| Objective:AKT plays an important role in cell proliferation,migration and cell survival.NF-?B is an important downstream target protein of AKT.Studies have confirmed that AKT/NF-?B signaling pathway is closely related to epilepsy.MK2206 is an AKT-specific allosteric inhibitor that specifically inhibits AKT phosphorylation,thereby inhibiting AKT activation.MK2206 can effectively inhibit the proliferation of various malignant tumor cells,promote cell apoptosis,and exhibit good anti-tumor activity.Necroptosis(Nec)is a novel cell death pattern discovered in recent years and regulated by a special death signaling pathway.Receptor-interacting proteins(RIP)1 and 3 are very important regulatory proteins in the necroptosis signaling pathway.Studies have shown that necroptosis occurs in a variety of injury-related diseases such as myocardial ischemia and cerebral ischemia.According to the above research background,the research of this thesis is as follows:1.Pretreatment of C57BL/6 male rats with AKT-specific allosteric inhibitor MK2206,lithium-pilocarpine(Li-Pilo)induced continuous epileptiform high-frequency bursts(continues epileptiform high-frequency bursts,SEs),explored the effect of MK2206 on AKT/NF-?B signaling pathway in a SEs mouse model.2 Establishing Spontaneous recurrent Epileptiform discharges(SREDs)in primary cultured hippocampal neurons induced by magnesium-free external fluid,and pretreatment with MK2206 to further explore the influences of AKT/NF-?B signaling pathway by MK2206 in rat SREDs model.3 The SREDs model pretreated by MK2206,the mRNA expression level of necroptosis signaling pathway-related protein was detected,and the necroptosis mechanism of epileptic hippocampal neurons was explored.MethodsPart ? Effect of AKT allosteric inhibitor MK2206 on seizures and AKT/NF-?B signaling pathway(1)Subjects and grouping:Healthy C57BL/6 male rats were randomly divided into experimental pilo90 group,control pilo90 group,and control pilo30 group.MK220650mg/kg was injected intraperitoneally 3 times/week for 2 weeks;SEs(pilo90mg/kg)was induced by intraperitoneal injection of Li-Pilo in experimental pilo90 group.The control pilo90 group and the control pilo30 group were injected intraperitoneally with 1%DMSO 0.1ml/kg 3 times/week for 2 weeks.The SEs were induced by intraperitoneal injection of Li-Pilo(control pilo90 group and control pilo30 group,pilo),the doses were 90 mg/kg and 30 mg/kg respectively.The Racine score of the seizure episode in mice reached grade IV and above and the SE lasted for 50 minutes,which was considered successful model.(2)Behavioral comparisons were performed for each group of mice(evaluation indicators included eclampsia rate,latency,SE duration,duration of a single seizure,Racine score at each time point).(3)Western blotting was used to detect the dynamic changes of AKT,p-Thr308-AKT,p-Ser473-AKT,p-IKKotp,IKK?? NF-?B(cytoplasm),p-NF-KB(cytoplasm),NF-?B(nucleus),p-NF-?B(nucleus)protein expression in hippocampus of mice,and to explore the relationship between AkT/NF-KB signaling pathway and epilepsy.(4)HE staining and NISSLE staining were used to observe the pathological changes of hippocampus in each group.Part ?:The effect of AKT allosteric inhibitor MK2206 on AKT/NF-?B signaling pathway and necroptosis induced by magnesium-free fluid in primary cultured hippocampal neuron epileptic discharge model.(1)Research objects and groupings:Primary culture of hippocampal neurons in birth rat within 24 hours.Divided into normal control group(On the 7th day culture on the physiological basic treatment solution for 24 hours);normal control+MK2206 group(on the 7th day culture on MK2206 lumol/L for 24 hours);SREDs group(on the 7th day culture on Mg2+-free treatment for 3 h;SREDs+MK2206 group(on the 7th day incubation with MK2206 1?mol/L for 24 hours,and continued to culture on Mg2+-free treatment for 3 h).(2)Western blotting was used to detect the expression levels of AKT,p-Thr308-AKT,p-Ser473-AKT,p-IKK??,IKK??,NF-?B and p-NF-?B in each group.(3)Real-time quantitative fluorescent PCR assay for detection of necroptosis-related protein gene RIP1 mRNA,RIP3 mRNA and MKLK mRNA expression levels,and the mechanism of neuronal cell necroptosis in epileptic hippocampal neurons was explored in a neuron epileptic discharge model.ResultsPart ? Effect of AKT allosteric inhibitor MK2206 on seizures and AKT/NF-?B signaling pathway1.Comparison of behaviors of each group:(1)Epileptic rate:In the experimental pilo90mg group,the seizure rate was significantly lower than that of the control pilo90mg(P=0.043)with statistical difference.There was no statistical difference between the other two groups.(2)Incubation period:The incubation period of the experimental pilo90mg group was significantly longer than that of the group pilo90mg(P=0.034).There was no significant difference in latency between the experimental pilo90mg group and the control group pilo30mg.The latency of the control pilo90mg group was significantly shorter than that of the control pilo30mg group(P=0.045)with statistical difference.(3)SE duration:The duration of SE in the experimental pilo90mg group was significantly shorter than that in the control pilo90mg group(P=0.03)with statistical difference.The duration of SE in the control pilo30mg group was significantly shorter than that in the control pilo90mg group(P=0.01)with statistical difference.There was no significant difference between the experimental pilo90mg group and the control pilo90mg group.(4)Single SE duration:The Single SE duration of SE was significantly shorter in the experimental pilo90mg group compared with the control group pilo90mg group(P=0.028).Single SE duration of SE was significantly longer in the control pilo90mg group compared with the control pilo30mg group(P=0.01).(5)Comparison of Racine scores between experimental pilo90mg group and control pilo90mg group at each point time:The Racine scores of the experimental pilo90mg group at different point time were compared with the control pilo90mg group.The P values were:P=0.037(5min);P=0.024(10min);P=0.030(15min);P=0.018(20min);P=0.011(25 min);P=0.014(30 min);P=0.027(35 min);P=0.031(40 min);P=0.029(45 min);P=0.016(50 min);P=0.027(55 min);P=0.016(60 min),and the P values were statistically different at each point time.2.Western blotting technique was used to detect the changes of important protein expression in AKT/NF-?B signaling pathway.(1)P-Thr308-AKT/AKT(P=0.019)and p-Ser473-AKT/AKT(P=0.023)were all decreased in the experimental pilo90mg group compared with the control pilo90mg group with statistical differences.There was no statistical difference between the other two groups.(2)p-IKK??/IKK?? were decreased in the experimental pilo90mg group compared with the control pilo90mg group(P=0.018)with statistical differences.(3)The experimental pilo90mg group had lower p-NF-?B/NF-?B(cytoplasmic)than the control pilo90mg group(P=0.038)with statistical differences.The experimental pilo90mg group had lower p-NF-?B/NF-?B(nucleus)than the control pilo90mg group(P=0.041)with statistical differences.There was no statistical difference between the other two groups.3.HE staining and NISSLE staining results in hippocampus on SE mouse model(1)HE staining(200 times)observed the hippocampal CA1 and CA3 neurons in each group:In the CA1 area and CA3 area of the pilo30 group,a small number of hippocampal neurons were swollen,the nucleolus was unclear,the cytoplasm was deeply stained,and the gap between some nerve cells and the surrounding brain interstitial was enlarged.The cells were arranged disorderly,some of the neurons were swollen,some cells were irregular in shape,fusiform or triangular,unevenly distributed,the gap around the cells was widened,the nucleus was pyknosis,the nucleolus was unclear,and the cytoplasm was deeply stained in the control pilo90mg group.In the experimental pilo90mg group,the damage of hippocampal neurons was lighter than that of the control pilo90mg group.Some neurons were swollen,some neurons were irregular in shape,nucleus condensed and deep-stained,surrounding vacuoles were formed,and a small number of cells were disordered.(2)NISSLE staining(200 times)counted neurons in CA1 and CA3 areas of hippocampus in each group:the number of neurons in CA1 area of experimental pilo90mg group was significantly higher than that of control pilo90mg group(P=0.023)with statistical difference.There was no statistical difference between the other two groups.The number of neurons in the CA3 area of the experimental pilo90mg group was significantly higher than that of the control pilo90mg group(P=0.039)with statistically different.There was no statistical difference between the other two groups.Part II:The effect of AKT allosteric inhibitor MK2206 on AKT/NF-?B signaling pathway and necroptosis in primary cultured hippocampal neuron epileptic discharge model induced by magnesium-free fluid.1 Western blotting was used to detect the changes of important protein expression in AKT/NF-?B signaling pathway in hippocampal neuronal cell epilepsy model.(1)The SREDs+MK2206 group had lower p-Thr308-AKT/AKT(P=0.028)and p-Ser473-AKT/AKT(P=0.011)than the SREDs group with statistical difference.Compared with the normal control group and the normal control+MK2206 group,the p-Thr308-AKT/AKT and p-Ser473-AKT/AKT were significantly increased in the SREDs+MK2206 group and the SREDs group(P<0.01)with significant differences.(2)The SREDs+MK2206 group had lower p-IKK??/IKK??(P=0.021)than the SREDs group with statistical difference.Compared with the normal control group and the normal control+MK2206 group,the p-IKK??/IKK?? was significantly increased in the SREDs+MK2206 group and the SREDs group(P<0.01)with significant differences.(3)The SREDs+MK2206 group had lower p-NF-?B/NF-?B(P=0.030)than the SREDs group with statistical difference.Compared with the normal control group and the normal control+MK2206 group,p-NF-?B/NF-?B was significantly increased in the SREDs+MK2206 group and the SREDs group(P<0.01)with significant differences.2 Real-time fluorescent quantitative qPCR method for detecting mRNA related to necroptosis signaling pathway:(1)The relative expression of RIP1 mRNA in SREDs+MK2206 group was lower than that in SREDs group(P=0.036)with statistical different.Compared with the two control groups,the relative expression of RIP1 mRNA in SREDs+MK2206 group and SREDs group was significantly higher thanthe normal control group and the normal control+MK2206 group(P<0.01).(2)The relative expression of RIP3 mRNA in SREDs+MK2206 group was lower than that in SREDs group(P=0.017)with statistical difference.Compared with the two control groups,the relative expression of RIP3 mRNA in SREDs+MK2206 group and SREDs group was significantly higher than the normal control group and the normal control+MK2206 group(P<0.01).(3)The relative expression of MLKL mRNA in SREDs+MK2206 group was lower than that in SREDs group(P=0.018)with statistical difference.Compared with the two control groups,the relative expression of MLKL mRNA in SREDs+MK2206 group and SREDs group was significantly higher than the normal control group and the normal control+MK2206 group(P<0.01).Conclusion1 Pretreatment with MK2206 significantly reduced the seizure rate,prolonged the incubation period,shortened the duration of seizures,reduced the Racine score at each time point in the SE mouse model.Pretreatment with MK2206 in the SE mouse model alleviated SE symptoms.2 The AKT/IKK/NF-?B signaling pathway was activated early in the SE mouse model.P-NF-?B was detected both in the cytoplasm and nucleus,and nuclear translocation occurred after NF-?B was activated by SE.3 MK2206 inhibits activation of AKT/IKK/NF-?B signaling pathway in SE mouse models.MK2206 inhibits the phosphorylation of the 308th threonine residue and the 473th serine residue on AKT and inhibits AKT activation.MK2206 indirectly inhibits the downstream IKK complex of AKT and the early activation of NF-?B downstream of IKK.4 MK2206 can alleviate the damage of hippocampal neurons in SE mice and protect hippocampal neurons.5 The AKT/IKK/NF-?B signaling pathway was activated in the SREDs rat model.6 MK2206 inhibits the activation of AKT/IKK/NF-?B signaling pathway in the SREDs rat model.7 Necroptosis is one of the mechanisms of hippocampal neuronal death in the SREDs rat model.8 MK2206 inhibits the mRNA of RIP1/RIP3/MLKL-related protein in the necroptosis signaling pathway and reduces the degree of necroptosis. |
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