| IntroductionEpilepsy is a complex neurosystemic disease that affects more than 70 million patients worldwide?nearly 3 million people in the United States?.Epilepsy has a complex pathogenesis,and its mechanism is not clear,but it usually involves the imbalance between excitatory and inhibitory neurotransmission in multiple brain structures,including voltage-gated sodium channels?Voltage-gated sodium channel,?.The expression of many ion channels,including VGSC,Changes in localization and function occur in the period after the initial acute attack and may lead to epilepsy.VGSC is one of the common and important Na+channel types,which is composed of one?subunit and multiple?subunits??1-?4?.?subunit has 10 different subtypes?NaV1.1-1.9,Na X?.NaV1.1,Na V1.2,NaV1.3 and Na V1.6 subtypes are widely distributed in the central nervous system.VGSC is the action potential of priming neurons,and VGSC is also the main therapeutic target of antiepileptic drugs.Studies have shown that the expression and function of VGSC in epileptic brain tissue and epileptic animal model have changed.Ca2+/calmodulin dependent protein kinase CaMK??Calcium/Calmodulin-dependent protein kinase?,CaMKII?is a major member of Ca2+/calmodulin?Calmodulin,CaM?regulatory protein family.It plays an important biological role in the pathophysiological process of cells.Our previous results showed that the expression of CaMK?protein was down-regulated and the expression of NaV1.1 and NaV1.2protein was up-regulated in hippocampus of hereditary epileptic rats.The expression of CaMK?protein was down-regulated and the expression of NaV1.2 protein was up-regulated in the hippocampus of TRM treated with KN93 in lateral ventricle,while the expression of NaV1.2 protein was unchanged.These results suggest that the change of VGSC expression in CaM may be related to the pathogenesis or secondary manifestations of epilepsy.So far,the toxic mechanism of KN93 on epileptic cells is not clear.P-jnk protein exists in p-CaMK?and p-ERK pathway,phosphorylated JNK protein.C-Jun N-terminal kinase?JNKs signal?,initially identified as kinase binding and phosphorylation of the upper serine of c-Jun and its transcriptional activation domain of-63 and Ser-73.They belong to the mitogen-activated protein kinase family and control stress responses caused by cytokines,ultraviolet irradiation,heat shock and osmotic shock,and play a role in T cell differentiation and apoptosis.Studies have shown that the inflammatory response of mammals and insects can be activated by two MAP kinases MKK4 and MKK7,and JNK can be inactivated by Ser/Thr and Tyr protein phosphatase.Therefore,this study revealed the toxic effect of KN93 on epileptic cells and its mechanism by cell culture and molecular biological methods,and clarified the interaction between KN93 and p-JNK pathway,in order to elucidate the pathogenesis of epilepsy.2.Materials and Methods2.1.The culture of PC12 cell line.The frozen cell seeds were revived at 37?for 30 minutes and poured into a petri dish with culture medium.the morphology of the cells was observed every 6 hours.When the cells were filled with culture dishes,the cells were subcultured,the cell fluid in the culture dishes was poured out,and the cells were slowly poured into the preheated serum-free culture medium for cleaning three times.The cells were digested with trypsin for 3 minutes?observing the state of the cells at any time?.After complete digestion,the trypsin was poured into the serum culture medium to terminate the digestion and spread to a new petri dish.The cells were digested completely and then injected into 2 mL serum culture medium.the cell fluid was transferred to 5 mL EP tube and sealed,and the centrifuge was rotated for 5 minutes to make the cell stick to the bottom of the tube.The cell-free supernatant was poured out from the ultraclean table,and 100?L DMSO cell cryopreservation solution was taken,and 900?L 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.2.2.Primary hippocampal neuron culture.Within 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?.The hippocampus was dissected rapidly under microscope ice bath,and the hippocampal tissue was cleaned three times with refrigerated 5 mL cell culture medium?Hyclone DME/F-12?.The hippocampal tissue was digested with 2 mL Pectinase,gently blown twice,and the cell suspension was taken out to digest the remaining tissue with Pectinase again until the digestion was complete.After digestion,the cells were evenly mixed with the culture medium of hippocampal neurons and counted for lamination.Half the volume of fluid was changed every other day after 24 hours of adhesion.After 7 days of culture for 9 days,the state of neurons was observed and the experiment was carried out.2.3.Western blot?Western Blot,WB?assay.The prearranged six-hole cell culture plate was taken out,and 150?L 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 90minutes.VGSC subunit rabbit first antibody and apoptosis related index first antibody were incubated with blocking membrane,TBST was washed three times for 10minutes,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.2.4.CCK8?Cell Counting Kit 8?cytotoxicity test.The cell subculture process was repeated and the cell suspension was inoculated in96-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 of 0,0.1,0.3,1,3,10,30 and 100 for 24 hours,and the final concentration volume was 100?L.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 30minutes.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%.2.5.Flow cytometry.Under the condition of ice bath,the cells were digested with trypsin,and the cells were precipitated by centrifugation after termination of digestion.The 4 mL Binding buffer was mixed in 36 mL deionized water and the Binding buffer reagent was diluted 10 times.The cells were washed with 900?L PBS,the suspension was transferred into a 1.5 mL EP tube,and centrifuged for 5 minutes at 1000 turn.After centrifugation,the supernatant was removed and gently blown with 1000?L Binding buffer and centrifuged for 5 minutes.the above process was repeated twice.After centrifugation,the supernatant was removed by EP tube,and the cell suspension treated with 1000?L was retained in each tube.In addition to PI single staining and blank control group,5?L of FITC?green?was added to each tube,and the staining was avoided for 10 minutes.Add 5?L PI(red to each tube,mix well and test internally 1 hour after dyeing.Add 300?L PBS and the sample in the flow tubule,label the sample,blow well before measurement.Staining and data statistics:late apoptosis of PI,early apoptosis of FITC.Q4 early Q2 late+necrotic Q1 injury is generally dominated by q2+q4.2.6.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?L was applied in a 4?wet box for one night.The next day,PBS was washed three times in 10 minutes,and 50?L 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 10minutes,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.2.7.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.3.Results3.1.Effect of long-term KN93 administration on cell survival rate.KN93 is a competitive inhibitor of Ca2+/Calmodulin-dependent protein kinase II?CaMKII?inhibitors.Calmodulin?Calmodulin,CaM?can regulate voltage-gated pathway and affect the survival rate of nerve cells.Using CCK8 experimental method,the concentration curve of KN93 was first measured and the concentration was determined when the half lethal dose?LD50?was reached.The survival rate of cells decreased with the increase of KN93 concentration,and reached the half lethal dose?LD50?when the concentration of KN93 reached 25?mol?n?6?.On this basis,we reconfirmed the results at the protein level and cell morphological expression level.After long-term administration of KN93,the expression of cell staining in PC12decreased by Ni's staining experiment.the expression of DAPI staining nucleus and NeuN staining neurons in primary cultured hippocampal neurons were decreased by immunofluorescence staining.From the point of view of cell morphology,it is proved that KN93 can lead to apoptosis or death.On this basis,several apoptosis-related pathway proteins were verified by Western Blot assay.as shown in figure 3,the expression of Caspase-3 protein was increased and other downstream proteins related to apoptosis pathway?BCL-2,Bax?were found to be increased.Cytochrome-c also shows a downward or upward trend,It was proved that long-term administration of KN93 activated apoptosis pathway and induced apoptosis?n?6?.According to the expression of protein level,the degree of apoptosis and the percentage of apoptosis?n?6?were observed by flow cytometry in the experimental group and the control group.3.2.Effect of long-term KN93 administration on voltage-gated sodium channels.In order to further understand the effect of long-term KN93 administration on apoptosis and nervous system,we studied the effect of long-term on four subtypes of voltage-gated sodium channel?NaV1.1,NaV1.2,NaV1.3,Na V1.6?.Four subtypes of voltage-gated sodium channel changed at the protein level after long-term KN93administration.Compared with the control group,the two subtypes of NaV1.1,NaV1.2were up-regulated,the subtypes of Na V1.6 were down-regulated,and NaV1.3 changed slightly but had no statistical significance.This phenomenon indicates that calmodulin dependent protein kinase inhibitor CaMK?antagonist KN93 induces apoptosis by regulating voltage gated sodium channel subtype,inhibiting or activating voltage type sodium channel opening under long term administration?n=6?.3.Effect of KN93 on VGSC 24 hours after administration.In order to further understand the apoptosis induced by 24-hour administration of KN93 and its effect on the nervous system,we studied its effect on four subtypes of VGSC NaV1.1,NaV1.2,NaV1.3,NaV1.6.24 hours after administration of four subtypes of voltage gated VGSC KN93,we found changes in expression at the protein level.Compared with the control group,Na V1.1 and NaV1.2 subtypes were up-regulated,Na V1.6 subtypes were down-regulated,and NaV1.3 had weak changes,but there was no statistical significance.This phenomenon indicates that KN93,an inhibitor of calmodulin-dependent protein kinase II,changes the expression of VGSC24 hours after administration.4.p-JNK signaling pathway participates in apoptosis induced by CaMKII inhibitor KN93.KN93 could act on p-CaMKII pathway 24 hours after administration.P-JNK was the downstream protein of p-CaMKII pathway.P-JNK inhibitors were obtained by CCK8assay.Effects of p-JNK inhibitor and KN93 on cell survival rate at different concentrations and time.The effect of p-JNK inhibitor on apoptosis was observed by immunofluorescence technique.Western Blot assay showed that the expression of apoptosis representative protein Caspase-3 decreased and other upstream and downstream antibody proteins related to apoptosis channel also changed,and p-JNK inhibitor?SP600125?could inhibit the increase of p-JNK expression induced by KN93.5.p-JNK signaling pathway participates in abnormal expression of VGSC induced by CaMKII inhibitor KN93.We used Western Blot assay to study the effects of CaMKII inhibitor KN93 on four subtypes of VGSC,NaV1.1,Na V1.2,NaV1.3 and NaV1.6.Compared with 24 hours after KN93 administration,p-JNK inhibitor could decrease the expression of Na V1.1and Na V1.2 and increase the expression of NaV1.6.DiscussionIt is reported that the regulatory effect of KN93 on voltage-gated sodium channel?VGSC?is related to its effect on calmodulin kinase,and has different effects on different voltage-gated sodium channel subunits.Based on previous studies,we further explored the mechanism of KN93?calmodulin kinase inhibitor?regulating the opening of voltage-gated sodium channels and inducing apoptosis by using different voltage-gated sodium channel subunit antibodies.First of all,we used CCK8 to test the cell survival rate at different concentrations.Then immunofluorescence Ni staining and Western Blot immunoblotting were used to observe the regulation and effect of administration on apoptosis at the morphological level and protein expression level,respectively.It was found that long-term administration of KN93could induce apoptosis,while different voltage-gated sodium channel subtypes showed different expressions of KN93 after long-term administration.In order to further explore the mechanism of long-term administration of KN93,and p-JNK inhibitors were used to verify the pathway mechanism of calmodulin kinase.The data showed that p-JNK inhibitors could reverse the apoptosis induced by long-term administration of KN93 and had different effects on different subtypes of voltage-gated sodium channels.These results suggest that p-JNK can act on different sodium channel subtypes to control the opening of voltage-gated sodium channels and thus control apoptosis.In addition,we also found that it administration could not change the expression of p-JNK pathway.It has been proved that long-term administration of KN93 affects the opening of sodium channels.These results confirm that the effect of calmodulin kinase on the opening of sodium channels comes from the p-JNK pathway on different subtypes.In conclusion,we are concerned about the effect of long-term high concentration administration of KN93 on voltage-gated sodium channels and the reversal and mechanism of apoptosis induced by p-JNK inhibitors.In the next step,we will study the effects of different channel subunits and their mutated peptides on voltage-gated sodium channels.In conclusion,the results of this study will provide new insights into the molecular mechanism of KN93 regulating voltage-gated sodium channels,and provide theoretical basis for finding new targets for antiepileptic drugs. |
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