| Epilepsy is a common,serious chronic neurological disease with high morbidity.With the development of the disease,patients also have to suffer from comorbid symptoms such as anxiety,depression,mental decline,and memory loss,which bring an extremely heavy burden to society and family.Acid-sensing ion channel 1a(ASIC1a)is a class of proton-gated cation channels,which has become targets of interest in the physiopathological process of epilepsy,pain,fear,stroke,etc.due to its distribution in the central nervous system and calcium ion permeability.ASIC1 a is closely related to the occurrence and development of epilepsy,but its specific role and regulatory mechanism in epilepsy are still controversial.Based on the previous results of our research group,this study used paraffin sections of brain tissue from patients with Temporal lobe epilepsy(TLE),Kainic acid(KA)amygdala-ignited epilepsy rat model,and glutamate Acid(Glutamate,Glu)-induced cell injury model,to verify the localization and functional changes of ASIC1 a in epilepsy,its role in neuronal death,and to explore the regulatory mechanism of ASIC1 a.This research is divided into three parts:Experiment 1: Expression distribution of ASIC1 a in patients with temporal lobe epilepsy,animal models,and cellular glutamate injury modelsObjective: To verify the expression and distribution of ASIC1 a in epilepsy from clinical specimens,animal models,and cell models.Methods:(1)Collect paraffin specimens of brain tissue from patients with temporal lobe epilepsy and control group;(2)Construct KA amygdala-kindled TLE animal model and observe its behavioral and hippocampal histopathological changes;(3)Construct Glu-induced TLE model Primary hippocampal neuron cell injury model;(4)Localization and semi-quantitative analysis of ASIC1 a expression by immunofluorescence staining,membrane protein extraction,mitochondrial extraction,Western blot,and other techniques.Results:(1)Calponin3 in the brain tissue of TLE patients was disordered along the nerve fibers,the expression of ASIC1 a was increased,and there was obvious perinuclear aggregation;(2)The KA amygdala-kindled TLE animal model had a high attack rate and a low mortality rate;(3)Similar to the patient specimens,the fluorescence staining of brain sections of TLE rats also showed a disordered arrangement of nerve fibers,and the expression of ASIC1 a was significantly increased.Western Blot showed that the relative expression of ASIC1 a in total protein and mitochondrial protein in the hippocampus showed a trend of first increasing and then decreasing,reaching a peak at 24h;the ratio of mitochondrial protein and total protein ASIC1 a expression gradually increased with the prolongation of attack time;(4)In the glutamate model of primary hippocampal neurons: Western Blot showed that the total protein ASIC1 a first decreased and then increased with the prolongation of glutamate injury time.The expression of ASIC1 a in the cell membrane than in the cytoplasm also showed a trend of decreasing at first and then increasing with time,suggesting the transfer of ASIC1 a to the cell membrane.Conclusion: Elevated expression of ASIC1 a in epilepsy and transport from cytoplasm to cell membrane/mitochondria were verified in TLE patients’ temporal lobe cerebral cortex brain tissue,rat KA amygdala ignition model and primary neuron cell Glu injury model.Experiment 2: ASIC1 a plays a role in mediating KA/Glu-induced neuronal damageObjective: To explore the pathological processes involved in mediating KA or glutamate-induced neuronal damage after ASIC1 a is blocked.Methods:(1)After administration of Pc Tx1 to block ASIC1 a,the behavioral changes of KA amygdala-kindled TLE animals were observed;(2)After Pc Tx1 was administered to block ASIC1 a,the histopathological changes of the hippocampus of KA amygdala-kindled TLE animals were observed;(3)The HT22 cell line was cultured in vitro,and the Glu injury model was prepared;(4)After administration of Pc Tx1 to block ASIC1 a,fluorescence staining was used to observe the expression of ASIC1 a in the Glu cell injury model;(5)After administration of Pc Tx1 to block ASIC1 a,the apoptosis and autophagy of Glu cell injury model were observed.Results:(1)In KA amygdala-kindled TLE animals,administration of PcTx1 to block ASIC1 a slightly reduced the degree and incidence of epileptic seizures in rats,but there was no significant difference in the degree of pathological changes;(2)In the Glu cell injury model,Blocking ASIC1 a with Pc Tx1 can alleviate the mitochondrial damage caused by Glu;(3)In the Glu cell injury model,blocking ASIC1 a with Pc Tx1 can reduce apoptosis and autophagy caused by Glu.Conclusions: At the animal and cell levels,blocking ASIC1 a can alleviate KA/Gluinduced neuronal damage and exert neuroprotective effects,and its specific mechanism is related to mitochondrial damage,autophagy,and apoptosis.Experiment 3: The regulatory mechanism of ASIC1aObjective: To explore which signaling pathways are involved in regulating the expression and function of ASIC1 a during glutamate-induced neuronal injury.Methods:(1)The HT22 cell line was cultured in vitro,and the Glu cell injury model was prepared;(2)The expression of Rho in the Glu cell injury model at different time points was observed;(3)The Glu cell injury model was given Rho activator /inhibitor After treatment,the co-localization changes of ASIC1 a and Rho were observed;(4)The changes in the expression and localization of ASIC1 a were observed after administration of Rho activator/inhibitor in the Glu cell injury model;(5)The changes in the expression of ASIC1 a was observed in the Glu cell injury model after administration of PI3 K inhibitor.Results:(1)In the Glu cell injury model,Western Blot showed that the total protein Rho first increased,then decreased,and then increased again with the prolongation of glutamate injury time.The expression of ASIC1 a in the cell membrane compared to the cytoplasm showed a trend of decreasing at first and then increasing with time;(2)After administration of Rho activator the co-localization of ASIC1 a and Rho fluorescence was enhanced,and the fluorescence showed that the distribution of ASIC1 a in the membrane increased,and real-time PCR showed the expression of ASIC1 a m RNA increased,Western blot showed that the protein expression of ASIC1 a increased,and the ratio of the expression of ASIC1 a in the cell membrane to the cytoplasm increased;(3)After Rho inhibitor was administered,the co-localization of ASIC1 a and Rho fluorescence was weakened,and the fluorescence showed that the expression of ASIC1 a decreased,and real-time PCR showed that the m RNA expression of ASIC1 a decreased,Western blot showed that the protein expression of ASIC1 a decreased,and the ratio of the expression of ASIC1 a in the cell membrane to the cytoplasm decreased;(4)After ROCK inhibitor was administered,real-time PCR showed that there was no significant difference in the m RNA expression of ASIC1 a,Western blot showed that the expression of ASIC1 a protein was slightly decreased;(5)After administration of PI3 K inhibitor,the expression of ASIC1 a at both m RNA and protein levels decreased.Conclusion: Rho/ROCK signaling pathway and PI3 K signaling pathway are involved in the regulation of ASIC1 a expression and localization in HT22 cell Glu injury model. |
