Mechanism Of Dexmedetomidine Regulating NLRP3-mediated Central Inflammatory Response Through Ubiquitination-autophagy In Postoperative Cognitive Dysfunction

Postoperative cognitive dysfunction(POCD)is a disorder of central nervous system(Central Nervous System;CNS)that appears during the perioperative period.It is more common in elderly and critically ill patients.The clinical manifestations are mainly decreased memory and concentration,which are usually accompanied by emotional ups and downs.Current studies believe that there are many risk factors for POCD,such as age,surgical stress,depth of anesthesia,and length of sleep.Among the many risk factors of POCD,microglia-mediated neuroinflammation is considered to be the main factor that causes cognitive impairment.As the main executor of central inflammation,microglia are the inflammatory immune cells of the CNS.The main role is to remove harmful substances that phagocytize the CNS cell metabolism,and synthesize the nutritional factors needed for neuron development and the immune response to early danger signals.In neurodegenerative diseases,the imbalance of microglia function can lead to neuronal function damage,and then aggravate neuropathy.Therefore,the homeostasis of microglia is maintained in neurodegener-ative diseases and central inflammation-mediated diseases.Plays a key role in disease.According to existing studies,the activation and sensitivity of microglia in the CNS are closely related to neuroinflammation mediated by NLRP3 activation.The stimulation of the first signal of external inflammation activates NF-?B,promotes the transfer to the nucleus,and initiates increased transcription of NLRP3 related genes.Under the activation of the second signal of inflammation,the NLRP3 protein,ASC and pro-caspase1 assemble into NLRP3 inflammasomes.Although there are many studies on NLRP3-mediated inflammation in different diseases,the mechanism of NLRP3-mediated central nervous system inflammation in POCD is still unclear and needs further study.Dexmedetomidine(DEX)is a new type of high-efficiency,highly selective ?2 adrenergic receptor agonist,currently widely used in clinical anesthesia.Studies have shown that the sedative effect of DEX is close to that of natural sleep,so it is welcomed by clinical anesthesia.Therefore,this study intends to use 12-month-old mice to establish a laparotomy exploratory surgery to simulate the aging POCD model,to explore the effect of DEX on the postoperative cognitive function of aging mice and the role of NLRP3 inflammasome-mediated central nervous system inflammation in it.Part One: The role of NLRP3-mediated central inflammation in postoperative cognitive dysfunction and the impact of DEXObjective: Explore the role of different inflammasomes in mediating POCD,and clarify the effect of DEX pre-administration on inflammasomes in hippocampal microglia in mediating central inflammation and on POCD.Methods: First,a surgical model of laparotomy was established in 12-month-old C57 mice,and the protein in the hippocampus of the mouse was extracted.Western blotting(WB)was used to detect the expression of inflammasomes NLRP1,NALP2,NLRP3,NLRC4,and AIM2;immunofluorescence was used to determine Which glial cells mediate the expression of inflammasomes.At the same time,different doses of dexmedetomidine(DEX)were pre-prescribed 3 days in advance,and a laparotomy exploratory model was used to establish a postoperative cognitive impairment model,and the effect of dexmedetomidine(DEX)on the inflammasome was detected at the body level.WB was used to detect the expression of NLRP3/CAS1/IL-1? pathway protein and the expression of synapse-related protein PSD95 in the hippocampus;immunohistochemistry was used to detect the effect of DEX on the activation of microglia and astrocytes in the hippocampal DG area.Golgi silver staining detects the density and length of nerve spine in the hippocampus;finally,Y-maze and new object recognition experiments detect the effect of DEX on the cognitive function of mice..Results: Compared with the sham operation group,the protein content of NLPR1,NLPR2,and NLPR4 in the hippocampus of mice in the anesthesia operation group did not change significantly,and the expression of NLPR3 and AIM2 protein increased.Among them,NLRP3 increased significantly after anesthesia operation;NLRP3/CASP1 was also detected.The expression of /IL-1? pathway,the NLRP3/CASP1/IL-1? pathway in the hippocampus of aged mice was significantly activated after surgery,and the expression of IL-1? and Caspase-1 protein increased significantly.In the experiment of pre-administration of DEX to treat POCD,DEX can reduce the expression of NLRP3 in the hippocampus in a dose-dependent manner and increase the content of PSD95.At the same time,30 ?g/kg DEX can significantly improve the activation of astrocytes and microglia in the hippocampus caused by surgical anesthesia;it can increase the density of neurites.And in the behavioral experiment,the cognitive ability of the mice was significantly improved,which was manifested by the increase of the time to explore the new arm in the Y-maze and the increase of the new object recognition index in the new object recognition experiment.Conclusion: NLRP3 inflammasome is involved in the occurrence and development of POCD in aged mice.NLRP3 inflammasome is mediated by microglial cells.DEX pre-administration can improve the postoperative cognitive function of aged mice in a dose-dependent manner.Part II DEX reduces microglia activation and NLRP3-mediated neuroinflammationObjective: To explore the effect of pre-administration of DEX in vitro on the activation of microglia and NLRP3 inflammasome,and its effect on neurons;Methods: In vitro studies,LPS and ATP stimulated primary microglia to detect the expression of CASP1 and IL-1? in the cell supernatant;use different doses of DEX to detect the cell activity of primary neurons to find the best DEX concentration;WB was used to observe the effect of different doses of DEX on the expression of microglia,apoptotic protein BAX and anti-apoptotic protein BCL-2;flow cytometry and immunofluorescence were used to explore the effect of DEX on primary neuronal cell apoptosis;combined NLRP3 inhibitor MCC950,use WB to detect the effect of NLRP3 inflammasome and inflammatory factor secretion;Results: DEX at a dose of 10?M is the best dose,which has the effect of reducing the expression of apoptotic protein BAX and increasing the expression of anti-apoptotic protein BCL-2;compared with the simple stimulation group,DEX at a dose of 10?M can reduce early neuronal apoptosis 10?M DEX+LPS+ATP group neuron axon length increased;compared with the group not treated with NLRP3 inhibitor MCC950,adding DEX inhibited the expression of NLRP3 and also significantly reduced the expression of IL-1? protein;DEX presented a dose Dependently reduce the activation of CASP1,the secretion of IL-1? and the expression of ASC,thereby inhibiting the assembly of NLRP3,thereby reducing the occurrence of inflammation.Conclusion: 10?M DEX can reduce the activation of microglia and inhibit the inflammatory response mediated by NLRP3;at the same time,inhibiting the neuroinflammation mediated by NLRP3 can reduce the early apoptosis of neuronal cells.Part ? DEX promotes NLRP3 degradation through ubiquitination-autophagy to produce neuroprotectionObjective: To explore whether DEX pre-administration promotes NLRP3 degradation and neuroprotection through the ubiquitination-autophagy pathway;Method: Treat primary microglia with LPS to activate the proteins ASC,po-CASP1,and NLRP3 required for the assembly of NLRP3 inflammasomes;autophagy inhibitors and proteasome inhibitors were used to detect the pathway of NLRP3 degradation;WB detection Changes in the expression of autophagy-related proteins SQSTM1,ATG5,ATG7 and MAP1LC3 B.Transfection of fluorescent RFP-GFP-MAP1LC3 B autophagy dual fluorescent virus to detect changes in autophagic flux;immunofluorescence and COIP techniques were used to explore the effect of ubiquitination on DEX on NLRP3 inflammasomes;Results: Under the stimulation of LPS,DEX could not change the expression of ASC,pro-CASP1,which is required for the assembly of NLRP3 inflammasomes at the transcriptional level.Therefore,it is considered that the protein degradation level affects the degradation and clearance of NLRP3.10?M MG-132 and 5 m M 3-MA antagonized ubiquitination-protein lysosome and autophagy-lysosome,respectively,and found that 3-MA significantly inhibited the expression of NLRP3,suggesting that DEX affected the degradation of NLRP3 from the autophagy pathway.DEX can significantly increase the expression of ATG5 and ATG7 protein,while reducing the expression of SQSTM1 protein,and promote the conversion of MAP1LC3B-I to MAP1LC3B-II;DEX not only promotes the formation of early autophagosomes in microglia,but also promotes the late stage.The formation of autophagosomes;LPS and ATP inhibited the fluorescence signal of ubiquitin protein Ub in primary microglia,while DEX-treated cells can enhance the fluorescence signal of UB and inhibit the fluorescence signal of NLRP3.Conclusion: DEX accelerates the degradation of NLRP3 protein by promoting ubiquitination and autophagy to produce neuroprotective effects.