| Purposes:With the increasing incidence of diabetes and the social trend of aging population,diabetes-related cognitive impairment characterized by central insulin resistance has attracted much attention.Studies have shown that central insulin resistance plays an important role in diabetes-related cognitive impairment.The occurrence of central insulin resistance is often accompanied by chronic inflammation,oxidative stress and other processes,which are closely related to the activation of glial cells.Thus,glial cells of the brain,including astrocytes and microglia,may play an important role in the process of diabetes-related cognitive impairment,but the specific mechanism is not clear.The body is a whole,and the cells interact with each other.In the brain,neurons,astrocytes and microglia also have extremely close links,influence and support each other.They jointly maintain the physiological function of the brain and play an important role in the development of disease.However,at this stage,there are few related studies on intercellular connections,and the multi-cell culture model is not perfect.Therefore,this study intends to establish a model of insulin resistance in the tri-culture system of neuron-microglia-astrocytes,and to explore the effect of central insulin resistance on the activation and function of glial cells.To further explore the effect of microglial activation on astrocyte and the effect of astrocyte activation on neuronal morphology and function.As neuronal support cells,mitochondrial damage of astrocytes not only affects energy supply,but also plays an important role in neuronal damage caused by oxidative stress.Therefore,we chose to observe changes in oxidative stress levels and explore the role of central insulin resistance in glials activationmediated oxidative stress alteration in neuronal injury.Methods:(1)A modified "sandwich" co-culture and supernatant transfer co-culture method were combined to establish neuron-microglia-astrocyte tri-culture system and the control group neuron-microglia co-culture system.The model of insulin resistance was constructed by palmitic acid,western blot detection of insulin resistance pathway activation was used to verify the successful establishment of the model;(2)The effects of insulin resistance on neuronal morphology and Nissl body were observed by Nissl staining;neuronal activity was detected by MTT;neuron apoptosis was detected by immunofluorescence and flow cytometry;and changes of synaptic plasticity related proteins were detected by western blot;(3)Transcriptional changes of i NOS(M1)and Arg1(M2)markers of microglial polarization were detected by PCR;i NOS and Arg1 protein level changes of microglial cells were detected by immunofluorescence staining to clarify their polarization phenotypes;microglial cytokine secretion was detected by protein chip to assess changes in microglial function;(4)Transcriptional changes of C3,i NOS(A1)and Arg1(A2)markers of astrocytes were detected by PCR;C3 and i NOS protein levels in astrocytes were detected by western blot and immunofluorescence staining to clarify their polarization phenotypes;changes in transcription levels of astrocyte-related functional genes were assessed by q PCR detection;and expression of NF-k B pathway proteins in astrocytes was detected by western blot to explore the mechanism of astrocyte activation;(5)Griess reagent was used to detect nitrite levels in co-culture system and triculture system to assess NO level changes in the system;immunofluorescence was used to detect changes in i NOS expression levels of neurons;proteins associated with i NOS downstream apoptotic pathway were detected by western blot to assess the effect of changes in i NOS levels on apoptosis;and ROS expression levels in neurons were detected by flow cytometry to assess changes in oxidative stress levels of neurons.Results:(1)There was no significant difference in insulin resistance between neuronmicroglia-astrocyte tri-culture group and neuron-microglia co-culture group under 0.075 m M palmitic acid modeling.However,neuron-microglia-astrocyte tri-culture group decreased cell activity,decreased neurite outgrowth,downregulated expression of synaptic plasticity-related proteins(PSD95,synapsin1 and p CREB/CREB)and increased apoptosis compared to neuron-microglia co-culture group,which indicated that astrocytes played a role in the injury of central insulin resistance neurons;(2)The central insulin resistance can upregulate the M1 activation marker i NOS and downregulate the M2 activation marker Arg of microglia at the transcriptional and protein levels,and increase the secretion of nerve injury factors TNF-a,IL-6,MCP-1,RANTES and GM-CSF;(3)M1 activated microglia can activate NF-k B pathways in astrocytes,cause A1 activation of astrocytes to upregulate transcription and protein levels of i NOS,and downregulate antioxidant-related genes(Gpx8,Atox1),mitochondrial-related functional genes(Ucp2,Cox8 b,Atp5g1),synaptic excitability-related genes(Sparc),and synaptic clearance-related genes(Megf10,Mertk)in astrocytes;(4)The NO level,neuronal i NOS and ROS expression in tri-culture system were significantly increased by insulin resistance compared with co-culture system,and the activation level of i NOS downstream apoptotic pathway was increased.Conclusions:Using a combination of “sandwich” and supernatant transfer co-culture method,we constructed neuron-microglia-astrocyte tri-culture system,and control group neuron-microglia co-culture system,and gave palmitic acid to establish an insulin resistance model.(1)Astrocytes play an important role in the process of central insulin resistance injury(neuronal activity,synaptic plasticity and neuronal apoptosis).(2)Central insulin resistance can induce microglia M1 polarization;M1 microglial cells can further activate astrocytes into A1 phenotype,impair their neuronal protective function and induce neuronal damage function;(3)Glia cells activation mediated by central insulin resistance can cause neuronal damage by enhancing neuronal oxidative stress levels and activating BAD apoptotic pathway. |
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