| As a prevailing breathing and sleep disorder,obstructive sleep apnea hypopnea syndrome(OSAHS)is caused by the repeated collapse and obstruction of the upper airway during sleep[1].It is often accompanied by symptoms and signs such as intermittent hypoxemia,nocturnal awakening,daytime sleepiness and cognitive dysfunction.A large-scale meta-analysis has shown that OSAHS is more commonly found in males(4～29%)than in females(2～9%)[2].Among them,severe OSAHS were 49.7%and 23.4%,respectively,and the incidence was increasing day by day[3].OSAHS induces a large amount of damage to all body systems,including cardiovascular morbidity,hypertension,dyslipidemia,diabetes,obesity,and neurocognitive dysfunction[4-6]Among them,neurocognitive dysfunction has received particular attention.Several studies have revealed that OSAHS-related cognitive dysfunction contributes to a variety of symptoms,including attention decrease,executive function abnormality,decision-making ambiguity,as well as learning and memory decline.For children OSAHS with cognitive impairment,some scholars conducted preoperative and postoperative cognitive function assessment and found that the children recovered to normal after surgery,suggesting that the cognitive impairment of children OSAHS is reversible[7].For adult OSAHS,the study of Weaver and Zimmerman et al[7,8]found that after continuous and formal CPAP treatment,the cognitive function assessment of most patients showed no significant difference from that before treatment,indicating that even if OSAHS patients received the best treatment,cognitive impairment could not be completely reversed,leading to permanent brain damage[8].Therefore,it is urgent to explore the pathophysiological mechanism of OSAHS related cognitive impairment.Of many associated factors,IH,in particular,is one of the hallmarks in OSA.Chronic IH is defined as a long-term cycle of hypoxia-reoxygenation during sleep,which is irreplaceable in brain damage and apoptosis of neurons in the cortex and hippocampus[9].However,the precise mechanism underlying the influence of IH on neurocognitive deficits in OSAHS patients has not been fully demonstrated.Brain-derived neurotrophic factor(BDNF),as a member of the neurotrophic family,plays a key role in nerve growth and differentiation,and can keenly regulate synaptic transmission and plasticity[[10].The earlier study found in the brain and spinal cord ischemia reperfusion,BDNF expression,in order to protect the neurons[11],short time hypoxia of mice hippocampus BDNF expression significantly reduced[12],a cycle longer IH study,however,the expression of BDNF hippocampus enhanced[13],in another less severe daily intermittent hypoxia paradigm has increased the expression of BDNF in the spinal cord[14].These results suggest that the amount and frequency of hypoxic/reoxygenation cycles may be the main factors affecting BDNF expression.Therefore,what is the signaling pathway that regulates the expression and release of BDNF to regulate synaptic plasticity under the 1H condition?It is urgent to explore the pathophysiological mechanism of OSAHS related cognitive impairment.As a serine/threonine kinase,mTOR obtained from the growth factor,nutrients,amino acids,and environmental cues can regulate multiple cellular processes including protein growth,aging,metabolism,synthesis,regeneration,and autophagy.In addition,NF-kB,an oncogenesis-associated transcription factor,can regulate cell proliferation and apoptosis in various types of tumors[14].A large number of clinical investigations and experimental studies have shown that mTOR signaling pathway and downstream factor NF-kB,are essential for maintaining the normal physiological function of the nervous system,especially for learning and cognitive function[15].As an important transcriptional regulator,NF-kB can regulate the expression of pro-inflammatory factors.Many molecules involved in the early stage of immune response and all stages of inflammatory response are regulated by NF-κB,including TNF-α,IL-1β,IL-2,IL-6,IL-8,IL-12,iNOS,COX2,chemokines,adhesion molecules,and colony-stimulating factors.Its regulation of pro-inflammatory cytokines is closely related to the expression of BDNF.It has been reported that IL-1βcan reduce BDNF expression[16]and also lead to impaired BDNF signal transduction[17]In this study,applying the cycle of low oxygen ternary gas(1.5%O2+5%CO2+93.5%N2)and normal oxygen ternary gas(21%O2+5%CO2+74%N2),we established an IH rat model and used hippocampal neurons to explore the mechanisms of IH related damage by evaluating the effect of Rapa and PDTC.We gave a hypothesis that IH-induced hippocampal neuron impairment might be mediated by the mTOR/NF-kB signaling pathway.We detected the access key signaling molecule detection IH environment(IKKβ、IκBα、NF-κB)and proinflammatory factor,synaptic related protein expression;Then the Rapa was used to inhibit mTOR and PDTC were used to inhibit NF-κB.The changes of the above signaling molecules were observed to clarify the role of mTOR/NF-κB signaling pathway in the regulation of synaptic plasticity in IH,providing a new idea for the study of the pathogenesis of cognitive impairment caused by OSAHS.Objectives(1)IH rat model and hippocampal neurons model were established to explore the mechanisms of IH at the cellular and animal levels respectively.(2)Rapa was used to inhibit mTOR and PDTC to inhibit NF-κB,respectively,to clarify the regulatory role of mTOR on NF-κB signaling pathway,to explore the regulatory role of BDNF-mediated synaptic plasticity under this role,and to identify the mechanism of synaptic plasticity damage under chronic IH environment.(3)Based on the regulation of mTOR/NF-κB signaling pathway on BDNF expression,the mechanism of hippocampal synaptic plasticity injury caused by melatonin-modified chronic IH was elucidated to provide a new idea for the prevention and treatment of OSAHS.Research MethodsPart 1 The Hippocampal Neurons Level(1)The primary hippocampal neurons were identified by immunofluorescence staining,and the changes of synaptic ultrastructure under different conditions were observed under light microscope and electron microscope.(2)Primary hippocampal neurons were cultured in Acongtinue hypoxia(IH)and Acongtinue hypoxia(1H)respectively.The changes of synapses under different conditions were observed under light microscopy.The protein levels of BDNF,SYN and PSD-95 were detected by Western blotting(WB).(3)After PDTC pretreatment,primary hippocampal neurons were cultured in IH environment for 18 hours.Real-time quantitative PCR(RT-PCR)was used to detect the mRNA levels of NF-κB,IL-1β,TNF-α,BDNF,synaptic associated protein SYN and PSD-95.Western blotting was used to detect the mRNA levels of NF-κB,IL-1β,TNF-α,BDNF,SYN and PSD-95.WB)to detect the expression of NF-κB,IL-1β and TNF-α proteins,as well as BDNF,SYN and PSD-95 in hippocampus.(4)After Rapa pretreatment,primary hippocampal neurons were cultured in IH environment for 18 hours.The mRNA levels of IKKβ,IκBα and NF-κB were detected by RT-PCR,The protein expression of BDNF,IKKβ,IκBα and NF-κB in hippocampal neurons was detected by WB.Part 2 The Rat Level(1)Morris water maze test was used to evaluate the effects of Rapa and PDTC on learning and memory ability of rats,and H&E staining was used to detect the protective effects of RAPA and PDTC on hippocampus and hippocampal neurons.(2)Rats were randomly divided into CTRL group(Control group),Rapa group,PDTC group,IH group,IH+Rapa group,and IH+PDTC group(n＝10 in each group).The first three groups were exposed to room air while the others were exposed to IH for 8 weeks.The protein expressions of IKKβ,IκBα,BDNF and NF-λB in the hippocampus of rats were detected by WB method.The mRNA levels of IKKβ,IκBa,BDNF and NF-κB in rat hippocampus were detected by RT-PCR.(3)After melatonin pretreatment,the rats were exposed to room air or IH for 8 weeks.The expression of IKKβ,IκBα,BDNF and NF-κB in hippocampal tissues of rats was detected by WB method.The mRNA levels of IKKβ,IκBα,BDNF and NF-κB in rat hippocampus were detected by RT-PCR.ResultsPart 1 The Hippocampal Neurons Level(1)The purity of primary hippocampal neurons was above 90%by immunofluorescence staining.(2)After exposure to the IH environment,the original hippocampus neurons were clearly curted,the nucleus was severely deformed,and the pretreatment of Rapa and PDTC could improve these phenomena.(3)The protein levels of BDNF,PSD-95 and SYN were significantly decreased after treatment with CH and IH,and this effect was more obvious under IH than under CH.(4)The protein expression levels of NF-κB,TNF-α and IL-1β in 1H group were increased,while the expression levels of BDNF,PSD-95 and SYN were decreased.However,Rapa and PDTC pretreatment could counteract this change.(5)The expression of IκBα and IKKβ was inhibited by IH,but the expression of Iκβαand IKKβ in PDTC group was not significantly changed,while the expression of Rapa group was significantly increased.Part 2 The Rat Level(1)The results of Morris water maze experiment showed that under the condition of IH exposure,the incubation period and pathway length of platform crossing increased and the platform crossing time decreased on the 2nd,3rd,4th and 5th day of training,and this trend was inhibited after the intervention of Rapa or PDTC.(2)IH exposure resulted in neuronal atrophy,necrosis and disordered arrangement,while the hippocampal injury in IH+Rapa and IH+PDTC group was significantly reduced compared with that in IH alone group.(3)The protein expression levels of NF-κB,TNF-α and IL-1β were increased in IH group,while the expression levels of BDNF,PSD-95 and SYN were decreased.However,Rapa and PDTC pretreatment could counteract this change.(4)The expression of IκBa and IKKβ was inhibited by IH,but the expression of IκBαand IKKβ in PDTC group was not significantly changed,while the expression of RAPA group was significantly increased.(5)After MT pretreatment,the increase of NF-κB protein and mRNA and the decrease of BDNF,PSD-95 and SYN were significantly decreased under IH condition.Conclusion1.The synaptic plasticity of primary hippocampal neurons in rats was more impaired by IH than CH.2.IH causes damage to hippocampal tissue and affects the learning and memory of rats.3.PDTC and Rapa preconditioning can improve IH-induced hippocampal neuron damage,suggesting that mTOR/NF-κB signaling pathway is involved in the regulation of hippocampal synaptic plasticity under chronic 1H by regulating BDNF.4.PDTC,Rapa and melatonin can reverse IH-induced hippocampal neuron damage,and provide new ideas for the prevention and treatment of OSAHS by designing and synthesizing new melatonin derivatives with mTOR,NF κB and BDNF as targets in the future.Meaning1.In chronic IH environment,by inhibiting mTOR and NF-κB respectively,the mechanism of mTOR regulating NF-κB signaling pathway and NF-κB-regulated inflammatory response involved in BDNF-mediated decline in synaptic plasticity and ultimately leading to cognitive impairment was clarified at the cellular and animal levels.2.The mTOR regulated IKKβ//IκB/NF-κB signaling pathway regulates the expression of BDNF,and elucidates the mechanism of melatonin in improving synaptic plasticity injury caused by chronic intermittent hypoxia. |
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