| Objective: In this study,we used epilepsy cells and animal models to explore whether TNC affects the development of epilepsy by regulating TLR4 and how TNC regulates TLR4 signaling pathway to play a role in the process of epilepsy,so as to lay a foundation for further exploring the mechanism of epilepsy and provide new ideas for the prevention and early intervention of epilepsy.method:1.The epileptic cells and rat models were established to detect the expression of TNC and TLR4.Epileptic cell model : The successfully isolated primary SD rat hippocampal neuron cells were cultured.One week later,MAP-2 was tested by immunofluorescence,and the positive rate was 90%.The epileptic cell model was induced and cultured by magnesium free extracellular fluid.The model cells were divided into 2 groups: control group and EP group.The Patch-clamp was used to detect the difference of action potential frequency between the two groups to determine whether the epileptic cell model was constructed successfully.Western blot(WB)and real-time q PCR(RT q PCR)were used to detect the protein and m RNA levels of TNC,TLR4 and My D88.Epileptic rat model: rats were induced by intraperitoneal injection of Li Cl-PILO,and the control group was replaced by the same dose of normal saline.The rats were dissected 1 day and 3 days after SE,and the brain tissue samples were taken.HE staining was used to observe the morphology of hippocampal cells,and the expression of TNC and TLR4 Protein was detected by immunohistochemistry.2.Effect of TNC knockdown on epileptic cell model.The epileptic cell model was divided into four groups,the first group was control group,the second group was transfected with negative control siRNA as knockdown control,the third group was transfected with TNC-siRNA-1 to knock down TNC,the fourth group was transfected with TNC-siRNA-2 to knock down TNC;finally,the first four groups of cells were induced and cultured into epileptic cell model with magnesium free extracellular fluid.The expression of TNC in protein and m RNA levels of the four groups were detected to determine the knockdown efficiency.TNC siRNA with high knockdown efficiency was screened out for subsequent experiments,that is,TNC-siRNA-1.Then we divide them into three groups: EP group,EP + NC group,epilepsy model + TNC knockdown group(EP +siRNA-1)group.The expression of TLR4 and My D88 at protein and m RNA levels were detected.At the same time,the Patch-clamp was used to detect the changes of AP frequency,and the effect of TNC knockdown on epileptic cell model was analyzed.3.Effect of TLR4 agonist LPS on epileptic cell model in TNC knockdown group.First,TNC-siRNA-1 was transfected into epileptic cell model to knock down TNC.then we divided the model cells into 2 groups.Only one group was added with TLR4 agonist LPS.Finally,the epileptic cell model was induced and cultured by adding magnesium free extracellular fluid in both groups.Then we divided them into EP+siRNA-1 groupand EP+siRNA-1 + LPS group.The protein and m RNA levels of TNC,TLR4 and My D88 were detected in two groups.Patch-clamp was used to detect the changes of AP frequency of cell membrane,and to analyze the effect of TLR4 agonist LPS added on the basis of TNC knockdown on epileptic cell model.result:1.The epileptic cells and rat models were established to detect the expression of TNC and TLR4.Epileptic cell model : Hippocampal neurons of neonatal SD rats were successfully isolated and cultured,and identified by immunofluorescence MAP-2staining.The positive rate was 90%.The results of whole cell patch clamp electrophysiology showed that there was no significant difference in resting membrane potential(RMP)between control group and EP group(control group:-70.11 ± 1.44 MV and model group:-70.01 ± 3.01 MV)(n = 3),but the firing frequency of action potential increased significantly(control group: 0.6 ± 0.058 Hz and model group: 2.167 ± 0.088 Hz).Compared with the control group,the relative expression of TNC m RNA in EP group was significantly higher(P < 0.01),the relative expression of TLR4 m RNA was significantly higher(P < 0.05),and the relative expression of My D88 m RNA was significantly higher(P < 0.001).Compared with the control group,the relative protein expression of TNC,TLR4 and My D88 in EP group were significantly increased(P < 0.001).Epileptic rat model:HE staining results: The nuclei of neurons in CA1,CA3 and DG areas of hippocampus in control group and epilepsy model group were round,located in the center of cells,with obvious nucleolus and regular arrangement of neurons.There was no significant difference between the two groups under microscope.Compared with the control group,there was no significant difference in the expression of TNC and TLR4 in hippocampal neurons 1 day after pilo modeling;Compared with the control group,the positive rates of TNC and TLR4 expression in hippocampal neurons were significantly increased 3 days after PILO modeling,and the difference was statistically significant(P < 0.0001).2.Effect of TNC knockdown on epileptic cell model.Compared with EP group,the relative expression of TNC m RNA in EP + NC group was not significantly different,but the relative expression of TNC m RNA in EP+ siRNA-1 group was significantly lower than that in EP + siRNA-2 group(P <0.001).Compared with EP group,the relative protein expression of TNC in EP + NC group was not significantly different,but the relative protein expression of TNC in EP+ siRNA-1 group was significantly lower than that in EP group(P < 0.001),and the relative protein expression of TNC in EP+siRNA-2 group was significantly lower than that in EP group(P < 0.001).Comprehensive experimental results showed that TNC-siRNA-1 knockdown efficiency was better than TNC-siRNA-2.Compared with EP group,the relative m RNA expression of TLR4 and My D88 in EP + NC group had no statistical difference,the relative m RNA expression of TLR4 in EP+ siRNA-1 group was decreased,the difference was statistically significant(P < 0.001),and the relative m RNA expression of My D88 in EP+siRNA-1group was decreased,the difference was statistically significant(P < 0.01).Compared with EP group,the relative proteinexpressions of TLR4 and My D88 in EP + NC grouphad no significant difference;compared with EP group,the relative expression of TLR4 in EP + siRNA-1 group was decreased,the difference was statistically significant(P < 0.001),and the relative expression of My D88 in EP+siRNA-1 group was decreased,the difference was statistically significant(P < 0.01).There was no significant difference in resting membrane potential(RMP)among EP group,EP + NC group and EP + siRNA-1 group(EP group-68.35 ± 0.44 m V,EP +NC group-70.02 ± 0.84 MV,EP + siRNA-1 group-71.32 ± 0.81 m V,n = 3).Compared with EP group,the AP frequency of EP + NC group hadno significant difference(2.53 ± 0.42 Hz in EP group and 2.37 ± 0.09 Hz in EP + NC group)(n = 3).The frequency of AP in EP + siRNA-1 group was significantly lower than that in EP+ siRNA-1 group(1.02 ± 0.14 Hz in EP + siRNA-1 group)(n = 3).3.Effect of TLR4 agonist LPS on epileptic cell model in TNC knockdown group.In the relative TNC m RNA expression level,compared with EP + siRNA-1group,EP + siRNA-1 + LPS group had no significant difference.TLR4 molecule m RNA increased,the difference was statistically significant(P < 0.01),My D88 molecule m RNA increased,the difference was statistically significant(P < 0.01).In the relative TNC m RNA expression level,compared with EP + siRNA-1group,EP + siRNA-1 + LPS group had no significant difference.TLR4 molecular protein increased,the difference was statistically significant(P < 0.001);My D88 molecular protein increased,the difference was statistically significant(P < 0.001).The levels of RMPbetween EP + siRNA-1 group and EP + siRNA-1 + LPS group had no significant difference(EP + siRNA-1 group-67.16 ± 3.72 m V,EP +siRNA-1 + LPS group-71.04 ± 0.42 m V,n = 3).Compared with EP + siRNA-1group,the frequency of AP release in EP + siRNA-1 + LPS group was significantly higher(0.85 ± 0.22 Hz in EP + siRNA-1 group and 2.11 ± 0.26 Hz in EP + siRNA-1 +LPS group)(n = 3),and the difference was statistically significant(P < 0.01).Conclusion1.TNC and TLR4 are correlated with epilepsy.2.TNC can regulate the firing frequency of action potential in epileptic cell model.3.TNC may regulate epilepsy by activating TLR4 signaling pathway and depending on My D88 pathway. |
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