| Objective:Traumatic brain injury is an important public health problem worldwide.Secondary brain injury promotes the development of cognitive impairment.Microglia are immune cells resident in the central nervous system and are the first line of defense against disease or injury.Microglia have two polarization states and have a dual role: secondary nerve damage and neuroprotection.Therefore,intervention in microglia polarization state may be a key target for improving long-term cognitive impairment in rmTBI.TLRs are a family of receptors that mediate innate immunity,and in particular TLR4 is thought to be a major regulator of microglial activation.TLR4 is highly expressed in microglia and plays a crucial role in regulating chronic inflammatory response and initiating secondary damage after TBI.Our previous study found that M1 phenotype microglia increased,which promoted neuroinflammation and caused cognitive impairment in the chronic phase of TBI.This study will further elucidate the role and mechanism of TLR4 in regulating microglia polarization phenotype and improving TBI long-term cognitive impairment,which provide a theoretical basis for new strategies of TBI treatment.Methods: 1.Detect the expression of TLR4 using Immunofluorescence and Western Blot to verify that TAK242 inhibits TLR4 expression.Evaluate the effects of TLR4 on neurological function and long-term spatial learning and memory in rmTBI mice using modified neurological function score and Morris water maze.2.Evaluate the effects of TLR4 on pathological protein deposition in rmTBI mice using Western Blot.3.In vitro and in vivo,detecting the expression and quantitative analysis of M1 and M2 phenotype microglia using Immunofluorescence and Western Blot and detecting inflammatory factors by ELISA to investigate the effect of TLR4 on the microglia polarization and release of inflammatory factors in acute and chronic phase of rmTBI.4.In vitro and in vivo,detecting the expression of TLR4,Myd88 signaling pathway and downstream protein NF-?B using Immunofluorescence and Western Blot,to confirm that TLR4 and important downstream signaling molecules Myd88 and NF-?B are involved in the regulation of microglia polarization and neuroinflammatory response after TBI.Results: 1.(1)In the acute and chronic phase after rmTBI,TLR4 expression is elevated,while TAK242 can effectively inhibit the expression of TLR4.(2)The mice show obvious neurological deficits in the acute phase after rmTBI,and with the prolongation of injury time,neurological deficits gradually recover.Mice have a significant decline in learning and memory in the chronic phase after rmTBI.These results further confirm that rmTBI is an important risk factor for neurodegenerative diseases.(3)After rmTBI,inhibition of TLR4 expression can promotes repair of neurological deficits in mice.(4)After rmTBI,inhibition of TLR4 expression can improve spatial learning and memory in mice.2.(1)Pathological protein APP,p-Tau protein were increased in mice at 3 days after rmTBI.At 28 days after rmTBI,the expression of APP and p-Tau protein is significantly increased compared with the acute phase.(2)In the acute and chronic phase after rmTBI,inhibition of TLR4 expression resultes in a significant decrease in the expression of APP and p-Tau,compared with the PBS group.3.(1)In vitro,LPS activates microglia,promotes microglia polarization to M1,increases proinflammatory factor release,and reduces inflammatory factor release.TLR4 expression is inhibited,promoting microglia polarization to M2,increasing release of anti-inflammatory factors,and decreasing release of pro-inflammatory factors.(2)In vivo,microglia was activated,the expression of M1 and M2 are significantly increased in the acute phase after rmTBI,and the expression of inflammatory factors is imbalanced.M1 type microglia are still activated,and the release of pro-inflammatory factors is increased,which promotes the increase of pathological proteins and causes cognitive impairment of mice in the chronic phase after rmTBI.In the acute and chronic phase after rmTBI,inhibition of TLR4 expression can significantly down-regulate the M1/M2 ratio,promotes the polarization of microglia to M2,increases the release of anti-inflammatory factors,reduces pathological protein deposition,and improves cognitive function.4.(1)In the acute and chronic phase after rmTBI,more TLR4 are co-localized in microglia,compared with the sham group.TAK242 significantly reduce the number of TLR4 colocalized to microglia,with the PBS group,using immunofluorescence double-labeled TLR4 and microglia marker Iba1.(2)After LPS and rmTBI,the expression of TLR4 and downstream signaling molecules Myd88 and NF-?B are significantly increased.Compared with the PBS group,TAK242 significantly down-regulate the expression of TLR4 and downstream signaling proteins Myd88 and NF-?B,which suggests that TLR4/Myd88/NF-?B signaling pathway is one of the key signaling pathways in neuroinflammatory response after rmTBI.In conclusion,TLR4 and downstream signaling factors Myd88 and NF-?B are involved in the regulation of microglia polarization and neuroinflammatory response after rmTBI.Conclusion: 1.After TBI,inhibition of TLR4 expression can down-regulate the expression levels of downstream signaling molecules Myd88 and NF-?B,reduces the ratio of M1/M2 microglia and promotesmicroglia polarization to M2.Inhibition of TLR4 expression can promote the release of anti-inflammatory factors,thereby inhibiting chronic neuroinflammatory reaction,which further alleviates secondary brain damage resulting in the reduction of pathological protein APP,p-Tau expression,and ultimately improving TBI long-term cognitive impairment.2.TLR4 is an important pattern recognition receptor that regulates the polarization phenotype of microglia,and affects the progression of neuroinflammation and the development of neurodegeneration.Therefore,TLR4 may be a promising intervention target for improving rmTBI long-term cognitive impairment.3.TAK242 is a potential new strategy for the treatment of TBI. |
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