The Role And Mechanism Of ?7nAChR Agonist PNU-282987 In Cognitive Dysfunction Induced By Chronic Intermittent Hypoxia In Mice

Objective:Obstructive sleep apnea(OSA)is a sleep disorder characterized by recurrent upper airway obstruction during sleep,resulting in repeated decreases in oxygen saturation,sleep fragmentation,and increased sympathetic autonomic activity.OSA is associated with systemic multi-system diseases,including cognitive impairment,hypertension,diabetes,fatal and non-fatal coronary events,arrhythmias(such as atrial fibrillation and unsustainable ventricular tachycardia),congestive heart failure,stroke,and death.The effects of OSA on cognitive function include attention and alertness,memory and learning,psychomotor function,emotional regulation,and executive function.Multiple prospective studies have shown that patients with OSA at baseline are more likely to develop cognitive impairment or even dementia at follow-up.The cognitive impairment caused by OSA needs our attention.Possible mechanisms for neurocognitive impairment include intermittent hypoxemia,sleep deprivation and fragmentation,hypercapnia,which disrupt brain cells and chemical homeostasis.Intermittent hypoxemia is the main manifestation of OSA and one of the most important physiological mechanisms.Intermittent hypoxemia may cause damage to the body through the ischemia/reperfusion mechanism and oxidative stress response.Previous studies have shown that IH is associated with programmed cell death or apoptosis.A number of animal studies have found that chronic intermittent hypoxia(CIH)can cause cognitive dysfunction in rats and mice,but the mechanism is still unclear.The central cholinergic system is an important regulatory system for cognitive function,and it participates in the formation of learning and memory.Studies have found that IH can cause a decrease in spatial learning and memory ability in rats,which is related to the decrease in the expression of choline acetyltransferase in the basal forebrain.In neurodegenerative diseases such as Alzheimer's disease,it is found that the decrease of cholinergic neurons is related to cognitive impairment.The regulation of the excitability of these neurons and the release of neurotransmitters are regulated by the nicotinic acetylcholine receptor(nAChR).Animal studies have found that nAChR in the hippocampus is involved in the regulation of working memory.Blocking nAChR in the ventral or dorsal hippocampus leads to an increase in the number of work errors in the radial arm maze task.In the central nervous system,the most expressed nAChR are two subtypes:?4?2 and?7.Among them,?7nAChR is widely distributed in the central nervous system,such as the I and VI layers of the cerebral cortex,the CA1,CA3and dentate gyrus of the hippocampus,basal ganglia and reticular thalamic nucleus.A number of studies have found that?7nAChR has neuroprotective effects,which can improve cerebral ischemia,reduce hematoma caused by cerebral hemorrhage,and also improve cognitive dysfunction such as Alzheimer's disease and schizophrenia.However,the mechanism and signal transduction pathways of?7nAChR and cognitive function are still unclear.?7nAChR has high Ca²⁺permeability.Studies have found that it can activate the MAPKs signaling pathway extracellular signal-regulated kinase(ERK1/2)and cyclic adenosine phosphate response element binding protein(c AMP-response element bingding protein,CREB)signal transduction pathway.After ERK1/2 activated,nuclear translocation occurs and directly activates certain transcription factors.At the same time,ERK activates the ribosomal S6 kinase and activates the downstream transcription factor CREB.CREB enters the nucleus and initiates CRE-dependent gene transcription.Phosphorylated ERK1/2 can also directly phosphorylate CREB,which in turn activates the downstream transcription process.Phosphorylated CREB is a transcription factor that plays an important role in neuroprotection,neurotransmission,learning and memory.Studies have found that cognitive dysfunction caused by CIH is related to changes in ERK1/2 and CREB levels.Brain-derived neurotrophic factor(BDNF)is an important molecule that promotes the growth and survival of nerve cells and the communication between neurons.It plays an important role in long-term memory and cognitive functions.Studies have found that the reduction of BDNF expression in the hippocampus after intermittent hypoxia in mice is related to the weakening of long-term potentiation(LTP).It is considered that the reduction of BDNF is one of the mechanisms of cognitive deficits caused by CIH.CREB and BDNF are essential in the survival of neurons,the formation of synaptic plasticity,and the process of learning and memory.CREB can regulate the expression of peroxisome proliferator-activated receptor?coactivator 1?(PGC-1?)and BDNF.PGC-1?enhances long-term potentiation by regulating the expression of downstream membrane protein FNDC5,thereby improving learning and memory ability,and affects the expression of BDNF by regulating FNDC5.PGC-1?is an interacting factor of nuclear receptor transcription factor PPAR?.And as a highly conserved transcriptional co-activator,it can regulate mitochondrial biogenesis factors to maintain mitochondrial functions,mitochondrial metabolism,glucose and lipids and plays an important role in the maintenance of quality and energy homeostasis,protecting neurons from destruction.The expression of PGC-1?has a neuroprotective effect in neurodegenerative diseases and aging.PGC-1?can regulate recombinant fibronectin type III domain containing protein 5(FNDC5),and improve cognitive dysfunction caused by aging.Studies have also found that PGC-1?is involved in the formation and maintenance of neuronal dendritic spines.Multiple studies have found that depression,epilepsy,stroke and Alzheimer's disease all affect cognitive function through the above pathways.Methods:This experiment is divided into three parts.Part 1:Four-week-old 30male CD-1 mice(SPF grade)were chosen and randomly divide them into air control group(RA group),CIH-2 weeks group(CIH2W group),CIH-4 weeks group(CIH4W group).Novel object recognition(NOR)test was conducted after hypoxia on the 26th day of CIH and Morris water maze(MWM)test was then conducted to evaluate cognitive function of mice.Part 2:104 male CD-1 mice(SPF grade)4 weeks old were selected and randomly divided into 4 groups,104 CD-1 mice were exposed to CIH for 4weeks and randomly divided into four groups:CIH-4 weeks group(CIH4W group),CIH-4 weeks with dimethyl sulfoxide(DMSO)group(sham group),CIH-4 weeks+?7nAChR-specific agonist PNU-282987 group(PNU group),CIH-4 weeks+?7nAChR specific inhibitor methylcaconitine(MLA)+PNU-282987 group(MLA+PNU group).From the 26th day of CIH,mice in the PNU group were given intraperitoneal injection of PNU-282987(5mg/kg)before hypoxia,mice in the MLA+PNU group was given 5mg/kg MLA intraperitoneally for 15 minutes and then given PNU 5mg/kg intraperitoneally,mice in the sham group were given intraperitoneal injection of 2%DMSO,and both PNU and MLA were dissolved with 2%DMSO.Part 3:Take 10 mice from each group in the second part for NOR test and MWM test;then take 8 mice for TUNEL(terminal deoxynucleotidyl transferase d UTP nick end labeling)test respectively,and immunohistochemistry and western blotting were used to detect the expression of ERK1/2/CREB pathway protein and the expression of PGC-1?,FNDC5 and BDNF protein.Image J and SPSS statistical software were used for analysis,and P<0.05indicated that the difference was statistically significant.Results:1.Comparison of cognitive function of mice with different CIH duration:in the NOR test,compared with the normal air control group(0.337±0.165),it was observed that the mean discrimination index of CIH2W group(0.253±0.160)was lower,but there was no statistical difference,while the CIH4W group(-0.127±0.154)was significantly different.Compared with the CIH2W group,the mean discrimination index of the CIH4W group was reduced,and the difference was statistically significant.In MWM test,during the 4 days of directional navigation training,the escape latency for the mice to find the platform gradually shortened with the increase of training days.Compared with the normal air control group,there was no significant statistical difference in the CIH2W group,while in the CIH4W group,the escape latency of finding the platform was relatively longer on day 1-4,indicating that CIH had effect on the learning and memory ability of mice.Compared with the CIH2W group,the mice in the CIH4W group showed significant differences on day 1-4.After the platform was removed on the fifth day of the exploration experiment,the platform crossings which is the number of times that the mice crossed the platform in MWM was calculated.The results showed that,compared with the normal air control group(5.7±2.058),the platform crossings in the CIH4week group(2.10±1.197)were significantly reduced,with a significant statistical difference,while the CIH2W group(4.90±1.912)had no significant statistical difference.Compared with the CIH2W,the CIH4W group had significant lower platform crossings.2.Effect of PNU on cognitive function of CIH4W mice:in the NOR test,statistical analysis showed that there was no significant difference in the total exploration time of the four groups of mice(p>0.05),and there was significant statistical difference in the mean discrimination index of the PNU group(0.180±0.105)compared with the CIH4W group(-0.141±0.125),sham group(-0.082±0.161)and MLA+PNU group(-0.183±0.295).In the Morris water maze experiment,the mean escape latency of mice in the PNU group was shorter than that in the CIH group and sham group during the 4 days of directional navigation training.After the platform was removed on the 5th day,compared with the CIH group(2.10±1.37)and sham group(2.30±1.252),the mean platform crossings in the PNU group(4.7±1.418)was significantly increased.3.The TUNEL tests in the hippocampus of mice in each group indicated that the percentage of TUNEL positive cells in the PNU group was significantly lower than that in the CIH group,sham group,and PNU+MLA group.Immunohistochemical results of p CREB and BDNF in the hippocampal tissues of each group showed that the expression levels of PNU group in CA1,CA3 and DG regions were increased compared with those in CIH group,sham group and PNU+MLA group.Comparison of expression levels by western blotting in the hippocampal tissues of each group:compared with the CIH group and sham group,phosphorylated ERK1/2 and phosphorylated CREB were increased in the PNU group,showing a statistically significant difference(p<0.05).There was no significant difference between the groups in total ERK1/2 and total CREB levels.The phosphorylation of ERK1/2 and CREB decreased in MLA+PNU group,but there was no statistical difference.Compared with the CIH group and sham group,the expressions of PGC-1?,FNDC5 and BDNF in the PNU group were all increased,with significant statistical differences(p<0.05).The expression of PGC-1?,FNDC5 and BDNF decreased in MLA+PNU group,but there was no statistical difference.Conclusion:CIH for 4 weeks could cause cognitive impairment such as learning and memory ability in mice.Intraperitoneal injection of?7nAChR agonist PNU-282987could improve the cognitive dysfunction induced by CIH,and up-regulated the levels of p-ERK1/2,p-CREB,PGC-1?,FNDC5,and BNDF in the hippocampal tissues of mice after CIH.PNU-282987 possibly improved the cognitive dysfunction induced by CIH through ERK1/2/CREB signaling pathway and upregulating the expressions of PGC-1?,FNDC5 and BNDF.