Effect Of Dexmedetomidine On Postoperative Cognitive Dysfunction In Aged Rats Via Lncrna-loc102546895

Background and objective:The new concept of perioperative neurocognitive impairment(PND)covers all cognitive disorders that have occurred before operation,within 30 days after operation,and within 30 days to one year after operation.Postoperative cognitive dysfunction(POCD)is a reversible and fluctuating cognitive deterioration complica-tion after surgical anesthesia.The clinical manifestations are memory loss,mental state abnormalities,personality anxiety,and emotions.Low,social ability changes,and even serious personality changes.The incidence of elderly patients is signific-antly higher than that of young patients,which is a great challenge for aging China.POCD increases patient hospitalization,medical resources,medical costs,and even mortality.This greatly reduces the quality of life of patients and also burdens patients' families and society.At present,the pathogenesis of POCD is still unclear and the pathogenesis factors are complex.The main research direction is central nervous system inflammation in the hippocampus of the brain.The current treatment of POCD mainly through three types of methods,namely the antioxidant route,the antioxidant pathway and the protection of the neurogenic pathway.Dexmedetomidine(Dex)is a centrally acting presynaptic,highly selective alpha2 adrenergic receptor antagonist used as a sedative and an anesthetic adjuvant in the operating room.Its mechanism of action is that the binding to the ?2 receptor hyperpolarizes the blue-spotted neurons,reduces the release of norepinephrine,and inhibits the release of norepinephrine.The pathway by which Dex treats POCD is primarily to protect neurons and anti-inflammatory effects.Long non-coding RNA(lncRNA)is an important member of non-coding RNA,more than 200 nucleotides in length,with no obvious protein coding function.lncRNA is involved in a variety of cellular processes and diseases,including signaling,embryonic stem cell differentiation,brain function and development,chromatin remodeling,cancer,and neurodegenerative diseases.Studies have shown that lncRNA can affect the regulation of Alzheimer's disease by affecting A? protein;it can regulate learning and memory ability by affecting neuronal synaptic function and dendritic development;it can also regulate the expression of pro-inflammatory factors to improve inflammation of hippocampus in the brain.Happening.POCD patients have spatial learning and memory ability changes.There may be changes in inflammatory factors and tissue morphology in the hippocampus.There may also be specific changes in lncrna in the hippocampus.DEX may treat POCD by affecting lncrna expression in hippocampus.Part 1 The establishment of POCD animal model and the effect of dexmedetomidine on animal modelsObjective:The therapeutic effect of dexmedetomidine on the animal model of POCD.Methods:In this study,the old rat POCD model was established by splenectomy.Water maze test was used to detect the spatial memory and learning ability of rats.HE staining was used to observe the shape of hippocampus.The expression of TNF-? and IL-1? in hippocampus were detected by qRT-PCR.1.Construction of POCD animal model: 18-month-old SD rats were anesthetized by intraperitoneal injection of pentobarbital sodium and then underwent splenectomy.DEX group was intraperito-neally injected with DEX before operation,and other groups were normal saline.2.Water maze experiment: constructed the experimental platform,trained the rats to explore the platform,and detected the relevant indicators after the training.3.H&E staining: hematoxylin and eosin were used to stain the hippocampal sections of rats.4.qRT-PCR extract the total RNA of rat hippocampus and detect the expression of the target gene.Results:1.30 minutes before operation,DEX decreased the escape latency and increased the times of crossing the platform.2.More neurons in hippocampal area of POCD rats have obvious nucleolarstructure and slightly sparse and relatively regular arrangement.In addition,the nuclei of neurons were stained shallowly,but there was no obvious pyknosis.3.Significantly decreased TNF-? and IL-1 ? in hippocampus of POCD rats.Part 2 Transcriptome sequencing and analysis of rat hippocampusObjective:The changes of mRNA and lncRNA expression in hippocampus of rats after dexmedetomidine treatment.Methods:In this experiment,differentially expressed mRNA profiles and lncRNA profiles were analyzed by whole transcriptome sequencing of rat hippocampus.Analysis and prediction of differentially expressed mRNA and lncRNA functions and signaling pathways by GO and KEGG methods.qRT-PCR was used to verify the sequencing results in hippocampus.1.RNA extraction and qRT-PCR detection: Total RNA was extracted from rat hippocampus,and the expression of lncRNAs in hippocampus(LOC103692676,LOC102546895,LOC100911992,Ncl-ps1 and LOC103692397)were detected by qRT-PCR.2.Whole transcriptome sequencing: Total RNA was extracted from hippocam-pus of Dex and POCD rats,rRNA was removed,and cDNA was reversed.After fragmentation,PCR was enriched and sequenced.The raw data of the sequencing were quality-controlled,and the functions of differentially expressed mRNA and lncRNA of Dex and POCD were predicted by GO and KEGG methods.Results:1.The expression of mRNA and lncRNA in hippocampus of Dex and POCD groups were significantly different,and their signaling pathways were mainly enriched in NF-?B signaling pathway and p53 signaling pathway.2.The PCR results indicate the reliability of the differential lncRNA expression in the sequencing results.Part 3 Functional study of lnc-LOC102546895 in microgliaObjective:The cell function of LOC102546895 in microglia,mainly the effects on cell proliferation,apoptosis and expression of NPAS4,a key gene in hippocampus.Methods:In this experiment,we used siRNA knockdown microglia loc102546895;CCK8to detect cell proliferation;flow cytometry to detect apoptosis;WB and qRT-PCR to detect the expression of NPAS4 protein and mRNA.1.Detection of cell proliferation activity: The cells were added to 96 plates according to a certain number.After transfection,the cells were treated with CCK-8at 0h,24 h,48h,72 h,96h,and the OD value was detected by the microplate reader at a wavelength of 450 nm,and the growth curve was drawn.,observe the cell prolifera-tion activity.2.Apoptosis detection: The control cells and the transfected cells were collected,and Annexin V-FITC and propidium iodide staining solution were added,and apoptosis was detected by flow cytometry.3.NPAS4 expression assay: control cells and transfected cells were collected,total protein was extracted,and NPAS4 expression level was detected by Western blot and qRT-PCR.Results:1.After knocking down the expression of LOC102546895,the proliferative capacity of microglia was significantly enhanced.2.After knocking down the expression of LOC102546895,the apoptosis of microglia cells was significantly reduced.3.After knocking down the expression of LOC102546895,the expression of NPSA4 in microglia was significantly reduced.Conclusions:1.Dex can significantly improve the spatial learning and memory ability ofPOCD rats,decrease the number of neuron cells in hippocampus and increase the expression of pro-inflammatory cytokines IL-1? and TNF-?.Dex is closely related to the protection of neuronal cells and inflammatory response.2.The expression of mRNA and lncRNA in hippocampus of Dex and POCD groups were significantly different,and their function were mainly enriched in NF-?B signaling pathway and p53 signaling pathway,indicating that Dex may achieve the purpose of treating POCD through NF-?B signaling pathway and p53 signaling pathway.3.Dex can affect the apoptosis and proliferation of microglia by regulating the expression level of LOC102546895,participate in the process of inflammatory reaction in hippocampus,and alleviate the inflammatory response of hippocampus in POCD.