The Effects Of Kappa Opioid Receptor Agonist On Pyroptosis And Synaptic Plasticity Of Microglia In APP/PS1 Mouse Model And Its Regulatory Mechanism

Objective:Alzheimer’s disease is a degenerative disease of the central nervous system.It is characterized by progressive memory loss and cognitive dysfunction,accompanied by various mental symptoms and behavioral abnormalities,which seriously affect the daily life and work of patients.At present,it is believed that the inflammatory response induced by Aβactivation may be the most important pathological mechanism of Alzheimer’s disease.In addition,as the core element of inflammatory response,inflammasome plays a crucial role in the occurrence and development of nervous system diseases.Evidence suggests that Aβand other abnormally aggregated proteins can activate the inflammasome,and then promote the maturation and secretion of key pro-inflammatory factors,such as IL-1β,to participate in the intrinsic immune inflammatory response and cause cell pyroptosis,Activated NLRP3 inflammasome is involved in neurotoxicity caused by cell pyroptosis,which aggravates neurodegenerative diseases and causes progressive cognitive impairment.As the final manifestation of AD,synaptic loss of neurons is mainly related to abnormal signal transmission of excitatory glutamate of cytoskeleton.NMDAR is an important signal mediator and plays an important role in synaptic transmission and plasticity.Overexpression of NMDAR results in the opening of Ca²⁺channels binding to glutamate receptors,which increases intracellular Ca²⁺concentration and activates Ca²⁺/CaMKⅡ.Phosphorylated CaMKⅡ can directly activate CREB,further promote the synthesis of ARC and synaptic protein,participate in the formation of LTP and maintain the formation of long-term memory.Which suggests Ca²⁺/CaMKⅡ/CREB pathway is closely related to synaptic plasticity.Kappa opioid receptor agonists are widely used in perioperative analgesia.They have strong analgesic effect,and can also regulate emotional and cognitive functions.Some studies have found that U50488H can significantly reduce cognitive impairment.Objective is to observe the effects of U50488H,a kappa opioid receptor agonist,on spatial memory ability,synaptic plasticity and inflammasome in Alzheimer’s disease mice,and to explore the relationship between microglia pyroptosis and Ca²⁺/CaMKⅡ/CREB pathway in synaptic plasticity.Methods:1.To study the effects of kappa opioid receptor agonist U50488H on cognitive function and synaptic plasticity of APP/PS1 mice:C57BL/6 mice and APP/PS1 mice were divided into different groups according to body weight and age.Experimental groups:Control group（C57BL/6 mice）,Model group（APP/PS1 mice）and kappa opioid receptor agonist group（APP/PS1 mice+kappa opioid receptor agonist）.Morris water maze was used to evaluate the cognitive function of mice;immunohistochemistry was used to detect the deposition of Aβ;HE staining was used to detect the pathological changes of brain tissue;Golgi apparatus staining was used to detect the density of dendritic spines;Western blot was used to detect the levels of NCAM、NR2B and Glu R1.2.To study the inhibitory effect of NLRP3 inflammasome mediated byκopioid receptor agonist on microglia pyroptosis in APP/PS1 mice:The serum levels of IL-1βand IL-18 were detected by enzyme-linked immunosorbent assay;the co-expression of IBA-1 and NLRP3,IBA-1 and ASC was detected by immunofluorescence assay;the expression of pro-caspase-1,pro-IL-1β,ASC and NLRP3 were detected by Western blot.3.The first part:the expression of NMDAR,CaMKⅡ,p-CaMKⅡ,CREB and p-CREB were detected by Western blot withκopioid receptor agonist.The second part:κopioid receptor agonist regulates NLRP3 through Ca²⁺/CaMKⅡ/CREB pathway,inhibits microglia pyroptosis and improves synaptic plasticity in APP/PS1 mice:Experimental groups:U50488H group（APP/PS1 mice+kappa opioid receptor agonist）and KN93 group（APP/PS1 mice+kappa opioid receptor agonist+specific CaMKⅡ antagonist）.Morris water maze test was used to detect the cognitive ability of different groups of mice;Golgi apparatus staining was used to detect the density of dendritic spines;Western blot was used to detect the expression of NR2B,Glu R1 and NCAM in brain tissue;ELISA was used to detect the levels of IL-1βand IL-18 in serum;Immunofluorescence was used to detect the co-expression of IBA-1 and NLRP3,IBA-1 and ASC.Western blot was used to detect the expression of pro-Caspase-1,pro-IL-1β,ASC and NLRP3.Conclusion:1.Kappa opioid receptor agonist can alleviate the brain damage of APP/PS1 mice,and inhibit the further development of Alzheimer’s disease by enhancing the expression of synaptic plasticity related proteins such as NCAM,NR2B and Glu R1.2.Kappa opioid receptor agonist can inhibit IL-1βand IL-18 and inhibit the coexpression of IBA-1 and NLRP3,IBA-1 and ASC,reduce the expression of NLRP3 inflammasome and its downstream related molecules,and alleviate the occurrence of apoptosis of microglia.3.Kappa opioid receptor agonist can inhibit the expression of NLRP3 inflammasome,inhibit microglia pyroptosis and improve the synaptic plasticity of APP/PS1 mice,which are related to the regulation of Ca²⁺/CaMKⅡ/CREB signaling pathway.