| Central nervous system oxygen toxicity（CNS-OT）refers to the fact that the human body breathes oxygen with high pressure in a short period of time,which will lead to abnormal brain discharge,convulsive changes such as limb rigidity and muscle bundle fibrillation.Severe cases result in death.Among them,oxygen convulsion is the most serious and typical symptom of acute oxygen poisoning.Hyperbaric oxygen（HBO）has been widely used in submarine escape,diving operations and clinical disease treatment[1].At present,the incidence of CNS-OT is very low in civil saturated diving and clinical oxygen due to strict control of the pressure and duration of HBO exposure.However,in the field of sports and military,the incidence of CNS-OT is still high[2,3].CNS-OT is sometimes caused by inhaling high-pressure oxygen,and the preventive measures that can be taken are also limited and passive,which seriously affects the smooth completion of combat tasks.At present,the mechanism of CNS-OT mainly includes neurotransmitter imbalance theory,oxidative stress theory,adenosine metabolism theory,etc.,but these theories cannot fully clarify the molecular mechanism of CNS-OT.As China’s determination to safeguard maritime interests becomes increasingly prominent,it is imperative to enhance the military strength of the navy.Therefore,it is particularly important to clarify the occurrence mechanism of CNS-OT and find its prevention target for future underwater operations,so as to ensure the life safety of combatants and reduce the loss of non-war personnel.Mitochondria as a kind of complex organelles in the cell is not immutable,mitochondrial dynamics refers to the mitochondria by splitting（fission）fusion（fusion）and change the morphology and structure of mitochondria and number,can make the dynamic changes of mitochondria in the cytoplasm to a dot,fragmented,strip or linear such as different forms.Mitochondrial dynamics is closely related to mitochondrial functions,such as cell proliferation,metabolism and migration,and is regulated by a variety of chemical enzymes and proteins.Once the mitochondrial dynamic balance is broken,it will have a significant impact on the body’s life activities[4].Studies have shown that abnormal mitochondrial dynamics is associated with epilepsy,cerebrovascular disease,Alzheimer’s disease,Parkinson’s disease and other neurological diseases.However,whether mitochondrial dynamics is involved in the occurrence and development of CNS-OT has not been reported yet.Nicotinic acetylcholine receptor（nAChR）is a ligand-gated ion channel protein widely distributed in the peripheral and central nervous system and involved in the regulation of receptor function.Theα7 nicotinic acetylcholine receptor（α7nAChR）is composed of five homologousα7 subunits with hollow ion channels in the middle.α7nAChR is widely expressed in various cells of the central nervous system,including neurons,cerebrovascular endothelial cells,astrocytes and microglia.Currently,the neuroprotective effects ofα7nAChR have been widely reported,and previous studies in our group also found that activation ofα7nAChR on microglia alleviates autoimmune encephalomyelitis（EAE）and ischemic stroke by promoting autophagy.In addition,a large number of studies have shown that activation ofα7nAChR has a protective effect on hemorrhagic stroke,Parkinson’s disease,Alzheimer’s disease and other central nervous system diseases,suggesting thatα7nAChR May become a new therapeutic target for central nervous system diseases.Therefore,based on the above analysis,this study aimed to study the relationship between central nervous system oxygen poisoning and mitochondrial dynamics imbalance,and further elucidated whether activation ofα7nAChR has a protective effect on central nervous system oxygen poisoning and the relationship betweenα7nAChR and mitochondrial dynamics imbalance.The content of this study is divided into the following parts:Part one,to explore the mitochondrial kinetic mechanism of oxygen poisoning in central nervous system;In the second part,the protective effect of activatedα7nAChR on central nervous system oxygen poisoning mice and its related mechanism were observed in the whole animal level.In the third part,we elucidated the effects ofα7nAChR activation on HBO-induced oxidative damage,mitochondrial function and mitochondrial dynamics in cultured neurons.（一）Mitochondrial dynamics plays an important role in the pathophysiology of CNS-OT1.Effects of central nervous system oxygen poisoning on mitochondrial morphologyC57 mice were randomly divided into two groups:CON group and HBO group.CON did not do any treatments,HBO group mice were exposed to hyperbaric oxygen chamber under 6ATA for 30min to prepare mice hyperbaric oxygen model.After modeling,we took mouse brain tissue and observed the mitochondrial structure of mouse brain tissue by transmission electron microscopy.The results of electron microscopy showed that the morphology of mitochondria in normal group was long tubular and elliptic,and the structure of mitochondrial crest and bilayer membrane was clear.In the HBO group,mitochondria in the brain tissue of mice were swollen,mostly spheroidal,and most of them were in the state of division.Compared with the normal control group,the proportion of abnormal mitochondria was increased.2.Effects of central nervous system oxygen poisoning on mitochondrial dynamicsC57 mice were randomly divided into two groups:CON group and HBO group.CON did not do any treatment,HBO group mice were exposed to hyperbaric oxygen chamber under 6 ATA for 30 min to prepare mice hyperbaric oxygen model.The expression changes of mitochondrial dynamics related proteins Drp1,Drp1 S637,Opa1 and Fis1 in mouse brain tissues were detected at gene level and protein level respectively.RT-PCR results showed that the expression levels of mitochondrial dynamics-related proteins Drp1 and Fis1 m RNA in HBO group were higher than CON group,and in the level of m RNA,the expression of Opa1 were lower than CON group.Western blotting results showed that the expression of Drp1 and Fis1 in CNS-OT mice brain tissue was higher than that in control group,while the expression of Drp1 S637 and Opa1 was lower than that in control group.It was consistent with m RNA level expression.3.Effects of central nervous system oxygen poisoning on mitochondrial functionAt the cellular level,we used HT22 mouse neuron cell line,and placed HBO group cells in the cell hyperbaric oxygen chamber for 6 ATA and 30 min hyperbaric oxygen treatment to establish the cell hyperbaric oxygen injury model,and detected a series of mitochondrial functional indicators:JC-1,NAD+/NADH and ATP content.The results of JC-1 fluorescence probe showed that the mitochondrial membrane potential in HT-22 cells in HBO group was lower than that in normal control group.NAD+/NADP test results showed that HBO group had lower NAD+/NADP than normal control group.The intracellular ATP content was reduced compared with the normal control group.This indicates that in CNS-OT,mitochondrial membrane potential decreases,productivity efficiency decreases,and mitochondrial dysfunction.4.Central nervous system oxygen poisoning is positively correlated with inflammation and oxidative stressC57 mice were randomly divided into two groups:CON group and HBO group.CON did not do any treatments,HBO group mice were exposed to hyperbaric oxygen chamber under 6ATA for 30min to prepare mice hyperbaric oxygen model.The changes of oxidative stress level,ROS content and m RNA expression level of inflammatory factors after HBO exposure were detected.Oxidative stress test results showed that compared with normal control group,the activities of glutathione peroxidase（GSH）and superoxide dismutase（SOD）in HBO group were decreased,and the content of lipid oxidation（MDA）was increased.RT-PCR results showed that the m RNA expression of inflammatory factors（IL-6,IL-1βand TNF-α）were higher than CON group.5.Use Drp1 inhibitor to inhibit mitochondrial division and clarify the pathogenesis of central nervous system oxygen poisoningIn vitro,cultured with HT-22 cell,blocking mitochondrial division with mdivi-1,an inhibitor of mitochondrial mitotic protein Drp1,to clarify the pathogenesis of CNS-OT.The experiment was divided into three groups:CON group,HBO group and HBO+Mdiv I-1（25 m M）group.CCK8 results showed that,compared with HBO group,the cell viability of HBO+Mdi VI-1 group increased,oxidative stress level decreased,and mitochondrial function recovered,indicating that excessive mitochondrial division occurred in central nervous system oxygen poisoning,which is one of the important pathogenesis of central nervous system oxygen poisoning,which is consistent with the above experimental results.Conclusion:In central nervous system oxygen poisoning,mitochondrial morphology changes,excessive increase of mitochondrial division,resulting in mitochondrial dysfunction,increasing the level of oxidative stress and inflammation in the brain,resulting in body damage.Therefore,imbalance of mitochondrial dynamics is one of the important mechanisms of CNS-OT.（二）In vivo,explore the protective effect of activationα7nAChR on CNS-OT and its mechanism1.To investigate the changes ofα7nAChR expression in brain tissue of mice with CNS-OT,and the effects of activation ofα7nAChR on convulsion latency and survival rate of miceFirstly,C57 mice were randomly divided into CON group and HBO exposure group.The treatment was the same as above.After the exposure,the brain tissues of the two groups were taken,and then the changes ofα7nAChR m RNA in brain tissues were detected by QT-PCR.It is suggested thatα7nAChR is closely related to the occurrence of central nervous system oxygen poisoning.Secondly,to clarify whether activation ofα7nAChR has a protective effect on convulsion and prognosis of mice with central nervous system oxygen poisoning,we randomly divided C57 mice into HBO+Vehicle group and HBO+PNU preconditioning group.They were given normal saline or PNU（3 mg/kg）intraperitoneal injection 30min before entry.Then the animals in both groups were exposed to hyperbaric oxygen chamber（6 ATA,99%oxygen）for 30 min,during which the occurrence of convulsion in mice was observed,and the survival of mice was observed after the chamber.Compared with the Vehicle group,PNU pretreatment significantly prolonged the incubation period of HBO induced oxygen convulsion in mice,and also significantly improve the survival of mice.These data suggest that activation ofα7nAChR has a protective effect on HBO induced central nervous system oxygen poisoning.2.To investigate the effects of activation ofα7nAChR on the survival rate of central nervous system oxygen poisoning and the incubation period of convulsionTo explore the effects ofα7nAChR activation on mitochondrial morphology and mitochondrial dynamics in HBO-exposed mouse brain tissues at the animal level,C57mice were randomly divided into three groups:CON group,HBO+Vehicle group,and HBO+PNU（3 mg/kg,I.P.）group.As mentioned above,the brain tissues were collected after HBO exposure.The mitochondrial structure changes of mouse brain tissues were observed by transmission electron microscopy and the expression of mitochondrial dynamics-related proteins were detected by Western Blot.Electron microscope results showed that the proportion of abnormal mitochondria increased significantly after HBO exposure.Preconditioning with PUN significantly reduced the proportion of abnormal mitochondria elevated by HBO.In addition,WB results showed that compared with HBO exposure group,the expression of mitochondrial mitogenesis related proteins Drp1 and Fis1 was decreased in PNU pretreatment group,while the expression of fusion related proteins Drp1 S637 and Opa1 was increased,and the m RNA detection results were consistent with the protein level.3.To investigate the effects ofα7nAChR activation on mitochondrial function and oxidative stress level in mouse brain tissue after HBOAt To elucidate the effects ofα7nAChR activation on oxidative stress levels in HBO-exposed mouse brain tissues at a global animal level,C57 mice were randomly divided into CON group,HBO+Vehicle group,and HBO+PNU group.The mice were sacrificed immediately after the barn to take brain tissue for detection.The results showed that compared with CON group,the mitochondrial membrane potential and NAD+/NADP ratio of mice brain tissue were significantly decreased after HBO exposure,while the reduction of mitochondrial membrane potential and NAD+/NADP ratio induced by HBO exposure were significantly improved after PNU pretreatment.In addition,HBO exposure also resulted in decreased GSH,SOD and increased MDA levels in mouse brain tissue,and these changes were significantly inhibited after preconditioning of PUN.These results suggest that activation ofα7nAChR can significantly improve mitochondrial dysfunction in brain tissue induced by HBO exposure and reduce oxidative damage in brain tissue.Conclusion:At the overall animal level,we found that activation ofα7nAChR could inhibit mitochondrial excessive division,improve mitochondrial function and reduce oxidative damage of brain tissue in CNS-OT mice,thus prolonging the incubation period of oxygen convulsion in mice,improving survival rate and improving prognosis.（三）In vivo,explore the protective effect of activationα7nAChR on CNS-OT and its mechanism1.To explore the effect ofα7nAChR activation on HBO over exposed neuronsWe divided cultured HT22 mouse neurons into several groups:CON group,HBO+Vehicle treatment group,HBO+PNU（1 uM,10 uM,100 uM）treatment group.30min before HBO exposure,each group was pretreated with Vehicle and PNU at different concentrations.After 30 min HBO exposure,cells and culture supernatants were collected immediately for a series of tests.Our results showed that after HBO exposure,cell viability decreased significantly and LDH release increased.PNU pretreatment improved HBO-induced decline in cell viability and increased LDH release concentration-dependent.2.To investigate the effects of activation ofα7nAChR on mitochondrial dynamics-related proteins in HBO over exposed neuronsTo further explore whether activation ofα7nAChR improves mitochondrial function is related to inhibiting HBO exposure induced mitochondrial over division and improving mitochondrial dynamic imbalance,cultured HT22 mouse neurons were divided into four groups as follows:CON group,HBO+Vehicle group,HBO+PNU（10 uM）group and HBO+PNU（100 uM）group were treated as mentioned above.Cells were collected after exposure.Western Blot was used to detect the expression changes of mitochondrial dynamics related proteins Drp1,Drp1 S637,Opa1 and Fis1 in intracellular mouse brain tissues.The results showed that compared with the HBO exposure group,the expressions of mitochondrial mitogen-related proteins Drp1 and Fis1 were significantly decreased in PNU preconditioning group,while the expressions of fusion-related proteins Drp1 S637and Opa1 were increased,suggesting that activation ofα7nAChR can inhibit HBO-induced neuronal over division and improve mitochondrial dynamic balance.3.To investigate the effects ofα7nAChR activation on mitochondrial function and oxidative stress injury of neuronsWe divided cultured HT22 mouse neurons into several groups:CON group,HBO+Vehicle treatment group,HBO+PNU（1 uM,10 uM,100 uM）treatment group.Treatment as described above,cells and culture supernatants were collected after exposure and a series of tests were performed.Our results showed that after HBO exposure,cell viability decreased significantly and LDH release increased.PNU pretreatment improved HBO induced decline in cell viability and increased LDH release concentration-dependent.HBO exposure also significantly decreased the activities of GSH and SOD,while PUN pretreatment significantly increased the activities of GSH and SOD.In addition,PNU pretreatment also significantly reduced the intracellular ROS elevation induced by HBO exposure.Based on the above results,we preliminarily concluded that activation ofα7nAChR could significantly reduce the intracellular oxidative damage induced by HBO exposure and protect neurons.To further clarify whether activation ofα7nAChR reduces HBO induced oxidative damage is related to its improvement of mitochondrial function,cultured HT22 mouse neurons were divided into three groups as follows:CON group,HBO+Vehicle group and HBO+PNU（100uM）were treated as mentioned above.Cells were collected after exposure,and changes in mitochondrial functional indicators were detected using kits.The results show:The mitochondrial membrane potential,NAD+/NADP ratio and ATP content of HT-22 cells decreased significantly after HBO exposure,while PNU pretreatment significantly improved HBO induced mitochondrial membrane potential reduction,NAD+/NADP ratio reduction and ATP content reduction.These results suggest that activation ofα7nAChR inhibit HBO induced mitochondrial dysfunctionConclusion:In vitro,activation ofα7nAChR inhibits HBO induced mitochondria over division,improves mitochondrial function,alleviates oxidative damage,and protect neurons injured by HBO over explore. |
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