Research On The Mechanism Of Neuronal Apoptosis Induced By Activation Of NMDA Receptor Caused By Chronic Ethanol Exposure

Objective:Ethanol,commonly known as alcohol.Alcohol-containing beverages are popular all over the world and can be metabolized by dehydrogenase and oxidase in the human body.The final products are carbon dioxide and water.Ethanol and a series of intermediate products in the metabolism in the body have some effect on tissues and cells,resulting in a series of mental and physical abnormalities.Chronic alcoholism can cause damage to multiple systems and organs,such as the nervous system,cardiovascular system,digestive system,immune system,muscle and so on.Ethanol can also lead to loss of neurons in specific brain regions.Studies have shown that cell necrosis or apoptosis can be activated by ethanol through a wide range of mechanisms,such as enhancing tumor necrosis factor-α（TNF-α）,increasing the Fas/Fas L expression,inducing the transcriptional activity of NF-κB,activating caspasein mitochondrial and resulting in intracellular Ca²⁺transport disorders.Intracellular Ca²⁺plays an important role in cell life events.It can influence or determine cell necrosis or apoptosis independently.Cellular Ca²⁺overload is the hub of necrosis or apoptosis.Intracellular Ca²⁺originates from the L-type voltage-operated calcium channels,N-methyl-d-aspartate receptors（NMDAR）-mediated calcium influxing into the cells,Inositol 1,4,5-trisphosphate（IP3）-induced calcium releasing and ryanodine receptor（Ry R）-induced calcium releasing.Long-term alcohol exposure can make patients’chronic alcohol poisoning’s excitatory amino acid increasing,such as glutamate,aspartate,homocysteine,which can significantly improve the neurons against excitotoxicity and oxidative damage of vulnerability.Studies have reported that excitatory amino acids play a role on total biochemical cascade of relates to N-methyl-D-aspartate（NMDA）receptor overstimulation,oxidative stress,caspases activation,DNA damage and mitochondrial dysfunction.N-methyl-D-aspartate receptor（NMDAR）is a ligand-gated Ca²⁺channels which are closely related with central nervous system development,learning and memory.NMDA receptors are heteromeric which are composed of three subunits（NR1,NR2,NR3）.NR1 subunit is a functional subunit of NMDA receptors,which has a wide distribution from the cerebral cortex to the spinal cord in the mammalian central nervous system.After long-term ethanol exposure,NMDA receptors in the central nervous system increase the sensitivity to NMDA and the combination with NMDA.This is one of the main reasons of alcohol withdrawal symptoms（especially withdrawal of epileptic seizures）and the nervous excitement toxicity of the neurotoxicity of ethanol.Studies which have used autoradiographic method found that after long-term ethanol feeding,guinea pig cerebral cortical MK-801 binding was significantly higher than the control group.There may appear more expression of NR1 in cerebral cortex,hypothalamus and hippocampus in mouse.It indicated that the long-term ethanol exposure resulted in up regulation of NMDA receptor expression which can cause the excited state of NMDA receptors.We try to study the mechanism of nerve cell damage caused by chronic alcoholism on the basis of former research further.We intend to use MTT,sh RNA,RT-PCR,Western blot,the determination of intracellular free calcium ions,ethology experiment,immunofluorescence staining and other molecular biology techniques to investigate changes in SK-N-SH human neuroblastoma cells and C57BL/6 mouse after chronic ethanol exposure systematically to find out the relationship between neuronal apoptosis induced by chronic ethanol exposure and NMDA receptors,changes of intracellular[Ca²⁺]i.We will investigate the cause of chronic alcoholism neurobiological mechanisms of neuronal apoptosis and toxic dementia NMDA receptor mechanism of chronic ethanol-induced neuronal apoptosis to study the role of changes of intracellular calcium after chronic ethanol exposure.The project also intends to advance to an experimental cell and animal memantine observed to inhibit chronic ethanol exposure NMDAR activation and neuronal apoptosis induced to investigate the protective effects of memantine on chronic ethanol exposure caused by nerve damage and therapeutic value.Materials and methods:1.Grouping:Grouping in vitro experiments:According to the duration of ethanol treatment,SK-N-SH cells is divided into 24 h,48 h and 72 h groups,according to the ethanol concentration,SK-N-SH cells is divided into 0 m M,50 m M,100 m M,200 m M and 400 m M groups.0 m M,100 m M ethanol groups treated SK-N-SH cells for 48 h,according to whether memantine was added,SK-N-SH cells is divided into M（+）group and M（-）group,where concentration of memantine group was 4μM.According to whether the NR1 gene was transfected by sh RNA,SK-N-SH cells is divided into NR1-sh RNA（+）and NR1-sh RNA（-）group.Grouping in vivo experiments:mice were divided into two groups according to the concentration of ethanol that they were given to,control group and 20%ethanol group;mice were also divided into two groups according to whether they were given memantine,control group and memantine group.Experiment is divided into 4 groups of 10 mice.All treatment duration was 90days.2.MTT:MTS are colorimetric assays used to measure cell viability via non-specific redox enzyme activity.Cells（100μl,1×10⁵cells/ml）were seeded into a 96-well flat-bottomed plate（white for Titer-Glo）and incubated for 24 h at 37°C with 5%CO₂.The medium was replaced with Culture liquid containing increasing alcohol concentrations（0 m M,50 m M,100 m M,200 m M and 400 m M）（in quintuplicate）.The cells were incubated for 24 h,48 h and 72 h(for 48-and 72-hr incubations,the medium was replaced at 24 h and 48 h with fresh medium containing increasing alcohol concentrations（0 m M,50 m M,100 m M,200 m M and 400 m M）（in quintuplicate）.The cells were washed with PBS three times and replaced with fresh medium（100μl）.MTS（20μl）reagents were added to the wells,and the plate was incubated for 1 h protected from light.Absorbance（MTS）was recorded at 490 nm.3.Annexin-V/PI double-staining:SK-N-SH cells were seeded into 25 cm²tissue Culture flasks and incubated for 24 h at 37°C with 5%CO₂.The medium was replaced with Culture liquid containing increasing alcohol concentrations（0 m M,50 m M,100 m M,200 m M and 400 m M）（in quintuplicate）.The cells were incubated for 24 h,48 h and 72h（we added the amount of volatile ethanol to the culture medium every 24 h for 48 h and72-hr incubations）and were then collected by low speed centrifugation and washed with ice-cold PBS followed by recollection through centrifugation.After washing with PBS twice the cells were double stained with FITC-conjugated annexin V and PI for 15 min at20°C in a Ca²⁺-enriched binding buffer.They were immediately analyzed on the flow cytometer in their staining solution.Annexin V and PI emissions were detected in the FL-1（band pass 530 nm,band width 30 nm）and FL-2（band pass 585 nm,band width 42nm）channels.4.Detection of[Ca²⁺]i in SK-N-SH cells:SK-N-SH cells were seeded into 25 cm²tissue culture flasks and incubated for 24 h at 37°C with 5%CO₂.The medium was replaced with Culture liquid containing increasing concentrations of alcohol（0 m M,50 m M,100m M,200 m M and 400 m M）（in quintuplicate）.The cells were incubated for 24 h,48 h and 72 h（we added the amount of volatile ethanol to the culture medium every 24 hours for 48-and 72-hr incubations）.Preparation of cell suspensions,the test cells were washed twice with Hanks solution,trypsinized,centrifuged and resuspended containing Hanks solution within 20 g/L BSA,adjusting the cell concentration of 1×10⁶～1×10⁷cells/ml,the cells can be used for experiments while trypan blue exclusion test checking the survival rate was over 95%.SK-N-SK(1×10⁶～2×10⁶cells/ml were loaded with 3 m M fura 2 acetoxymethyl ester（fura 2/AM）in hanks at 37°C for 1h.The cells were then washed and resuspended in Hepes/Tyrode’s buffer in a stirred cuvette.The fura2/AM-loaded cells were stimulated with Dnp–HSA at 37°C.Then we used fluorescence spectrometry measure intracellular[Ca²⁺]i.5.Western Blotting:The cells or Brain tissue were washed with 1×phosphate-buffered saline（PBS）,and lysed in radioim Munoprecipitation assay（RIPA）buffer supplemented with protease and phosphatase inhibitor cocktail,and centrifuged at 14,000 rpm for 15min at 4°C.After protein concentration was determined by the bicinchoninic acid（BCA）protein assay,equal amounts of proteins were subjected to 10%SDS-PAGE,and the separated protein was transferred onto nitrocellulose membranes.The membranes were blocked for non-specific binding with 5%non-fat dry milk in Tris-buffered saline containing 0.05%Tween 20（TBS-T）for 1 h at room temperature and then probed with primary antibodies overnight at 4°C,followed by incubation with horse radish peroxidase（HRP）-conjugated immunoglobulin G（Ig G）for 1h at room temperature.Chemiluminescence was detected with Pierce ECL Western blotting substrateand visualized by Chemi Doc MP Imaging system.6.RNA Extraction and Real-Time RT-PCR:Total cellular RNA was extracted from cells using the RNeasy Plus Mini Kit from（Qiagen）.Quantitative realtime polymerase chain reaction（QPCR）was done using SYBR Green PCR Master Mix（Applied Biosystems）in a total volume of 20μl on a 7500 Real-Time PCR System（Applied Biosystems）:95°C for 30 s,40 cycles of 95°C for 5 s,60°C for 34 s,95°C for 15 s,60°C for 60 s,95°C for 15 s.Beta-actin was used as the reference gene.The relative levels of gene expression were represented asΔCt-Ct gene-Ct reference,and the fold change of gene expression was calculated by the 2^-ΔΔCtMethod.Experiments were repeated in triplicate.Double fluorescein may report gene analysis.7.Morris water maze:Hidden Platform Test:Test for water maze learning and memory ability is to obtain measurements in mice.Day 1 and day 2 were visible platform training experiment,day 3 to day 6 were Hidden platform experiment.We will stick mouse stone into the water at the same time tracking system affect locomotor activity recorded with a computer during the experiment in mice,including escape latency and pathway length.If the platform is not found by mice within 60 seconds,the record is 60 seconds.Probe test:Mouse may Climb the platform owing to touch platform accidentally when they were searching the hidden platform.We removed the platform at day 7 in order to eliminate the randomness.We put mice into water atthe first quadrant,and the mice search platform with 60 s in the pool.We observed frequency that mice across the position corresponding to the platform which were used as indicators that evaluate spatial cognition and memory in mice.8.Immunofluorescent staining of NR1 and NSE:After hydration of tissue sections,Sudan black staining and a closed hot fix of antigen,rabbit anti-mouse dropping NR1antibody（1:200）and goat anti-mouse NSE antibody（1:50）,4℃moisturizing box incubated overnight,then treated with donkey anti-rabbit/goat secondary antibody incubation 90 min,nuclei using DAPI staining.Observe under a fluorescence microscope at last.9.Immunofluorescent staining of TUNEL and NSE:After hydration of tissue sections,Sudan black staining,proteinase K treatment,and blocking,goat anti-mouse NSE antibody（1:50）,4℃moisturizing box incubated overnight,then treated with donkey anti-goat secondary antibody incubation 90 min,join TUNEL working solution,nuclei using DAPI staining.Observe under a fluorescence microscope at last.10.Statistical analysis:All statistical analyses were performed using SPSS 17.0.Means of Each group were compared using single factor analysis of variance（ANOVA）and perform S-N-K pairwise comparisons.T test was used to detectgray value of Western Blot results and the like.p<0.05 as statistically significant.P values<0.05 were considered statistically significant.Results:1.Chronic ethanol exposure reduce the cell viability of SK-N-SH cells:MTT results showed that compared with the control group,AU of the chronic ethanol exposure group was significantly reduced.It showed that chronic ethanol exposure can reduce cell viability of SK-N-SH cells and inhibit cell proliferation of SK-N-SH cells.Ethanol concentration of cells acting on the greater of SK-N-SH shows a more severe degree of inhibition of cell proliferation,the longer the time the ethanol effect on SK-N-SH cell proliferation is more severe.2.Chronic ethanol exposure increases the apoptosis rate of SK-N-SH cells:Seen from the results of flow cytometry,compared with the control group,apoptosis rate of chronic ethanol exposure group increased.It suggests that chronic ethanol exposure can increase the apoptosis rate of SK-N-SH cells.The greater concentration of ethanol effect is on the SK-N-SH cells,the higher apoptosis rate of SK-N-SH cells was.The longer the role of alcohol is in SK-N-SH cells,the higher apoptosis rate of SK-N-SH cells was.3.Chronic ethanol exposure increases[Ca²⁺]i in SK-N-SH cells:After chronic ethanol exposure,the average calcium concentration in SK-N-SH cells was increased.The greater concentration of ethanol effect is on the SK-N-SH cells,the higher average calcium concentration was in SK-N-SH cells.The longer the role of alcohol in SK-N-SH cells,the higher average calcium concentration was in SK-N-SH cells.4.Chronic ethanol exposure makes the protein expression of NR1 in SK-N-SH cells and brain tissue of mice increased:Immunofluorescence experiments in vivo results show that compared with the control group,NR1 staining intensity in brain tissue of ethanol group mice was significantly enhanced.Results of western blot showed that compared to the control group,relative expression levels of NR1 in brain tissue of ethanol group mice increased.Western blot results in vitro showed that compared with the control group,relative expression levels of NR1 increased in the experimental chronic ethanol exposure groups.The greater concentration of ethanol effect is on the SK-N-SH cells,the more NR1 protein were expressing in SK-N-SH cells.The longer the role of alcohol is in SK-N-SH cells,the more NR1 protein were expressing in SK-N-SH cells.RT-PCR results also show that compared with the control group,m RNA levels of NR1 ethanol group also increased significantly.5.Memantine has protective effect on apoptosis induced by chronic ethanol exposure:in vivo,compared to the control group,mice in the ethanol group showed they have longer escape latency and the number of crossing platform reduced which means the spatial reference and memory skills damage in water maze test.Compared with ethanol group,damage of the ethanol and memantine group was significantly smaller.Western Blot results in vivo experiments suggest that NR1 protein expression in the brain of mice after chronic ethanol exposure a substantial increase while NR1 protein in ethanol and memantine group increased less.We can see from immunofluorescent staining of TUNEL and NSE results,compared to the control group and ethanol（+）/memantine（+）group,there were significantly more NSE and TUNEL double staining cells in brain of the mice in ethanol group.In vitro,compared to memantine negative experimental group,cell proliferation of SK-N-SH cells treated with chronic ethanol exposure which was added memantine was increased.The average calcium concentration and apoptosis rate was significantly reduced.Activation fragments of caspase-3 protein expression was reduced,too.These have confirmed that memantine has a protective effect on ethanol-induced neuronal injury.This also suggested that apoptosis caused by chronic ethanol exposure in neurons correlated with NR1.6.Cell damage reduced after chronic ethanol exposure on NR1-sh RNA-transfected SK-N-SH cells:After applying si RNA down NR1 receptor gene expression,with respect to the cell negative group,cell proliferation of SK-N-SH cells of positive group was increased.The average calcium concentration and apoptosis rate was significantly reduced.Activation fragments of caspase-3 protein expression was reduced,too.These also confirmed that the apoptosis of ethanol exposure-induced in neurons correlated with NR1.Conclusion:1.Chronic ethanol exposure causes neuronal apoptosis,inhibition of neuronal cell viabilit.2.Damaging effects of chronic ethanol exposure on neuronal cells are related to Ethanol exposure concentration and exposure time.3.Chronic ethanol exposure which can increase the protein expression of NR1 in neurons increases intracellular calcium concentration in neurons by activation of NMDA receptor,and then lead to apoptosis in neurons.4.Memantine has a protective effect for nerve damage caused by chronic ethanol exposure.