| Aims:In recent years,the incidence rate of diabetes has increased year by year,and has become an important disease that seriously affects people's health.At the same time,diabetes-related cognitive disorders are attracting increasing attention.Studies have shown that insulin signaling pathway is key to cognitive function.Abnormal brain insulin signaling pathway,or named brain insulin resistance,may be an important part of diabetes-related cognitive impairment.However,the mechanism of nerve injury caused by brain insulin resistance is unclear and needs to be further explored.Endoplasmic reticulum is an important organelle in eukaryotic cells,which is responsible for protein folding,translation,lipid synthesis,calcium storage and transfer to mitochondria.In the face of various pressures,endoplasmic reticulum homeostasis is often broken and endoplasmic reticulum stress occurs.Studies have shown that the neuron injury could be improved via regulating endoplasmic reticulum stress.In addition,insulin resistance is associated with endoplasmic reticulum stress.Some studies suggest that insulin resistance can destroy the homeostasis of endoplasmic reticulum environment and induce the occurrence of endoplasmic reticulum stress,which further aggravates insulin resistance.Thereinto,IRE1?/ XBP1 is the key pathway of endoplasmic reticulum stress and is known as the "central regulator" of endoplasmic reticulum stress.However,there are few studies on the changes of this pathway in neurons and its impact on neuronal function under brain insulin resistance.Therefore,this study intends to construct the co-culture model of neurons and astrocytes to maintain the microenvironment of neurons,and establish the brain insulin resistance model by the palmitic acid stimulation,in order to observe the effect of brain insulin resistance on the morphology and function of neurons,and explore the role of IRE1?/XBP1 pathway in neuronal injury induced by brain insulin resistance;Then we intervened with hypoglycemic drugs,which are used to sensitized insulin signal,to observe the improvement of hypoglycemic drugs on endoplasmic reticulum stress and neuronal injury.Methods:(1)Establishment of insulin resistance model: The co-culture system of neurons and astrocytes was established;The model of insulin resistance was established by 0.1 mm palmitic acid,and the insulin signal pathway-related proteins were detected by Western blot,to evaluate the success of the insulin resistance model.(2)Detection of neuronal morphology and synaptic function: After the establishment of brain insulin resistance,we observed the morphology of neurons by immunofluorescence staining;Neuronal apoptosis was detected by flow cytometry and TNNEL method;The changes of synaptic plasticity associated proteins in neurons were evaluated by Western blot.(3)Endoplasmic reticulum stress pathway and morphological detection: The expression of endoplasmic reticulum stress protein and endoplasmic reticulum morphology-related protein were detected by Western blot;The spliced variant of XBP1 was analyzed by PCR;The nuclear translocation of XBP1 s and the morphology of endoplasmic reticulum were observed by immunofluorescence staining.(4)The intervention of hypoglycemic drugs: The GLP-1 receptor agonist,exenatide and TZD drug,pioglitazone was used.The insulin signal related proteins were detected by Western blot,in order to evaluate the improvement of neuronal insulin resistance;The morphological alterations of neurons were evaluated by immunofluorescence staining;Neuronal apoptosis was detected by flow cytometry and TNNEL method;The expression of synaptic plasticity related proteins and endoplasmic reticulum stress-related proteins were detected by Western blot.(5)The effects of IRE1?/XBP1 pathway on endoplasmic reticulum stress and neuronal synaptic plasticity,via XBP1 was knocked down or overexpressed in neurons.The expressions of endoplasmic reticulum stress-related protein and synaptic plasticity related protein were detected by Western blot;The morphology of neurons was detected by immunofluorescence staining.(6)The effect of FoxO1 on neuronal synaptic plasticity and the regulatory effect of XBP1 on FoxO1: after the establishment of brain insulin resistance model,the expression of FoxO1 protein in neurons was detected by Western blot;MG132was used to inhibit the ubiquitin proteasome degradation pathway,and the expression of FoxO1 protein in neurons was detected by Western blot;The ubiquitination modification of FoxO1 in neurons was detected by immune-coprecipitation.When FoxO1 was overexpressed in neurons,the expression of synaptic plasticity related proteins was detected by Western blot;Immunofluorescence staining was used to detect the morphology of neurons;After knockdown or overexpression of XBP1 in neurons,the expression of FoxO1 protein in neurons was detected by Western blot;The expression of E3 ubiquitin ligase STUB1 in neurons was detected by Western blot;STUB1 was knocked down in neurons,and the expression of FoxO1 protein was detected by Western blot;Finally,the transcriptional targets of XBP1 s were analyzed by chromatin immunoprecipitation.Results:(1)After administration of 0.1 m M palmitic acid for 24 hours,neurons developed the insulin resistance,presenting that the expression of p IRS1 and p Akt decreased significantly.(2)Under brain insulin resistance,neuronal morphology changed significantly.The length of neuronal axons decreased significantly;The percentage of neuronal apoptosis increased;The expression of synaptic plasticity-related proteins,decreased significantly including PSD95,Synapsin 1,Synaptopodin and p CREB.This suggested the synaptic function of neurons was damaged.(3)Brain insulin resistance could induce the occurrence of endoplasmic reticulum stress,the expression of related markers increased significantly,IRE1?/XBP1 pathway was significantly activated,and the nuclear translocation of XBP1 increased significantly;endoplasmic reticulum membrane of neurons exhibited swelling and vacuolation alterations.(4)By sensitizing the insulin signal,hypoglycemic drugs can significantly inhibit ER stress,then improve the morphology,apoptosis and synaptic function of neurons;At the same time,ER stress was significantly inhibited,mainly manifested in the decreased expression of IRE1?/XBP1 pathway and the decreased nuclear translocation of XBP1.(5)Regulation IRE1?/XBP1 pathway can significantly improve endoplasmic reticulum stress and neuronal synaptic plasticity.Under brain insulin resistance,inhibition of XBP1 expression can significantly improve endoplasmic reticulum stress and neuronal synaptic plasticity;When XBP1 is overexpressed,endoplasmic reticulum stress and neuronal synaptic plasticity will be further aggravated.(6)Under brain insulin resistance,the expression of FoxO1 in neurons decreased significantly,and FoxO1 was degraded by ubiquitin proteasome pathway.After overexpression of FoxO1,the synaptic function of neurons was partially improved,suggesting that FoxO1 plays a key role in neuronal synaptic plasticity.Inhibition of XBP1 expression can increase the expression of FoxO1 protein in neurons,suggesting that XBP1 can regulate FoxO1 expression.As a transcription factor,XBP1 could combined with STUB1 promoter to promote the gene transcription of E3 ubiquitin ligase STUB1,which induced the degradation of FoxO1 by ubiquitination.the expression level of FoxO1 protein in neurons can be significantly improved via regulation of E3 ubiquitin ligase STUB1.Conclusions:We established the co-culture system of neurons and astrocytes,and gave palmitic acid to construct the central insulin resistance model.The following conclusions were obtained:(1)Brain insulin affects the morphology and synaptic function of neurons and causes neuronal damage.(2)Brain insulin resistance can damage the homeostasis of endoplasmic reticulum environment and cause endoplasmic reticulum stress.(3)GLP-1 receptor agonists and TZD hypoglycemic drugs can significantly improve the endoplasmic reticulum stress,morphological and functional damage of neurons induced by brain insulin resistance.(4)Regulation IRE1?/XBP1 pathway can improve the endoplasmic reticulum stress and neuronal injury induced by brain insulin resistance.Therefore,IRE1?/XBP1 pathway may be a new target for the treatment of neuronal injury which is caused by brain insulin resistance.(5)FoxO1 in neurons plays a very important role in maintaining neuronal synaptic function.As a transcription factor,XBP1 s improves the gene transcription of E3 ubiquitin ligase STUB1,which activates the ubiquitin proteasome pathway and leads to FoxO1 protein degradation.Therefore,brain insulin resistance could induce neuronal injury,through IRE1?/XBP1/FoxO1 pathway. |
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