| Neurological damage and cognitive impairment caused by electromagnetic pulse(EMP)radiation have received much attention,but the mechanism is still unclear.Previous studies have shown that M1-type microglia activation-mediated central inflammatory response is one of the important causes of neuronal apoptosis,synaptic damage and cognitive impairment.TLR4/NF?B activation can cause inflammatory reactions;EMP Increased neuronal apoptosis in the brain,impairing cognitive function;isoflurane preconditioning(IP)can inhibit the activation of TLR4/NF?B after cerebral ischemia-reperfusion injury,and reduce the inflammatory response to play a neuroprotective role.Therefore,it is speculated that EMP may induce inflammatory reaction and increase neuronal apoptosis by increasing the activation of M1-type microglia through TLR4/NF?B pathway;while IP regulation of microglia transite from M1-type to M2-type,reducing inflammatory response.Reduce neuronal apoptosis.This study clarified the effect of EMP on the polarity transition of M1/M2 and the TLR4/NF?B pathway in microglia.It was elucidated that IP can up-regulate the expression of SOCS1 and regulate the transformation of microglia from M1-type to M2-type after EMP irradiation.Inflammatory response.It provides a new basis for the study of the mechanism of EMP neuronal damage and provides a strategy for its prevention and treatment.Experiment 1 The role of microglia polarization and TLR4/MyD88/NF?B signaling pathway in brain damage and recovery induced by electromagnetic pulseAIM: To determine the effects of EMP irradiation on neuronal apoptosis,microglia polarity and activation of TLR4/MyD88/NF?B signaling pathway.Method: Male SD rats were randomly divided into 4 groups,6 in each group: control group,EMP12 h group,EMP24 h group,EMP3 d group.The control group received the fake treatment and the rats in the irradiated group received 400kV/m,200 P EMP irradiation,and were taken at 12 h,24 h and 3 d after EMP irradiation.The cleaved-Caspase-was detected by Western blot.3,Bcl-2,Bax,TLR4,MyD88 and I?B-? protein expression,using Real-Time PCR,Western blot and immunofluorescence staining to detect microglia M1 labeled iNOS and M2 labeled Arg1 changes The mRNA expression of inflammatory related cytokines(TNF-?,IL-6,IL-1?,IL-10,BDNF and TGF-?)was detected by real-time quantitative PCR.N9 microglia and HT-22 neurons were co-cultured in Transwell system and randomly divided into 4 groups(n = 6): control group(false photos),CLI group(false photos),EMP12 h group and CLI + EMP In the group,HT22 cells were scraped at the same time to detect the apoptotic rate.Result:At 12 h and 24 h after EMP irradiation,the expression of caspase-3 and Bax in the cortex of rats increased and the expression of bcl-2 decreased(P <0.05).The expression level of iNOS was significantly increased(P<0.05).<0.05),while the expression level of Arg 1 did not change significantly,TLR4 and MyD88 protein expression was up-regulated,I?B-? protein expression was down-regulated,M1 microglia-related molecules(IL-1?,IL-6 and TNF-?)The mRNA level increased(P <0.05);in EMP3 d group,cleaved caspase-3,Bax and iNOS expression decreased compared with EMP 12 h and 24 h groups,while bcl-2 and Arg 1 expression increased,TLR4 and MyD88 protein expression Down-regulation,I?B-? expression is up-regulated,mRNA levels of M1 microglia-related molecules(IL-1?,IL-6 and TNF-?)are decreased,and mRNA expression levels of M2-related molecules(IL-10,TGF-?,BDNF)are down-regulated.Significantly elevated(P <0.05).Compared with the Con group and the CLI group,the expression of iNOS was up-regulated in the EMP group,the expression levels of IL-1?,IL-6 and TNF-?,TGF-? and BDNF mRNA were increased,and the apoptosis rate of HT22 cells was increased(P < 0.05).Compared with EMP group,iNOS expression was down-regulated in EMP + CLI group,IL-1?,IL-6,TNF-? and TGF-? m RNA levels were decreased,IL-10 and BDNF mRNA levels were significantly increased,and HT22 cells were withered.The mortality rate decreased(P <0.05).Compared with the Con group,the CLI group and the EMP group,there was no significant difference in the mRNA and protein expression levels of Arg1 in the EMP + CLI group(P > 0.05).Conclusion: In the early stage after EMP irradiation(<24h),microglia changed from resting state to M1 type,activated TLR4 / MyD88 / I?B-? signaling pathway,producing inflammatory cytokines IL-1?,IL-6 and TNF-? increases and induces neuronal apoptosis.In the late stage of EMP irradiation(> 3d),in the self-repair stage,microglia is mainly M2 type,inflammatory mediators IL-1?,IL-6 and TNF-? are produced,while neuroprotective IL is produced.-10,TGF-? and BDNF increased,and apoptosis decreased.Experiment 2 Isoflurane preconditioning reduces the nerve damage caused by electromagnetic pulse by up-regulating SOCS1 to regulate the polarity conversion of microglia M1/M2.AIM: To determine the effects of isoflurane pretreatment on microglia polarity and neuronal apoptosis after EMP irradiation.Method: Male Sprague-Dawley rats were randomly divided into 4 groups,6 in each group: control group,EMP group,IP group,IP+EMP group.The control group received the fake treatment and received the material;the IP group and IP+EMP were treated with isoflurane before the first irradiation,and the IP group was not irradiated;the IP+EMP group and the EMP group received 400kV/m per day.400 P repeated 3d EMP irradiation,24 h after the last EMP irradiation;Western blot analysis of cleaved-Caspase-3,Bcl-2,Bax,TLR4,MyD88,I?B-? and SOCS1 protein expression,using Real-Time PCR Western blot and immunofluorescence staining were used to detect changes in iNOS and Arg1,and inflammatory related cytokines(including TNF-?,IL-6,IL-1?,IL-10,BDNF and TGF)were detected by real-time quantitative PCR.-?)mRNA expression changes.N9 microglia were randomly divided into 4 groups(n = 6): control group,EMP group,IP+EMP group and IP+EMP+siRNA group.The IP+EMP+siRNA group was transfected with microglia by SOCS1 siRNA,pretreated with isoflurane,and then subjected to repeated 3d EMP irradiation;the four groups of cells were tested at the same time point.The expression levels of iNOS and Arg1 were detected by real-time quantitative PCR and Western blot.Western blot was used to detect the expression of TLR4,MyD88,I?B-? and SOCS1 protein.mRNA levels of inflammatory related cytokines including TNF-?,IL-6,IL-1?,IL-10,BDNF and TGF-? were detected by real-time quantitative PCR.Result:Compared with the Con group and the IP group,the expression of cleaved caspase-3 and Bax in the cortex of EMP group and IP+EMP group was increased,the expression of bcl-2 was decreased,and the expression level of iNOS mRNA and protein was significantly increased(P < 0.05),the mRNA level of Arg 1 decreased(P <0.05)and the protein expression level did not change significantly.The expression of TLR4 and MyD88 protein was up-regulated,the expression of I?B-? protein was down-regulated,and the M1 type microglia-related molecule(IL-1?)The mRNA levels of IL-6 and TNF-? were increased(P <0.05),while the expression of SOCS1 was not significantly changed(P <0.05).Compared with EMP group,celeved caspase-3 and the cortex of rats in IP+EMP group The expression of Bax decreased,the expression of bcl-2 increased,the expression of iNOS mRNA and protein decreased(P <0.05),and the mRNA and protein expression levels of Arg 1 were significantly increased.M1 microglia-related molecule(IL-1?,The mRNA levels of IL-6 and TNF-? decreased,and the mRNA expression levels of M2-related molecules(IL-10,TGF-?,BDNF)were significantly increased(P <0.05),while the expression of TLR4 and MyD88 protein did not change significantly(P<0.05).<0.05).Compared with EMP group,iNOS mRNA and protein expression levels were decreased in IP+EMP group,IL-1?,IL-6 and TNF-? mRNA levels were decreased,Arg1 mRNA and protein expression levels were up-regulated,IL-10,TGF-? and The mRNA level of BDNF increased,the expression of TLR4 and MyD88 protein in IP+EMP group did not change significantly,and the expression of I?B-? was up-regulated(P <0.05).Compared with IP+EMP group,iNOS mRNA and protein expression in IP+EMP+siRNA group The levels of IL-1?,IL-6 and TNF-? mRNA increased,the expression levels of Arg1 mRNA and protein decreased significantly,the mRNA levels of IL-10 and BDNF decreased(P <0.05),and the mRNA levels of TGF-? were not increased.There was no significant change in TLR4 and MyD88 protein expression(P > 0.05),and I?B-? expression was significantly down-regulated(P > 0.05).Conclusion: 400gV/m 400 P repeated 3d EMP irradiation,microglia activation mainly M1 type,activation of TLR4 / MyD88 / NF?B signaling pathway,inflammatory cytokines IL-1?,IL-6 and TNF-? production Increase and induce neuronal apoptosis.After IP,I received EMP irradiation.The microglia activation phenotype was mainly M2 type,and the production of inflammatory mediators IL-1?,IL-6 and TNF-? was reduced,while neuroprotective IL-10,TGF Increased production of ?-and BDNF,reduced neuronal apoptosis,and neuroprotective effects of IP production may be associated with up-regulation of SOCS1 expression. |
PDF Full Text Request