| Objective:Epilepsy,as a complex neurological disease,is caused by abnormal discharge of neurons in the brain and produces nerve excitability.It has a high incidence,many types of seizures,and the pathogenesis is more complicated.At present,the research on its pathogenesis mainly focuses on the abnormal aspects of ion channels,glial cells and neurotransmitters,and the specific mechanism is not clear.Calcium/Calmodulin-dependent protein kinase II(CaMKII)belongs to a multifunctional serine/threonine protein kinase.Its total enzyme consists of four subunits,which are ?,?,?,and ? form more than 30 subtypes.Therefore,CaMKII has a wide range of functions and plays a non-negligible role in the development of various diseases in the body.KN93 is a CaMKII-specific inhibitor that penetrates cells and inhibits the phosphorylation activity of CaMKII.The study found that the expression of phosphorylated CaMKII protein was significantly reduced in hippocampal tissue of hereditary epileptic rats(Tremor,TRM)and tremor rats.But so far,the effect of CaMKII inhibitor KN93 on nerve cells and its mechanism are not clear.Transcription factor NF-E2 related factor 2(Nrf2)is one of the transcription factors of the cap and collar(CNC)family,which also includes Nrf1,Nrf3,p45,p53,Bach1 and Bach2,and Nrf2 is a family The most active transcriptional regulator among members.Nrf2 is a protein with a relative molecular weight of 66,000 encoded by a 2.2kb base pair.It has six red blood cell-derived CNC homologous protein(ECH)domain names,Neh1 to Neh6.Nrf2 is reported to play a wide range of roles in various organs and tissues,including the heart,kidneys,liver,and brain.Most importantly,Nrf2 plays an important role in neuroprotection.Previous studies have shown that Nrf2 is regulated by several upstream factors,such as substances represented by sulforaphane(SFN),dimethyl fumarate(DMF),and synthetic triterpenoids.At the same time,Nrf2 can also regulate many protective proteins downstream of it,common are heme oxygenase-1(HO-1),quinone oxidoreductase-1(NQO1),NAD(P)H and sulfur Oxycyclines(Trxs)These second-stage antioxidant enzymes bind to drug metabolites or endotoxins.These results indicate that Nrf2 mediates complex signaling pathways involved in neuroprotection.Nrf2 has been shown to protect several types of cells from acute and chronic oxidative stress injury.Oxidative stress is a ubiquitous biological process that participates in the physiological and pathological states of many organs and tissues.Tasaki reported that the toxic substances produced by chemically generated oxidative stress reactions may promote their proliferation and pre-tumor lesions to tumor processes,and to a greater extent in Nrf2-deficient mice.In addition,chronic oxidative stress has proven to be a major mediator of chronic kidney disease(CKD),and the restoration of Nrf2 activity may reduce glomerulosclerosis,interstitial fibrosis,and inflammation in CKD rats.Recently,Hyeon studied the differentiation of Nrf2 in osteoclasts,and then participated in the production of reactive oxygen species(ROS),showing that Nrf2 may inhibit the receptor activator of nuclear factor ?B ligand(RANKL)-by controlling the disruption of oxidative responses in cells Expression of genes that induce osteoclast differentiation.In addition,evidence suggests that oxidative stress helps improve stroke,and activation of Nrf2 can improve oxidative stress-induced damage to brain endothelial cells,prevent tight junctions,reduce protein,and protect the blood-brain barrier(BBB).The relationship between the Nrf2 pathway and the effect of CaMKII inhibitor KN93 on nerve cells is currently unknown.Based on this,this study revealed the effects of KN93 on nerve cells and their excitability changes through cell culture,patch clamp technology,and a series of molecular biological methods,and clarified the toxic effects of KN93 on nerve cells and KN93-induced toxic effects The relationship with the Nrf2 pathway further clarifies the mechanism of KN93-induced toxicity and provides a theoretical basis and experimental basis for the pathogenesis of epilepsy.Methods: 1.Primary hippocampal neuron culture.Wistar rats that were 18 days pregnant were subjected to surgery to remove fetal rats,anaesthetize them,and remove brain and brain tissues,and immerse them in Hank's solution at 4 degrees.Hippocampal tissue was dissected under a dissecting microscope,and the tissue was washed two to three times with cell culture medium(Hyclone DME/F-12).According to the number of tissues,take an appropriate amount of trypsin for digestion,perform 30 minutes at 37 ?,and shake it thoroughly every 10 minutes.Finally,add the same amount of the planting solution(20% serum culture solution)at 1: 1 to terminate the digestion.Centrifuge at 1000 rmp for 5 min to obtain a cell pellet,add neuronal culture solution,and plant the cells in a culture plate after counting is complete.After most cells adhered,the medium was changed every 24 hours with a half-feed.Follow-up experiments were performed after 7-9 days of culture to observe the neuron status.2.Neuronal cell line culture The frozen cells were resuscitated at 37 ? for 30 minutes,and the cell culture solution was added.Passage when the cell density reaches 80-90%,discard the original cell culture solution,slowly add the rewarmed medium and wash it two or three times to remove necrotic cells and their metabolites.Add trypsin to digest until the cells fall off the wall,add serum culture medium to end the digestion process,and passaging.3.CCK8(Cell Counting Kit 8)cytotoxicity test The digested cell suspension was counted,and then planted in a 96-well plate with about 3000-5000 cells per well and mixed uniformly.After the cells are cultured to maturity,they are administered at a KN-93 concentration gradient of 0,2,5,20,100,500,1000 ?M for 12 or 24 hours.CCK-8 reagent(100 ?L/mL)was added,and the incubation was continued at 37 ? for 2 hours.The absorbance value of the sample at 450 nm was measured with a microplate reader,and the cell survival rate of each experimental group was calculated by the absorbance value and formula.4.Immunofluorescence When the neurons were cultured,the cells were planted in cell slides(WBS).After maturation,the cells were fixed with 4% paraformaldehyde for 30 min.After the fixation,the surface residues were washed away with PBS.Block again with 3% BSA.After 30 min,take 50 ?L of NeuN Primary Antibody Diluent(Abcam)and drop evenly on the cell slides.Incubate overnight at 4 ? in a wet box.After the incubation of the primary antibody,wash the residue with PBS,add FITC fluorescent secondary antibody dilution solution(Nakasugi Golden Bridge)and incubate for 1.5 hours.Repeat the above washing step again,stain the nucleus with DAPI stock solution for 10 min,remove the cell climbing slide,mount with a mounting plate,and photograph the location and expression of NeuN and cell nucleus in neurons under a confocal microscope.5.Western blot(WB)Remove the cell sample from the CO2 incubator,remove the culture medium,and add the cell lysate(RAPI: PMSF = 1000: 1)prepared in advance to the six-well plate,150 ?L per well,using a cell scraper to make most The cells were detached from the inner wall of the culture plate,lysed at 4 ? for 30 minutes,the sample was added to a pointed EP tube,and centrifuged at 12,000 rpm for 20 minutes in a cryogenic centrifuge,and the resulting supernatant was subjected to protein concentration measurement.Add a quarter of the sample volume to 5 × Loading Buffer and place it on a dry thermostat at 100 ? for 10 min to denature the protein.Configure acrylamide gel for sample loading,electrophoresis,and transfer.After the wet transfer,the PVDF membrane was immersed in 5% skim milk,blocked at room temperature with shaking for 30 minutes,and the surface residue was washed with the membrane washing solution TBST.The diluted primary antibody solution was added,and the primary antibody was incubated at 4 ? overnight.After the end,the excess antibody is washed away with TBST solution,and the secondary antibody corresponding to the primary antibody is incubated.Usually,it is incubated at room temperature for 2 hours,and the excess antibody is washed away again with TBST solution.The ECL luminescent liquid(Biorad)was prepared,that is,the A liquid and the B liquid were mixed 1: 1,and evenly dropped on the PVDF film,and the result photos were taken by the imaging system.6.ELISA kit Take out the six-well cell culture plate that was planted in advance,and pipette the supernatant to prepare a sample.Dilute the standard according to the instructions,then add the blank control and sample to the microplate,incubate at 37 ? for 30 minutes,discard the sample,wash it five times with a wash solution configured in advance,and pat dry.Add 50 ?L of enzyme-labeled reagent to each well,except for the blank.Incubate and wash.Add 50 ?L of developer A50 ?L + 50 ?L of developer B.Avoid light for 10 min at 37 ?.Then add 50 ?L of stop solution to each well to stop the reaction.During the measurement,the blank hole was adjusted to zero,and each empty absorbance(OD value)was measured at a wavelength of 450 nm.7.Patch clamp technology Action potential studies were performed using the whole-cell patch-clamp technique in the current clamp mode.In ramp mode,the action potential is induced by injecting a 1-s depolarized current with a maximum current of 200 pA.Membrane potential is clamped at-70 m V for evoked action potential excitation measurement.Records are low-pass Bessel filtered at 5 kHz and digitized at 50 kHz.For voltage clamp recording,rat hippocampal neurons(DIV 15-21)cultured on coverslips were ruptured using a Multiclamp 700 B amplifier and pClamp 10.2 software(Molecular Devices)and maintained at-70 mV at room temperature.Use a P-97 extractor(Sutter Instrument)to pull out the recording pipette(1.5 ? 2.5M?)according to a four-step procedure,and perform fire polishing with the MF-900 micro forge(Narishige).All experiments were performed at room temperature(22 ± 2 ?).8.Statistical analysis.The PVDF film obtained in the Western Blot immunoblot experiment was taken with an imaging technique,and it was converted into a gray value by Image J and standardized.Use Prism 6 software to draw statistical graphs and annotate the multiple comparison results of the experimental groups.The statistical methods are mainly Student's t-test and analysis of variance.The extracted data are mean + standard deviation.The results show that P <0.05 is statistically significant,and P <0.01 is significant.Results: 1.KN93 administration has toxic effect on nerve cells Using the CCK8 kit,KN93 was administered at a concentration gradient of 0,2,5,20,100,500,1000 ?mol for 12 or 24 hours to determine the concentration and cell survival rate.With the increase of KN93 concentration,the cell survival rate continued to decline,and from the KN93 concentration of 5 ?mol,it can be seen that there was a statistical difference in the decrease in cell survival rate.It was found by immunofluorescence staining that when 5 ?mol of KN93 was administered,NeuN stained positive cells in primary hippocampal neurons were significantly reduced.Therefore,KN93 administration can have a toxic effect on cells.2.Effect of KN93 administration on action potentials and nerve excitability of primary hippocampal neurons Using the whole-cell patch-clamp technique to detect the primary hippocampal neuron control group,KN93 concentration was 5 ?mol for 1 h,KN93 concentration was 5 ?mol for 24 h,and KN92 concentration was 5 ?mol for 24 h.The excitability of the cells given 5 ?mol for 24 h increased and was statistically significant.3.Expression of inflammation-related factors 12 hours after KN93 administration In order to further understand the toxic effect of 12 hours of KN93 administration on nerve cells,we tested the protein expressions of inflammation-related factors NF-?B,IL-6,TNF-?,and IL-1? in each experimental group.In nerve cells treated with KN93 for 12 hours,we found that its inflammation-related expression was significantly increased.This indicates that the neurotoxicity induced by KN93 may be related to the inflammatory response.4.Effects of 12 hours after KN93 administration on Nrf2 expression Based on this,verification by Western Blot experiments revealed that Nrf2 protein expression was increased.This result was consistent in hippocampal neurons,N2 a,and SH-SY5 Y cells,proving that the Nrf2 signaling pathway(n = 6).5.Effects of ML385 administration on the expression of Nrf2 and inflammation-related proteins The expressions of inflammation-related proteins in KN93 and ML385 treatment groups were down-regulated and statistically significant.The negative control group,that is,the ML385 administration group alone,had no change compared with the control group.This phenomenon indicates that the calmodulin-dependent protein kinase II inhibitor KN93 changes the expression of inflammation-related proteins 12 hours after administration,and the Nrf2 inhibitor ML385 can reverse this change.Conclusion: First,we tested the survival rate of cells at different concentrations of KN93 using the CCK8 kit,then performed fluorescent NeuN staining and whole-cell patch clamp to reveal the effect of KN93 on neural cells.Finally,from the protein expression level,Western Blot immunoblot was used.And ELISA kit to detect the expression of related proteins.The results show that CaMKII inhibitor KN93 induces neuron cells to up-regulate the expression of inflammation-related indicators 12 hours after administration.In order to further explore the mechanism of action 12 hours after KN93 administration,Nrf2 and its inhibitor ML385 were introduced to verify the mechanism of toxic action of KN93 on nerve cells,and it was found that KN93 caused excitability changes in nerve cells.The data show that Nrf2 inhibitor ML385 has a reversal effect on the cellular inflammatory response induced by KN93 for 12 hours,suggesting that ML385 can regulate the neurotoxicity of KN93.Therefore,we pay attention to the relationship between KN93 administration and Nrf2 for 12 hours and the reversal and action mechanism of Nrf2 inhibitor ML385 on its inflammatory response in neurons.In the next work,we will further explore the effect of co-administration of Nrf2 inhibitor ML385 and KN93 on the phosphorylation activity of CaMKII protein.In summary,this study revealed that KN93's toxic effect on nerve cells is related to inflammatory response.The involvement of Nrf2 pathway provides theoretical and experimental evidence for the study of epilepsy pathogenesis and the search for new targets for antiepileptic drugs. |
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