Role Of Neuronal Nitric Oxide Synthase In Epilepsy Models And Its Toxicity Mechanism

Objective:Epilepsy is a chronic disease in which sudden abnormal discharges of neurons in the brain can cause transient brain dysfunction.According to the latest epidemiological data in China,the overall prevalence of epilepsy in China is 7.0 ‰,the annual incidence is 28.8 / 100,000,and the prevalence of active epilepsy that occurs within one year is 4.6 ‰.According to this estimate,there are about 9 million epilepsy patients in China,with that 5-6 million are patients with active epilepsy.At the same time,about 400,000 new epilepsy patients are added each year.In China,epilepsy has become the second most common neurological disease after headache.Nitric oxide(NO)is widely distributed in various tissues of the human body,especially neural tissue.It is a new type of biological messenger molecule and was selected as a star molecule by Science magazine in 1992.NO is a very unstable biological free radical with small molecules and a simple structure.It is a gas at room temperature,and slightly soluble in water and fat,which has a biological half-life of only 3 to 5 seconds,and can quickly diffuse through Biofilm.Nitric oxide synthase(NOS)has critical biological functions in the aspects of heart,cerebrovascular regulation,nerve and immune regulation.Therefore,it has received widespread attention.In the central nervous system,nitric oxide has a number of functions,such as regulating synaptic plasticity,sleep-wake cycles,and hormone secretion.The role of nitric oxide as a Janus molecule in the cell death or survival mechanism of brain cells is of particular interest.Nitric oxide regulates synaptic transmission and its level increases during epilepsy in animal models of epilepsy.However,the role of nitric oxide in the development and maintenance of epilepsy is controversial.However,in recent studies,it has been found that NO and NOS have different phenotypes and effects in epilepsy caused by different causes.In the past research,it has been proved that oxidative stress plays an important role in epilepsy.At the same time,oxidative stress is also closed related to inflammation in epilepsy,so it can inhibit oxidative stress and have an important impact on the study of epilepsy.In the inflamed state,the nitric oxide produced is significantly increased,and together with other Reactive oxygen species(ROS),it causes oxidative stress,so nitric oxide can be used as a sign of oxidative stress.In this study,Western Blot,cell culture,immunofluorescence,SOD,and LDH kits were used to reveal the role of nitric oxide synthase in epilepsy models and its toxicity mechanism,and to reveal that nitric oxide synthase can regulate cell survival in epilepsy models.The rate and the effects of oxidative stress lay a solid theoretical foundation and experimental basis for the pathogenesis of epilepsy.Methods: 1.The culture of cell line.(N2A,SH-SY5 Y cell lines)Culture the frozen stored cell seeds were resuscitated at 37 ° C for 30 minutes,and then pour them into a petri dish with culture medium,and observed cell morphology every 6-12 hours.Passage when the cells are full of the petri dish,pour out the cell fluid from the petri dish,and slowly pour into the pre-warmed serum-free medium and wash three times with PBS.Pour trypsin for 3-5 minutes into the plates(observe the state of the cells at any time).After the digestion,pour the serum medium into the plates to terminate the digestion and transfer it to a new culture dish.Take the grown cells and repeat the cell passaging process.After the digestion,type 2 mL of serum-containing culture solution,transfer the cell solution to a 5 mL EP tube and seal it.The cell-free supernatant was poured out from the ultraclean table,and 100 μL DMSO cell cryopreservation solution was taken,and 900 μL bovine serum and horse serum suspension was blown to evenly blow the cells until they were transferred to the cell cryopreservation tube to win the marked date and time,4 ℃ for 30 minutes,-20 ℃ for 2 hours,-80 ℃ gradient cryopreservation.2.Primary hippocampal neuron cultureWithin 2 days after birth,the newborn Wistar rat cubs were anesthetized and decapitated,and the brain was quickly removed and soaked in Hank’s solution stored at 4 ℃.Quickly dissect the hippocampus under a microscope ice bath,and wash the hippocampal tissues three times with refrigerated 5 mL cell culture medium(Hyclone DME / F-12).Digest the hippocampal tissues with 1-2 mL of Pectinase,gently pipette 1-2 times,remove the cell suspension,and digest the remaining tissue with pectinase again until digestion is complete.The digested cells were uniformly mixed with hippocampal neuron culture medium and counted for lamination.After 24 hours of adhesion,change the fluid every 2 days.After 7 days of culture for 9 days,the state of neurons was observed and the experiment was carried out.3.Western blot(Western Blot,WB)assay.The prearranged six-hole cell culture plate was taken out,and 150 μL cell lysate was added to each plate,and the cell scraper evenly scraped the culture plate until the cell was completely mixed with the lysate and placed for 30 minutes.The cell lysate was absorbed into a 12000 mL EP tube,centrifuged at 4 ℃ for 20 minutes,and the impurities such as cell membrane organelles were centrifuged to the bottom of the tube.The supernatant was transferred to a new 1.5 mL EP tube and the protein concentration was measured.Mix the sample with Loading Buffer 4:1 and heat it in a sample heater for 10 minutes to denaturalize the protein.After gel preparation,electrophoresis was carried out,75 V low voltage electrophoresis for 30 minutes and switching voltage 110 V electrophoresis for 1 hour.Marker electrophoresis to the bottom of the glass plate to stop electrophoresis,remove glue.Make a transmembrane sandwich and put it in an ice bag.turn to macromolecules for 300 mA for 6 hours and small molecules for 200 mA for 90 minutes.VGSC subunit rabbit first antibody and apoptosis related index first antibody were incubated with blocking membrane,TBST was washed three times for 10 minutes,sheep anti-rabbit second antibody was incubated,and TBST was washed three times for 10 minutes.1:1,ECL luminous solution was configured to emit light.4.CCK8(Cell Counting Kit 8)cytotoxicity test.The cell subculture process was repeated and the cell suspension was inoculated in 96-well plate with 5000-7000 cells per pore(note mixing).After adhering to the cell wall,the drug was administered according to the concentration gradient for 24 hours,and the final concentration volume was 100 μL.CCK-8 reagent was added at 1:10 and incubated in the cell incubator for 2 hours.the absorbance at 450 nm was measured by enzyme-linked immunosorbent assay(Elisa).If the absorbance was not up to standard,the absorbance was remeasured every 30 minutes.The survival rate of CCK8 cells was calculated by absorbance according to the formula of cell survival rate(%)= [A(added)-A(blank)] / [A(0 added)-A(blank)] × 100%.5.Tissue immunofluorescenceThe pretreated rats were anesthetized by intraperitoneal injection of chloral hydrate,and the rats were perfused with heart.First,infuse 15 ml of normal saline,and then infuse 15 ml of 10% paraformaldehyde until the tail of the mouse trembled and stiffened.The rats were then decapitated,and then peel off the rat brains,and then the hippocampus was partially peeled off and wash them with physiological saline.Then fix it with paraformaldehyde,using 30% sucrose to sink them to the bottom at 4 ° C overnight,and shaking it evenly on a shaker.The next day,place it on a frozen microtome,and place the brain slices on glass slides.Then drop blocking solution made of 3% BSA to block for 2 hours,and then blot it with a paper towel or filter paper.Drop the diluted primary antibody and put it in a wet box at 4 ° C overnight,wash it with PBST for 10 minutes three times the next day.Then evenly drop the fluorescent secondary antibody and DAPI staining solution,protecting it from light for two hours,then wash with PBST for 10 minutes three minutes.Take out the treated cell slides,cover the slides with glycerol,and photograph the expression of NOS and nuclei by fluorescence microscopy.6.Immunofluorescence.When the cultured cells were cultured,the cultured neurons were taken from each dish by(WBS),fixed with 4% paraformaldehyde for 30 minutes,and cleaned with PBS for 10 minutes and 3 times.The 3%BSA sealant was sealed for 30 minutes,and the primary anti-NeuN diluent(abcam)50 μL was applied in a 4 ℃ wet box for one night.The next day,PBS was washed three times in 10 minutes,and 50 μL of FITC fluorescent antibody(Zhongshan Jinqiao)was diluted 100 times for two hours.After PBS cleaning for 10 minutes and three times,DAPI solution was stained for 10 minutes,and the cleaning steps were repeated again.the treated cell slides were taken out,sealed with glycerol,and the expression of NeuN and nucleus was photographed by fluorescence microscope.7.Pretreatment of cell samples.Digest the cells with trypsin,or scrape the cells with a cell scraper,centrifuge the culture solution at 1000 rpm at room temperature for 10 minutes,and discard the supernatant to retain the cell pellet.Add 0.5-1ml PBS(Isotonicity)to the cell pellet,gently invert and mix.Centrifuge the culture solution at 1000 rpm at room temperature for 10 minutes.Discard the supernatant and leave the cell pellet.Times.Then use an ultrasonic cell disruptor,300 W power,each ultrasound for 3 to 5 seconds,with an interval of 30 seconds,repeat 4 to 5 times to obtain a suspension.After that,make the ratio according to the instructions.Mix well,leave it at room temperature for 3 minutes,440 nm,adjust the light diameter of 1cm with double distilled water,and measure the absorbance of each tube.Definition and calculation formula of lactate dehydrogenase in cells: LDH activity =(measured OD value-control OD value)/(standard OD value-blank OD value)* standard concentration / protein concentration of the test sample Definition and calculation formula of lactate dehydrogenase in cells: SOD activity in homogenate =(control absorbance-measurement tube absorbance)/ control tube absorbance / 50% * total reaction volume / sampling volume / protein content in the tissue8.Statistical analysis.SPSS software 22.0 was used to evaluate the significant difference in the data.All the results were expressed as mean ± SEM.Significance of differences was evaluated using ANOVA followed by Tukey’s post test and Student’s t-test.P<0.05 was considered statistically significant.Results:1.nNOS expression and localization in hippocampus of spontaneous epilepsy ratsWe used Western Blot to compare hippocampal proteins in spontaneous epilepsy rats and Wister rats,and detected up-expression of nNOS protein.Next,we performed fluorescence localization of the nNOS protein in adult rats(1 year)and found that NOS was concentrated in the CA1 and CA3 regions.In the young rats(4 months),we issued the CA1 and CA2 regions.The expression level of NOS is more in the same time,and the expression of NOS is also significantly increased in the DG region.2.Establishment of hippocampal neuron epilepsy model and detection of nNOS expressionWe used pilocarpine to establish an epilepsy model in vitro,and we found that the expression of nNOS protein in the epilepsy model group was significantly up-regulated.We performed the same pilocarpine culture on N2 A cells and found similar results.Using the WST-8 test method,we also used CCK8 to test the cell survival rate of N2 A cells.After 3 hours of pilocarpine treatment 21 hours,we found that N2 A cells had apparently died.We also performed immunofluorescence on the cells and observed the localization and expression of nNOS in hippocampal neurons and N2 A cells.It was found that the expression of nNOS in cellular epilepsy models increased significantly.3.Effect of NOS inhibitor on nNOS expression in epilepsy cell modelWe used a NOS inhibitor,7-Nitroindazole(7-NI).We used CCK8 to detect the cell viability with 7-NI concentration gradient on N2 A and SH-SY5 Y cells through WST-8 method,and 7-NI was administered at a concentration gradient of 0.01 μM,0.1 μM,1 μM,10 μM,100 μM,1000 μM for 24 hours.After performing concentration-effect curve determination,we found that 7-NI was most suitable at a concentration of 100 μM.We used Western Blot to test cell proteins and found that 7-NI can effectively reduce the expression of NOS in epileptic cell models.At the same time,we also found that 7-NI did not reduce the expression of NOS protein in the control group.4.Effect of NOS inhibitors on cell survival in epilepsy cell modelsCCK8 was used to observe the effect of 7-NI on cell death caused by pilocarpine epilepsy cell model.In the results obtained,7-NI was found to be effective in reducing cell death due to epilepsy.5.NOS inhibitors can reduce the effects of oxidative stress in epilepsy cell modelsWe used SOD and LDH kits to test N2 A cells in the Control group,the epilepsy model group,the epilepsy model group,and the control group,and found that nitric oxide synthase inhibitors can indeed reduce the oxidative stress damage.Conclusion: Compared to Wister rats,we found that nNOS protein expression up-regulated in the hippocampus of spontaneous epilepsy rats.We also conducted immunofluorescence experiments and found that as the rats grew,nNOS decreased significantly in the DG region of the hippocampus,and nNOS in the CA1 and CA2 regions decreased with age.We used hippocampal neurons and N2 A cells to establish a pilocarpine epilepsy cell model.In the cell model,we found that the expression of nNOS was up-regulated.At the same time,we also performed the WST-8 experiment and found that the cell survival rate of the epilepsy model group was significantly reduced.By immunofluorescence technology,nNOS protein also reduced in the model group.To the further study the mechanism of nNOS in epilepsy,we used nitric oxide synthase inhibitors,7-NI and Western Blot technology.We found that 7-NI could effectively reduce the expression of nNOS protein in epilepsy model group.Through WST-8 experiments on N2 A cells,we found that 7-NI can also significantly reduce cell death caused by epilepsy.We found that inhibition of nNOS in the epilepsy cell model group reduced oxidative stress in the epilepsy model.Through our research,we have shown that the expression of nNOS is up-regulated in in vivo and in vitro models of epilepsy,indicating that nNOS plays an important role in epilepsy.By inhibiting NOS,we found that inhibiting NOS can reduce cell death caused by epilepsy and oxidative stress damage caused by epilepsy.This study lays a solid theoretical foundation and experimental basis for exploring the pathogenesis of ion channel-related neurological diseases such as epilepsy and the interaction between nitric oxide synthase and oxidative stress.