoretical basis for the mechanisms of cognitive dysfunction caused by hypoglycemia.Methods:1)Establish a co-culture system of mouse hippocampal neurons and astrocytes in vitro to simulate the microenvironment of brain cells,give a glucose-free medium to build a model of hypoglycemia of nerve cells,and use MTT method to detect viability of neurons after glucose-free treatment for 2h,4h and 6h,looking for the best time for modeling hypoglycemia;2)Use HE staining to observe the effect of hypoglycemia on neuronal morphology;Use Hoechst / PI fluorescence staining to detect the effect of hypoglycemia on neuronal apoptosis;3)Detect the effects of hypoglycemia on the concentration of excitatory amino acid glutamate in neurons,astrocytes,and culture medium through the kit;Test the effects of hypoglycemia on the activity of glutamine synthetase in astrocytes and the activity of glutaminase in neurons through the kit;Western blot was used to detect the effects of hypoglycemia on the changes of glutamate transporter EAAT2 in astrocytes;So as to clarify the effect of hypoglycemia on the glutamate-glutamine cycle;4)Western blot was used to detect the effects of hypoglycemia on the expression of learning and memory related signaling pathway proteins NMDA,p ERK / ERK,p CREB / CREB,and its downstream synaptic plasticity proteins BDNF,synapsin1 and Arc on neurons.Results:1)MTT results showed that the glucose-free medium treatment of neuron-astrocyte co-culture system for 6h is the best modeling time for hypoglycemia model.2)HE staining results showed that neuronal morphology changed significantly after treatment with hypoglycemia.Hoechst / PI fluorescence staining results suggested that hypoglycemia can significantly increase neuronal apoptosis.3)Hypoglycemia can significantly increase the content of excitatory amino acid glutamate in neurons,astrocytes and culture medium;The activity of glutaminase in neurons is enhanced;The activity of glutamine synthetase in astrocytes is enhanced,and EAAT2 protein expression in astrocytes is enhanced.4)Hypoglycemia caused a decrease in the expression of NMDA receptor-ERK-CREB signaling pathway-related proteins NMDA,p ERK / ERK,p CREB / CREB and downstream synaptic plasticity proteins BDNF?synapsin1 and Arc.Conclusions:The neuron-astroglial co-culture system was replaced with glucose-free medium for 6 hours to establish a hypoglycemia model,and the following conclusions were drawn.1)Hypoglycemia can affect neuronal morphology and synaptic plasticity,and accelerate neuronal apoptosis.2)Hypoglycemia can cause neuronal-astroglial glutamate-glutamine cycle disorders.On the one hand,astrocytes enhance glutamate uptake during hypoglycemia and reduce the excitotoxic damage suffered by neurons,but this effect of astrocytes is limited and cannot remove glutamate-mediated excitatory amino acid toxicity of neurons;on the other hand,excitatory amino acid toxicity participates in hypoglycemic neuronal damage and affects learning and memory-related signaling pathways and the expressions of plasticity proteins,resulting in decreased learning and memory ability and cognitive dysfunction.