Astrocyte-derived Exosomes Enriched With MiR-873a-5p Inhibit Neuroinflammation Via Microglia Phenotype Modulation After Traumatic Brain Injury

Objective: The interaction between astrocyte and microglia plays a vital role in the damage and repair of brain lesions after traumatic brain injury(TBI).Recent studies have shown that exosomes act as potent mediators between intercellular communication.We study the effects of miR-873a-5p in astrocyte exosomes on microglial activation and its inflammatory response after TBI and the possible mechanisms were investigated.Methods: An in vitro brain injury model was established to detect the release of astrocyte exosomes and their effects on microglia polarization.Subsequently,miRNA sequencing technology was used to analyze the main components of miRNA in astrocyte exosomes.Specific miRNA mimics were used to detect the neuroprotective function and inhibition of neuroinflammatory response of the miRNA and its mechanism in vitro and in vivo trauma models.Furthermore,western blot and q RT-PCR were used to detect the expression of miRNA in human brain trauma samples.Results: The exosomes secreted by astrocytes increased significantly after traumatic brain trauma.These exosomes can activate microglia and promote the transformation to M2 phenotype.According to miRNA sequencing,819 miRNAs were found in the astrocyte exosomes,of which 135 were upregulated,including miR-873a-5p,miR-1224-5p,miR-708-5p,etc.miR-873a-5p is a major component that was highly expressed in human traumatic brain tissue.In vitro TBI model,miR-873a-5p mimics inhibited the transformation of primary microglia to the M1 phenotype,and further studies found that ERK and NF-κB p65 signaling pathways were significantly inhibited.In the mice model of TBI,miR-873a-5p significantly improved m NSS score and reduced edema in brain tissue after TBI.Exogenous injection of miR-873a-5p agomir inhibited M1 activation and promoted M2 activation in microglia after TBI.In addition,ERK and NF-κB p65 signaling pathways were significantly inhibited in TBI mice treated with miR-873a-5p.Conclusions: After traumatic brain injury,miRNA secreted by astrocyte exosomes,represented by miR-873a-5p,can regulate the activation of microglia and mediate neuroinflammation,then reduce brain tissue edema and improve neurological function score.Its molecular signaling mechanism may be associated with by inhibiting ERK and NF-κB signaling pathways.Part1: Characteristics of astrocyte-derived exosomes and its effect on microglia phenotype after traumatic brain injury Objective: To study the exosomes secreted by astrocytes and their effects on microglia after traumatic brain injury.Methods: After establishing the TBI model in vitro,exosomes secreted by astrocytes were detected by immunofluorescence,western blot,and transmission electron microscopy.After co-culture of the labeled exosomes with microglia,the inflammatory cytokines of microglia were detected by immunofluorescence,western blot and q RT-PCR.Differential changes of astrocyte exosomes were analyzed by miRNA sequencing.What’s more,we collected clinical traumatic brain injury specimens and detected the expression of miR-873a-5p and inflammatory factors by q RT-PCR.Results: Compared with the control group,astrocytes secreted a large amount of exosomes with a diameter of about 50-100 nm after TBI.Exosomes secreted by astrocytes were absorbed by microglia.Exosomes secreted by astrocytes promoted the expression of microglial M2 phenotypic markers including Arg1,IL-4,and IL-10 after TBI.By miRNA sequencing analysis,135 kinds of miRNA in astrocyte exosomes were significantly upregulated(upregulated by more than 2 times,P<0.05)after TBI,and those changed most significantly were miR-1224-5p,miR-708-5p,miR-873-5p,miR-218-2-3p,and miR-551b-3p.In addition,miR-873a-5p,Arg1,IL-4,i NOS,IL-1β,and IL-6 were increased in the necrotic areas of patients with brain trauma.Conclusion: Exosomes secreted by astrocytes can promote the microglia to M2 phenotype after TBI,and miR-873a-5p may play a key role.Part2: Effects of miR-873a-5p carried by astrocyte exosomes on phenotypic transformation and function of microglia and the molecular mechanism Objective: To study the effect and the underlying mechanism of miR-873a-5p on microglial inflammation in vitro.Methods: Microglia transfected with miR-873a-5p mimic were stimulated with LPS.The inflammatory factors including HMGB1,IL-1β,i NOS,TNF-α,IL-6,NF-κB and ERK signal pathway were detected by western blot and q RT-PCR.The experiments are grouped as follows,con group: negative control;NC group: microglia transfected with negative control mimic;873 mimic group: microglia transfected with miR-873a-5p mimic;LPS: microglia stimulated with LPS alone;LPS + NC group:microglia were transfected with negative control mimic and stimulated with LPS;LPC + 873 mimic group: microglia were transfected with miR-873a-5p mimic and stimulated with LPS.Results: After in vitro LPS treatment,the expressions of proinflammatory factors HMGB1,IL-1β,i NOS,TNF-α,and IL-6 in microglia were increased,and the NF-κB and ERK signaling pathways were activated.After transfection with miR-873a-5p mimic,the expressions of HMGB1,IL-1β,i NOS,TNF-α,IL-6 expression were decreased,and phosphorylation of NF-κB and ERK signaling pathways were inhibited.Conclusion: miR-873a-5p may inhibit the proinflammatory factors by inhibiting NF-κB and ERK signaling pathways.Part3: The effect of miR-873a-5p carried by astrocyte exosomes on the structure and function of the central nervous system after traumatic brain injury Objective: To study the effects of miR-873a-5p on neurological function,cerebral edema,inflammatory factors and possible mechanisms after traumatic brain injury in mice.Methods: After establishing the mice model of TBI,miR-873a-5p agomir was injected into the lateral ventricle.We measured the lesion area,cerebral oedema and m NSS score after traumatic brain injury.Immunofluorescence and q RT-PCR were used to detect the expression of M1 phenotypic proinflammatory factors CD32,i NOS,IL-1β and M2 phenotypic anti-inflammatory factors CD206,Arg1 and IL-4.The NF-κB and ERK signaling pathway were detected by western blot.The experiment was grouped as follows,Sham group: sham operation group;Sham + miR-873a-5p agomir group: sham operation treatment + lateral ventricle injection miR-873a-5p agomir;TBI group: CCI model group;TBI + miR-873a-5p agomir group : CCI model+ lateral ventricle injection of miR-873a-5p agomir.Results: Compared with the control group,the miR-873a-5p level in the Sham +miR-873a-5p agomir group,the TBI group and the TBI + miR-873a-5p agomir group were significantly increased.TBI + miR-873a-5p agomir group had lower neurological scores,brain damage area,and cerebral edema than the TBI group.The m RNA expressions of CD32,i NOS,and IL-1β in the TBI + miR-873a-5p agomir group were lower than that in the TBI group,while the expressions of CD206,Arg1,and IL-4 were higher than that in the TBI group.The results of immunofluorescence showed that the percentage of i NOS+ Iba1+ positive cells in the TBI + miR-873a-5p agomir group was lower than that in the TBI group,while the number of Arg1+ Iba1+positive cells was higher than that in the TBI group.In addition,the phosphorylation of NF-κB and ERK signaling pathways in the TBI + miR-873a-5p agomir group were also lower than that in the TBI group.Conclusion: miR-873a-5p inhibited inflammatory response via inhibiting NF-κB and ERK signaling pathways,and improved neurological function and cerebral edema.