Research On The Mechanism Of Neuronal Apoptosis Induced By Chronic Ethanol Exposure Via Na⁺/Ca²⁺Exchanger

Objective:Drinking is a common social behavior in the global.The heavy economic burden and social harm that people bring to the country,society,families and individuals due to drinking.In particular,drinking poses a very serious threat to human health.The WHO released the 2018 Alcohol and Health Report"Global status report on alcohol and health 2018",which reported that about 3 million people die each year from drinking,accounting for 5.3%of all deaths worldwide.In forensic practice,there are significant proportions of cases of acute and chronic poisoning and death caused by drinking and ethanol-related cases.More and more studies have shown that many diseases,especially neurological diseases,are related to drinking.Although ethanol-related population is still huge,and involving many young people especially,the threat of ethanol to humans is becoming more and more serious.As the main ingredient in drinking water,ethanol（EtOH）is an organic compound that acts directly on brain nerve cells pass the blood brain barrier（BBB）.Long-term ethanol exposure causes damage to central nervous system,leading to Wernicke encephalopathy,Korsakov syndrome,chronic alcoholic dementia,alcoholic mental and behavioral disorders,among which cognitive dysfunction is the most widely.The exact toxicological mechanism of ethanol is not fully understood.Ethanol induces neuronal degeneration and apoptosis through various mechanisms.Intracellular Ca²⁺overload-induced apoptosis is a particularly important pathway and a common pathway for the collection of various mechanisms.Intracellular Ca²⁺overload activates calcium-sensitive proteins,including apoptosis-associated factors such as caspase-3 and calpain-1,to trigger apoptosis.In addition,excess Ca²⁺can also affect mitochondrial ATP synthesis and disrupt DNA structure.Calcium ion is an important ion in the body,which is of great significance for maintaining intracellular and extracellular ion concentration,signal transduction,neurotransmitter release,synaptic plasticity,and learning and memory.Usually,the intracellular free Ca²⁺concentration is very low,about 10^-710^-8 mol/L;the intercellular fluid Ca²⁺concentration is high,about 5×10^-3 mol/L.Extracellular Ca²⁺,even if it is influx into the cell,causes a significant change in the concentration of cytosolic free Ca^2+,resulting in a series of physiological responses.In order to maintain the balance of Ca²⁺concentration inside and outside the cell,the cells will discharge the Ca²⁺that fulfills the“messenger”task through the plasma pump（PMCA）and the sodium-calcium exchanger(Na⁺/Ca²⁺exchanger,NCX).Calcium pump is a plasma membrane one-way calcium ion transporter,which is characterized by strong affinity with Ca²⁺and weak transporting ability.It is mainly responsible for its role in resting state.Unlike calcium pumps,the plasma membrane NCX is very sensitive to changes in Ca²⁺concentration.It has nine transmembrane transporters and has a very high transport capacity for Ca²⁺,which is10-50 times that of PMCA.Three subtypes of NCX1,NCX2 and NCX3 have been found in mammals by now.NCX1 is widely expressed in various tissues,while NCX2 and 3 are expressed only in brain and skeletal muscle.In the brain,the three subtypes of NCX have different degrees of distribution and expression.NCX activates when the intracellular Ca²⁺rises suddenly（up to 500 mM）,activates the Ca²⁺transport mode,and excretes more than 2/3 of the Ca²⁺in the cells.NCX has a particularly important role for the Ca²⁺rises sharply and then rapidly decreases to the basal level to nerve cells.Chronic ethanol exposure can cause cognitive dysfunction in humans and animals.The cytoplasmic Ca²⁺overload in nerve cells leads to degradation and apoptosis.However,there is no relevant research reports about why the neuronal plasma membrane NCX cannot prevent ethanol from inducing intracytoplasmic Ca²⁺overload by Ca²⁺clearance,still no studies have been made on whether or not the expression of NCX can be changed after ethanol exposure.We speculate that it is possible that the rate of Ca²⁺influx in ethanol is faster than the rate of calcium excretion in NCX,or that ethanol inhibits the expression of NCX through certain mechanisms that prevent it from functioning adequately.In order to investigate the changes of NCX expression and dysfunction during the induction of neuronal apoptosis by chronic ethanol exposure,this experiment used animal model to observe the effect of chronic ethanol exposure on cognitive function of experimental animals,hippocampal neuronal changes,NCX isoforms expression changes and their causal relationship;At the same time,cell model was used to observe the expression changes of NCX1,NCX2 and NCX3 in cultured neurons induced by ethanol exposure,and the NCX isoforms were analyzed in chronic ethanol-induced neuronal apoptosis.To explore the new mechanism of chronic ethanol exposure-induced neuronal apoptosis,provide basic data and new ways for the prevention and treatment of nerve damage caused by chronic alcoholism,and provide experimental data for forensic identification.Methods:Chronic ethanol exposure animal experiment,using C57BL/6 male mice,divided into 30 d-CON group,30 d-10%EtOH group,30 d-20%EtOH group,60 d-CON group,60 d-10%EtOH Group,60 d-20%EtOH group,90 d-CON group,90 d-10%EtOH group,90 d-20%EtOH group,10 mice per group.Cognitive dysfunction in different groups of mice was observed by morris water maze.To the preset ethanol exposure time,weighed,the mice were sacrificed by ether anesthesia,and the brain tissue was taken.The distribution and expression of NCX isoforms in the hippocampus of mice were observed by immunohistochemistry.The hippocampus of mice were observed by Western blot and qPCR.Expression of NCX1,NCX2,NCX3 and calpain-1,cleaved-caspase 3,phosphorylation of Akt and expression of CREB1,analysis of the relationship between the changes of NCX isoforms and cognitive dysfunction in mice after chronic ethanol exposure.Cell experiments:ethanol group:1 d-CON,1 d-100 mM,1 d-200 mM,2 d-CON,2 d-100mM,2 d-200 mM;and LY294002,NaCl added to the ethanol group Corresponding groupings after transfection of NCX1,NCX2 and NCX3,respectively.Western blot was used to observe the expression changes of NCX1,NCX2,NCX3,calpain1,caspase3,Akt,p-Akt and CREB1 at different time of ethanol exposure,and the relationship with ethanol exposure was observed.The changes of NCX isoforms and apoptosis of nerve cells induced by ethanol exposure were observed after inhibiting the Akt phosphorylation pathway.After silencing NCX1,NCX2 and NCX3 isoforms by siRNA technique,the apoptosis of nerve cells after ethanol exposure was observed,and the specific effects of the three isoforms on chronic ethanol exposure were determined.Select a NCX isoform which plays a major role,was overexpressed,and its effect on apoptosis of nerve cells induced by ethanol exposure was observed,and its effect was further confirmed from both positive and negative aspects.Results:Animal experiments:The spatial memory capacity of mice after chronic ethanol exposure was severely impaired with time and the increase concentration.The expression of cleaved-caspase 3 and calpain-1 in mouse hippocampal neurons increased with the prolongation of ethanol exposure and dose.Three isoforms of NCX were expressed in mouse hippocampal neurons.When ethanol was exposed for a short time,the expression of NCX1 was up-regulated,and that of the 10%EtOH group was significantly up-regulated.There was no significant difference between the expression of NCX1 and the CON group at 60 d and 90 d EtOH.The expression of NCX2 was up-regulated with increasing concentration at 30 d after ethanol exposure.There was no significant difference between NCX2 expression and CON group at 60 d and 90 d after ethanol exposure.The expression of NCX3 had obvious regularity.The expression level of the 10%EtOH group was the highest,and there was no significant difference between the CON group and the 20%EtOH group.With the prolongation of exposure time and the increase of ethanol concentration,the level of NCX3 was significantly down-regulated.The immunohistochemical staining of NCX3 showed that the positive expression of NCX3 in the CA1,CA3 and DG areas of the hippocampus of the 10%EtOH group was higher in the 30 d group.The number of positive cells of NCX3 in the 20%EtOH group was significantly reduced compared with the CON group.After exposure to ethanol for 60-90d,the number and intensity of NCX3 positive cells in CA1,CA3 and DG areas of mice hippocampus were significantly down-regulated with time and dose,and apoptosis appeared in cells,which was severely changed in CA3 area.The level of Akt phosphorylation and the level of CREB1 upstream of NCX3 are consistent with the expression trend of NCX3.Cellular experiments showed that ethanol exposure promoted the increase of intracellular Ca²⁺and the increase of apoptosis,and the expression of apoptosis-related factors caspase-3 and calpain-1 increased.The expression of NCX1 was inhibited by high concentration of ethanol,and the concentration of ethanol was reduced to NCX1.With the increase of ethanol concentration and the prolongation of exposure time,the expression of NCX2 increased gradually;NCX3 also increased with the increase of ethanol concentration and exposure time.The degree of phosphorylation of Akt increased with increasing concentration at 1 d of ethanol exposure and decreased significantly at 2d.When LY294002 inhibits the phosphorylation pathway of Akt,the expression of NCX1 and NCX3 is decreased,the pro-apoptotic effect of ethanol is enhanced,and the intracellular Ca²⁺overload is severe.Using siNCX to down-regulate the levels of NCX1,NCX2 and NCX3,respectively,it was found that NCX1 has protective effects on nerve cells exposed to ethanol for a short period of time,and has poor long-term exposure;down-regulation of NCX2 may be achieved by compensatory up-regulation of NCX3levels and protect nerve cells.Silencing NCX3 directly led to a significant increase in Ca²⁺fluorescence signal in neurons exposed to ethanol,and the expression of apoptotic factors cleaved-caspase 3 and calpain-1 was significantly up-regulated,and the apoptotic rate was increased.When overexpressing NCX3,it was found that the degree of apoptosis of nerve cells exposed to ethanol was significantly reduced.Increasing the concentration of Na⁺in the neuronal medium exposed to ethanol,the degree of apoptosis of nerve cells was significantly reduced,and the mechanism was achieved by up-regulating the expression of NCX3.When the level of NCX3 was down-regulated,the increase of extracellular Na⁺concentration could not be protected.The role of nerve cells.Conclusions:1.Chronic ethanol exposure leads to spatial learning and memory dysfunction in mice;2.Decreased expression of NCX3 in mouse hippocampal neurons has relation to cognitive dysfunction of mice afrer chronic ethanol exposure;3.Chronic ethanol exposure can cause mouse hippocampus neurons injury,particularly to CA3region;4.NCX3 protein expression changes are expected to provide a reference for the pathological diagnosis of nerve damage caused by chronic ethanol poisoning;5.NCX3expression enhances inhibition of chronic ethanol exposure induced neuronal apoptosis.Increasing the concentration of extracellular sodium ions is expected to be a therapeutic target for nerve cell damage caused by ethanol.