The Role Of TLR4/NF-?B Inflammatory Pathway In CX3CR1 Mediated Over Synaptic Pruning Of Microglia In Brain Injury After Epilepsy In Rats

Objective:Developmental epilepsy is a common acute and severe disease in infants,some of which are refractory epilepsy or epileptic status.Epilepsy can lead to different degrees of brain damage during development,and even life-threatening cases.In recent years,it has become a key problem for pediatricians to treat brain damage after epilepsy and improve their cognitive function.At present,there is no effective treatment,and excessive synaptic pruning is an important cause of this condition,but the specific mechanism is not clear.Our previous experimental study found that the expression of CX3CR1,a key protein in synaptic pruning,was increased in animal models of epilepsy,along with the expression of inflammatory factors.Therefore,we propose the hypothesis that microglia(MG)mediated TLR4/NF-?B signaling pathway increases IL-1?,promotes the expression of CX3CR1,aggravates synaptic pruning,and thus promotes brain injury after epilepsy.This project first reveal microglia in epilepsy after microglia activation and polarization,and pilocarpine epileptic rat model of small and medium-sized glial cells in the expression of TLR4,the NF-?B and Iba increased 1 protein present positive correlation,while microglia increased expression of chemokine receptor CX3CR1 expression may be involved in excessive synaptic pruning,lead to the occurrence of epilepsy and aggravating,thereby aggravating epilepsy head injury;Secondly,at the cellular level,the inhibition of microglia activation and the expression of CX3CR1 in microglia by down-regulating the TLR4/NF-?B inflammatory pathway resulted in antiepileptic effects.Finally in the body for further verification,down TLR4/NF-?B inflammatory pathways through inhibition of microglia activation polarization,and inhibit CX3CR1 high expression of microglia,promote synaptic protein expression,inhibit the decrease in the number of synapses,improve the synaptic ultrastructure,thus inhibiting excessive synaptic pruning,inhibit the occurrence of epilepsy and aggravating,thus improving epilepsy head injury,this research to important scientific problem-synaptic pruning effect and mechanism of epilepsy head injury as the breakthrough point,through the methods of molecular biology,cell culture research microglial cells secrete inflammatory factor,Further studies on the role and mechanism of up-regulation of CX3CR1 in promoting synaptic pruning in post-epileptic brain injury provide new ideas for the pathogenesis of epileptic brain injury.Methods:This study is divided into two parts:in vivo animal and in vitro cell experiments.1.The first part is in vivo animal Experiments:This research adopts the 21st day after the birth of juvenile Sprague-Dawley(SD)rats,male,weight 45 g to 60 g,12 in each group,and randomly assigned to normal control group and the pilocarpine induced group status epilepticus SD rats,and to choose 6 different time points(4 h,12 h,1 d and 2 d,3 d,7 d),12 only each time point,USES the abdominal cavity injection pilocarpine model status epilepticus and observe behavior,recording EEG change,HE staining and TUNEL staining observation of hippocampal CA1,CA3 and DG area neuronal damage,by immunohistochemical,Activation and polarization of microglia in the hippocampus after status epilepsy were detected by Western blot,and the expression of M1 and M2-type related factors was detected by RT-PCR.The expression of TLR4,NF-?B and CX3CR1 in microglia in the hippocampus was detected by fluorescence double labeling,and the correlation between the expression of microglia and Iba-1 was detected.2.The second part is in vitro cell Experiments:In this study,male Sprague-Dawley(SD)newborn rats,1-3 days after birth,were used.Primary microglia were isolated and subcultured,and the cells were divided into 4groups for the experiment.Group I normal control group(Ctrl group);In group II,microglia monolayered rats treated with magnesium free solution(Mg²⁺free group):primary microglia cells were cultured to the third day and treated with magnesium free cell solution for 3 hours;Group III:the microglia monolier of rats treated with magnesium free solution was added to TLR4siRNA group(Mg²⁺Free+TLR4siRNA group):Primary microglia cells were taken and cultured for the third day.TLR4-siRNA(100pmol/m L)was added to the culture medium in vitro for 48h,and then the cells were added to the magnesium-free cell solution for further culture for 3 hours.Group IV:microglia monolayers of rats treated with magnesium free solution were added with NF-?B blocker(Mg²⁺Free+Bay11-7082group):primary microglia cells were taken and cultured to the third day for 1 day,then 5?mol of NF-?B blocker(Bay11-7082)was added for 6 hours,and the cells were added with magnesium free cell solution for 3 hours.The expression of M1-type related molecules in microglia in each group was detected by RT-PCR.The expression changes of CX3CR1 in microglia in each group were detected by RT-PCR and Westenblot,and the expression changes of TLR4,NF-?B and I?B in microglia were detected by Westenblot after the down-regulated treatment with magnesium-free solution in TLR4/NF-?B inflammation pathway.3.The second part is in vivo animal experiments:juvenile male SD rats(weight 45g-60g)21 days after birth were selected in this study,with 22rats in each group.They were randomly divided into I normal control group;II.SD rats with epileptic status induced by pilucapine;III.SD rats were treated by intraperitoneal injection of NF-?B inhibitor(BAY11-7082)with solvent;SD rats were treated with IV solvent by intraperitoneal injection of TLR4 inhibitor(TAK-242).Intervention group in building after 1 h,1 d and 2d by intraperitoneal injection of 1 times a day the NF-?B blockers BAY11-7082 and TLR4 inhibitors(TAK-242),after the success of the building after the intervention of 2 d materials,TUNEL and Nissl staining to detect each hippocampus brain injury,fluorescence staining and Westen blot detection observation group the hippocampus microglia and M1,M2 factor expression,double immunofluorescence staining,Westen blot detection and PCR detection the expression of TLR4 and NF-?B,CX3CR1,The expression of proteins before and after synapses was detected by Westen blot,and the ultrastructure of synapses was observed by electron microscopy.Seven days after the model was established,10 mice in each group were tested for cognitive function,and general motor ability,intelligence and anxiety related emotional behavior were tested in open field by Morris water maze.All data using SPSS19.0 software for data processing and Graph Pad Prism 7.0statistical analysis software,for the results of the measurement data in the experiment to mean±standard deviation to represent(mean±SD),such as comparing two groups of data are analyzed by t test results,such as multiple sample data comparison,with a mean comparison using the method of single factor variance analysis results.P<0.05 was considered statistically significant.Results:1.The expression of Iba-1 protein was the lowest in Ctrl group,which was slightly increased at 4h,gradually increased at 12h(P<0.05),reached the peak at 1d(P<0.001),and slightly decreased at 3d(P<0.01),and was close to Ctrl group at 1w.The m RNA levels of CD86,CD14,CD206 and Arg-1 in microglia of juvenile rats in epilepsy group increased gradually with time after 12h,1d,2d and 3d.Compared with the corresponding normal control group,the m RNA levels of CD86,CD14,CD206 and Arg-1 in microglia of juvenile rats in epilepsy group were significantly different,and the expression reached the peak on the 2nd day.2.TUNEL staining showed that the TUNEL positive cells reached the peak at 2 days after SE in all areas of the hippocampus.3.In this study,it was found that the chemokine receptor CX3CR1 expressed in microglia cells increased to varying degrees after epileptic status,and the presynaptic protein expression decreased to varying degrees after epileptic status.4.The expression of TLR4/NF-?B in microglial cells of pilucapine epileptic rat model was positively correlated with the expression of Iba-1 protein.5.At the cellular level,the expression of inflammatory cytokines IL-1?(P<0.001)and TNF-?in microglia of rats treated with magnesium free solution increased(P<0.01),the expression of inflammatory cytokines IL-1?(P<0.01)and TNF-?decreased after Bay11-7082 treatment(P<0.01),and the expression of inflammatory cytokines IL-1?(P<0.001)and TNF-?decreased after siRNA-TLR4 treatment(P<0.01).Compared with normal rat microglia,the expression level of CX3CR1 protein in microglia of rats treated with magnesium free solution was significantly increased(P<0.01),the expression level of CX3CR1 protein in microglia of rats treated with Bay11-7082 was significantly decreased(P<0.05),and the expression level of CX3CR1 protein was significantly decreased after siRNA-TLR4 treatment(P<0.01).Compared with normal rats,the protein levels of TLR4 and NF-?B in microglia of rats treated with Mg-free solution were significantly increased(P<0.01),while the expression of IF-?B was decreased(P<0.01).Compared with Mg-free group,the protein levels of TLR4(P<0.05)and NF-?B were decreased(P<0.01)after treatment with NF-?B inhibitor Bay11-7082(P<0.01),while the expression of I?B was increased(P<0.05).After siRNA-TLR4 treatment,the protein levels of TLR4(P<0.01)and NF-?B were decreased(P<0.05),while the expression of I?B was increased(P<0.01)compared with that of the magnesium free solution treatment group.6.Frequency of seizures:Compared with the EP group,the number of 2d convulsion in the EP+BAY group was reduced(P<0.001).Compared with the EP group,the number of 2d convulsion in the EP+TAK group decreased(P<0.001);Seize grade:Compared with the EP group,the intensity of 2d convulsion in the EP+BAY group was decreased(P<0.01),and the intensity of 2d convulsion in the EP+TAK group was decreased compared with the EP group(P<0.001).Mortality:The mortality rate was about 5.5%in the EP group,while no death occurred in the EP+BAY and EP+TAK groups.7.The neuronal injury of each group was observed by TUNEL and Nissl staining,and the expression of TUNEL positive cell count and Nissl corpussal cell count in the Epilepsy group was the most obvious,which showed statistical difference compared with the Ctrl group,EP+BAY group and EP+TAK group,respectively;The expression of Neu N was the least in the Epilepsy group,and there was a statistical difference with the Ctrl group,EP+BAY group and EP+TAK group,respectively,P<0.001.8.TLR4,NF-?B and CX3CR1 were the most expressed in the Epilepsy group,respectively,and there were statistical differences with Ctrl group,EP+BAY group and EP+TAK group,P<0.001;The expression of SYP and PSD95 was the least in the Epilepsy group,with statistical difference compared with the Ctrl group,EP+BAY group and EP+TAK group,respectively,P<0.001.9.The expression of SYP and PSD95 was the least in the Epilepsy group,with statistical difference compared with the Ctrl group,EP+BAY group and EP+TAK group,respectively(P<0.001).Synapses were observed under electron microscope Epilepsy group decreased,synaptic ultrastructure was blurred,the postsynaptic membrane became thin,twisted or interrupted,and the density decreased.Compared with Ctrl group,the synaptic gap became narrow and irregular.After the intervention,the synaptic structure tended to be in the normal group,and the number of synapses in the hippocampus in each group was statistically less in the Epilepsy group than in the Ctrl group,and there was statistically significant difference between the Epilepsy group and the Ctrl group,EP+BAY group and EP+TAK group,respectively.10.In the Morris water maze test,there was no statistical difference in the escape latency of each group during the 1-4 days.On the 5th day,the escape latency of EP group was longer than that of the control group(P<0.05),the escape latency of EP+BAY 11-7082 group was shorter than that of the EP group(P<0.05),and the escape latency of EP+TAK-242 group was shorter than that of the EP group(P<0.001).Compared with the Ctrl group,the number of crossing platform in EP group was decreased by P<0.001,the number of crossing platform in EP+BAY 11-7082 group was increased by P<0.05,and the number of crossing platform in EP+TAK-242 group was increased by P<0.01.Compared with the control group,the target quadrant movement time of EP group was shortened(P<0.001),the target quadrant movement time of EP+BAY 11-7082 group was increased(P<0.05),and the target quadrant movement time of EP+TAK-242 group was increased(P<0.01).11.In the open field experiment,compared with the Ctrl group,the residence time of EP group in the central region was delayed(P<0.001),the moving distance of the central region was prolonged(P<0.001),the total distance was prolonged(P<0.001),and the average velocity was increased(P<0.001).Compared with the EP group,the residence time of EP+BAY 11-7082 in the central region was shortened(P<0.01),the moving distance of the central region was shortened(P<0.01),the total distance was shortened(P<0.001),and the average velocity was decreased(P<0.001).Compared with EP group,EP+TAK-242 reduced central area residence time(P<0.01),central area movement distance(P<0.01),total distance(P<0.001),average velocity(P<0.001),and EP+BAY 11-7082 and EP+TAK-242 improved in different degrees after treatment.Conclusion:1.the organization level to justifying the microglia in epilepsy after microglia activation polarization,and 2 days after status epilepticus peak,the M1 and M2 factor to express different degree increased,the pilocarpine epileptic rat model of small and medium-sized glial cells express TLR4,the NF-?B increased protein present positive correlations with Iba-1,at the same time microglia expression of chemokine receptor CX3CR1expression present positive correlations increased,reduced presynaptic protein expression,which can be speculated that TLR4/NF-?B signaling pathway may be involved in activation of microglia in epilepsy and polarization,It may be involved in excessive synaptic pruning,leading to the occurrence and aggravation of epilepsy,and thereby aggravating brain damage after epilepsy.2.At the cellular level,it was demonstrated that Mg-free solution treatment produced antiepileptic effects by down-regulating the TLR4/NF-?B signaling pathway to inhibit microglia activation and decrease the expression of CX3CR1.3.It has been demonstrated at the tissue level that down-regulation of the TLR4/NF-?B inflammatory pathway in epilepsy can inhibit the activation and polarization of microglia,and inhibit the expression of M1 and M2 cytokines;At the same time,the expression of chemokine receptor CX3CR1 was inhibited,the expression of presynaptic membrane protein and postsynaptic membrane protein were promoted,the number of synapses was increased,the synaptic structure was improved to be orderly,the imbalance of synaptic E/I caused by excessive synaptic pruning was reduced,the occurrence and aggravation of epilepsy were inhibited,and the brain injury after epilepsy was improved.The downregulation of the TLR4/NF-?B inflammatory pathway can improve the cognitive and emotional abilities of brain injury after epilepsy in young rats,according to water maze and open field experiments.