Toll-like Receptor 4 Modulate Microglia Polarization After Traumatic Brain Injury And Its Mechanism

Objective: To study the role and mechanism of Toll-like Receptor 4(Toll like Receptor 4;TLR4)in traumatic brain injury.Methods: After 24 h brain injury,comparative study TLR 4 knockout(TLR4(-/-))/ wild type mice(TLR4(+/+)).Edema area change of different groups is compared by MRI scan.Use RT-q PCR and WB method to compare microglia M1 / M2 phenotypic markers like inos,arg-1,TNF-a,IL-1 b,IL-10,and VEGF change between different groups.In vitro experiments,TLR4 gene knockout and wild type primary microglia cells was cultured,using immunofluorescence observations microglia morphological changes,RT-q PCR comparison microglia M1 / M2 phenotypic marker expression.To compare important downstream signaling molecules Myd88,ERK1/2,NF –k B,RAC-1 and AKT changes in different groups,we use Western Blot.Results: After 24 h of traumatic brain injury,significant difference were observed in different groups in Morris water maze test(p < 0.05).TLR4 KO mice group showed shorter incubation period(p < 0.05),and TLR4 KO mice group showed more times across over the platform than WT mice(p < 0.05).Comparing to the edema area of WT mice group,the area of TLR4 KO mice was smaller(p < 0.05).Comparing to the WT mice,the expression of IL-10,arg-1 and VEGF increased dramatically in TLR4 KO mice(P<0.05,respectively),while inos,TNF-a,IL-1βdecreased significantly(P<0.05,respectively).The purity of primary microglia was more than 90%.After stimulation with brain injury model in vitro,TLR4 gene knockout type primary microglia in tend to M2 type more obviously,the expression of i NOS,TNF alpha and IL-1 beta significantly lower than the wild type primary microglia cells(p < 0.05,respectively),while Arg-1,IGF-1,BDNF and IL-10 significantly raised(p < 0.05,respectively).The TLR4 gene knockout type primary microglia in tend to M2 type in shape more obviously.The important downstream signaling molecules of TLR4,we found that activation of Myd88,ERK1/2 and NF-κB in TLR4 KO mice is significant lower than that in WT mice(P<0.05,respectively),whereas the RAC-1activity and AKT phosphorylation of was obviously higher than that that in WT mice(P<0.05,respectively).Conclusion: After traumatic brain injury,the activation of TLR4 promoted inflammatory response in cerebral,aggravating cerebral edema and secondary,by acceleration the microglia M1 polarization.In traumatic brain injury,the downstream TLR4 Myd88,ERK1/2,nf-kappa B and signaling pathways activated obviously,but inhibited the activity of the RAC-1.Part 1: Toll-like receptor 4 inhibits the recovery of neurological function after traumatic brain injuryObjective: Comparative study of TLR4 gene knockout(TLR4(-/-))and wild type(TLR4(+ / +))in 24 h after controlled cortical impact brain injury model in neurological function,learning ability and the change of edema area.Methods: After 24 h brain injury,comparative study TLR 4 knockout(TLR4(-/-))/ wild type mice(TLR4(+/+)).Free fall brain injury model group are using 2.5 mm diameter blow head accurately hit the mouse cortex(depth: 1.0 mm;weight: 10.0 g;blow height: 2cm).Sham besides no impact,are the same as the CCI group.With Morris water maze experiment to compare each group neurological function and the learning ability;the variation of edema area between groups is compared by MRI scan.Results: Compared with the sham group,TLR4(+ / +)CCI group and TLR4(-/-)CCI group needed more time to look for platform,and incubation period of TLR4(-/-)CCI group was obviously shorter than the TLR4(+ / +)CCI group(p < 0.05).Compared with the sham group,the number of across over the platform of CCI group were decreased significantly,TLR4(-/-)CCI group across over the platform was more frequentiy than TLR4(+ / +)CCI group(p < 0.05).Sham group did not find edema in MRI,and TLR4(+ /+)CCI group and TLR4(-/-)CCI group were visible edema area(P < 0.01).Comparing to the edema area of WT mice group,the area of TLR4 ko mice was smaller(p < 0.05).Conclusion: In traumatic brain injury,the activation of TLR4 inhibit the recovery of neurological function and the learning ability,and aggravate the injury after brain edema.After knockout TLR4 can obviously improve the outcome of traumatic brain injury by promoting recovery of neurological function and relieving brain edema after trauma.Part 2: Toll-like receptor 4 promote microglia to M1 polarization after traumatic brain injuryObjective :Comparative study of M1 / M2 microglia phenotypic markers of expression changes between TLR4 gene knockout(TLR4(-/-))/wild type(TLR4(+ / +))in controlled cortical impact brain injury after 24 h,3 d,7 d.Methods : TLR4 gene knockout and wild type mice grouped into three group: the control group(sham),wild type mice brain injury model group(free fall(TLR4(+ / +)CCI)and TLR4 gene knockout mice free fall brain injury model group(TLR4(-/-)CCI).Free fall brain injury model group are using 2.5 mm diameter blow head accurately hit the mouse cortex(depth: 1.0 mm;weight: 10.0 g;blow height: 2 cm).Sham besides no impact,are the same as the CCI group.Within 24 h,3 d,7 d after the modeling,taking brain tissue surrounding injury site respectively,protein and m RNA was tested by RT-q PCR and Western Blot,to comparison microglia M1 / M2 phenotypic markers(M1 phenotypic markers: i NOS,TNF-alpha,beta,IL-1 M2 phenotypic markers: Arg-1,VEGF,IL-4,IL-10)expression changes between groups.After 24 h,3 d,7 d the modeling,brain was collected,fixed,slice and did immunofluorescence staining respectively,to contrast activated microglia cells number change surrounding injury site.In vitro experiments,TLR4 gene knockout and wild type primary microglia cells was cultured,using immunofluorescence observations microglia morphological changes,RT-q PCR comparison microglia M1 / M2 phenotypic marker expression changes.Results: In the m RNA level,TLR4(+ / +)CCI group and TLR4(-/-)CCI group within 24 h,3 d,7 d of injury,i NOS,Arg-1,IL – 1 beta,IL-10 were significantly higher than that in the control group.After injury,the expression of IL-1 beta in TLR4(-/-)CCI group as obviously lower than that of TLR4(+ / +)CCI group(P < 0.05).After injury 3 d,7 d,TLR4(-/-)CCI group the expressed lower i NOS than the TLR4(+ / +)CCI group(P <0.05).In addition,after injury,at three time points,expression of inflammation inhibiting factor like IL-10 was obviously higher in TLR4(-/-)CCI group(P < 0.05).The expression of Arg-1 in TLR4(-/-)CCI group was obviously higher than that of TLR4(+ / +)CCI group(P < 0.05).In the protein level,CCI group within 24 h,3 d,7 d of injury,i NOS,TNF alpha,Arg-1,VEGF were significantly higher.Within 24 h,3 d,7 d of injury,the expression of TNF alpha and i NOS in TLR4(-/-)CCI group was obviously lower TLR4(+ / +)CCI group(P < 0.05;respctively).The purity of primary microglia was more than90%.After stimulation with brain injury model in vitro,TLR4 gene knockout type primary microglia in tend to M2 type more obviously,the expression of i NOS,TNF alpha and IL-1 beta significantly lower than the wild type primary microglia cells(p < 0.05,respectively),while Arg-1,IGF-1,BDNF and IL-10 significantly raised(p < 0.05,respectively).The TLR4 gene knockout type primary microglia in tend to M2 type in shape more obviously.Conclusion: In traumatic brain injury,the activation of TLR4 activates and promotes the inflammatory response,accelerating the M1 polarization microglia,restraining its to M2 polarization.Through the knockout TLR4 gene can obviously reduce the inflammatory response after traumatic brain injury,reduce the microglia switch to M1 phenotype,promote its polarize to M2 phenotype.In vitro culture of microglia,the TLR4 gene knockout microglia polarize to M2 more obvious.Part 3: The function of Myd88 / ERK/NF-kappa B signaling pathway and the RAC-1Objective: Comparative study of Myd88 / ERK/nf-kappa B signaling pathway and the expression of RAC 1 change in TLR4 gene knockout(TLR4(-/-))and wild type(TLR4(+/ +))mice in 24 h after controlled cortical impact brain injury model.Methods: TLR4 gene knockout and wild type mice grouped into three group: the control group(sham),wild type mice brain injury model group(free fall(TLR4(+ / +)CCI)and TLR4 gene knockout mice free fall brain injury model group(TLR4(-/-)CCI).Free fall brain injury model group are using 2.5 mm diameter blow head accurately hit the mouse cortex(depth: 1.0 mm;weight: 10.0 g;blow height: 2 cm).Sham besides no impact,are the same as the CCI group.In 24 h after brain injury of modeling,taking brain tissue surrounding injury site respectively,protein was tested by Western Blot,to comparison Myd88 / ERK/NF-kappa B signaling pathway and the expression of RAC 1 change between groups.Results: Compared with the sham group,CCI groups after injury My D88,p ERK1/2,p NF-kappa B p65 were significantly increased(P < 0.01).TLR4(-/-)CCI group My D88 significantly lower than the relative expression of TLR4(+ / +)CCI group(P < 0.05).The relative expression level of My D88,p ERK1/2,p NF-kappa B p65 in TLR4(-/-)CCI group was significantly lower than the TLR4(+ / +)CCI group(P < 0.05,respectively).Compared with the sham group,CCI groups 24 h after injury,the RAC-1activity were significantly increased(P < 0.01).Activity of RAC-1 in TLR4(-/-)CCI group was obviously higher than that of TLR4(+ / +)CCI group(P < 0.05).At the same time,in TLR4(-/-)CCI group,the activation of AKT was obviously higher(P < 0.05).Conclusion: In traumatic brain injury,the activation of TLR4 downstream Myd88 depending on signaling pathway,further promoting the phosphorylation of ERK1/2 and the activation of the NF-kappa B p65,lead to the NF-kappa B p65 translated into the nucleus,starting the transcription of inflammatory cytokines,and promoting inflammatory response.In addition,the activation of TLR4 inhibits the activity of the RAC-1,thus blocking the brain damage protective signaling pathway PI3K/AKT.By knocking out TLR4 can reduce activation of Myd88 and the downstream ERK1/2,leading less transcription of the NF-kappa B,at the same time,promote the activation of RAC – 1 and starting the PI3K/AKT signaling pathway.