Astrocyte Derived Exosomal Non-Coding RNA Attenuate Neuroinflammation Post Tramatic Brain Injury Via Regulating SMAD7/IκB-α Signaling Pathway

Background: Traumatic brain injury(TBI)poses a huge threat to human health and productivity.The pathogenesis of TBI has long been a hot topic in research field.The cellular interactions between in central nerve system and their associated secondary neuroimmune inflammatory response are discovered to be important factors in neurological impairment after a TBI.In the present study,multiple technologies including lentivirus,AAV gene editing technology,RNA sequencing(RNA-seq),Quantitative Real-time PCR(RT-q PCR),transmission electron microscopy(TEM),Nanoparticle Tracking Analysis(NTA),protein immunoprecipitation(IP),enzyme linked immunosorbent assay(ELISA),western blot assay,immunofluorescence(IF),hematoxylin-eosin staining(HE),flow cytometry(FCM),and 9.4T MRI,phagocytosis assay,transwell assay,agarose spot assay,were used to investigate the role of SMAD7/IκB-α signaling pathway in the neuroinflammation response after TBI.Besides,morris water maze(MWM)and Rotarod test,modified Neurological Severity Score(m NSS)were utilized to determine the neurological function post TBI.This study is divided into four sections:(1)Characterizing protective effects of astrocyte exosome derived lnc RNA 49 RIKon neurovascular units after TBI;(2)Identification the mechanism of astrocyte exosome derived lnc RNA 49 RIK regulating phenotypic transformation of microglia through miRNA-10a-5p/SMAD7 signaling pathway after TBI;(3)Illustrating the role of nc RNA mediating the function of microglia through SMAD7 signaling pathway to protect neurovascular units;(4).Revealing that SMAD7/IκB-α signaling pathway inhibits neuroinflammatory response to protect the neurological function through negatively regulating NF-κB p50/p65 activation after TBI.Therefore,this study focuss on astrocyte exosomes that activate microglia SMAD7/IκB-α inhibiting neuroimmune inflammatory response after TBI and exploring a potential therapeutic target for TBI.Section Ⅰ: Characterizing protective effects of astrocyte exosome derived lnc RNA 49 RIK on neurovascular unit after TBI Objective: The purpose of this study was to examine the impact of astrocyte derived exosomal lnc RNA 49 RIK on neurovascular unit recovery post TBI.Methods: TBI in vitro model were generated by CCE treatment and TBI in vivo model were conducted by CCI;The exosome were extracted by ultracentrifugation and verified by electron microscope,western blot,and NTA;RNA-seq was used to detect the expression of nc RNA in astrocytes and exosome;HE staining was used to determine the cerebral lesion after TBI;IF assay was utilized for structural changes of neurovascular units;The expression of inflammatory factors released by microglia was detected by ELISA;The phagocytosis of microglia was examined by cell phagocytosis assay;the neurological and motor function of TBI mice were assessed by MWM and Rotarod test.Results: TBI mice model and CCE model were successfully established;exosomes were extracted and identified,and I verified that microglia could phagocytize them;lnc RNA 49 RIK was identified and confirmed;lnc RNA 49 RIK attenuated the expression of microglial proinflammatory factors and protected neurovascular units;lnc RNA 49 RIK improves memory and motor function in TBI mice model.Conclusions: Exosomal lnc RNA 49 RIK derived from astrocytes might improve neurovascular unit recovery post TBI.Section Ⅱ: Identification the mechanism of astrocyte exosome derived lnc RNA 49 RIK regulating phenotypic transformation of microglia through miRNA-10a-5p/SMAD7 signaling pathway after TBI Objective: To investigate whether astrocyte derived exosomal lnc RNA 49 RIK that regulates microglia phenotype switch post TBI via regulating miRNA-10a-5p/SMAD7 signaling.Methods: RNA-seq was used to evaluate the expression of nc RNA in astrocytes and exosomes,and its downstream target gene was analyzed using a database and literature;HE staining was used to observe the cerebral lesion focus after TBI;RT-q PCR was used to detect the expression of nc RNA and inflammatory factors,as well as downstream target genes;The expression level of downstream target protein was detected using a western blot assay;IF was used to observe the expression of microglia and downstream target proteins;ELISA was used to assess the production of pro-inflammatory factors in microglia,and a double luciferase reporter gene assay was used to confirm the link between nc RNA and target protein;The SMAD7 site controlled by E2F7 and TFAP2 C was detected using a double luciferase fragment deletion experiment;MWM and Rotarod tests were utilized to assess the neurological and motor function of TBI animals.Results: The expression of lnc RNA 49 RIK was increased,whereas the expression of miRNA-10a-5p was down-regulated,and its downstream target proteins E2F7,TFAP2 C,as well as SMAD7 were up-regulated;miRNA-10a-5p increased the expression of proinflammatory factors and down-regulated anti-inflammatory factors,whereas lnc RNA 49 RIK has the opposite function;lnc RNA 49 RIK improved memory and motor function recovery by up-regulating the expression of E2F7,TFAP2 C and SMAD7 in TBI mice model,while the function of miRNA-10a-5p has the opposite function;Besides,SMAD7 expression level was negatively correlated with m NSS score.Conclusions: The astrocyte derived exosomal lnc RNA 49 RIK may regulate microglia phenotypic change through the miRNA-10a-5p/SMAD7 signaling pathway after TBI.Section Ⅲ: Illustrating the role of non-coding RNA mediating the function of microglia through SMAD7 signaling pathway to protect neurovascular unit Objective: To investigate the impact of SMAD7 on microglial properties in regulating neurovascular unit recovery and their related mechanisms.Methods: HE staining and MRI imaging were used to assess the cerebral lesion after TBI.RT-q PCR was used to detect inflammatory factor m RNA expression;Western blot was used to detect downstream target protein expression;Immunohistochemistry staining was used to examine the cellular proportion of microglia,astrocytes,and neurological myelin sheath marker proteins in TBI brains;ELISA was used to detect the expression of inflammatory factors released by microglia;MWM and Rotarod tests were used to eveluate the neurological and motor function of TBI mice model;Transwell assay and agarose spot assay were utilized to determine the migration and chemotaxis of microglia;The phagocytic ability of microglia was detected by phagocytosis assay.Results: Activated SMAD7/IκB-α signaling pathway can significantly attenuate the upregulation of inflammatory factors in microglia;SMAD7 inhibited migration and chemotaxis while enhancing microglia phagocytosis;SMAD7 can significantly alleviate brain edema,promote neurovascular unit recovery after TBI,improve BBB reconstruction as well as neuron regeneration,and promote memory and motor ability in TBI mice model.Conclusions: nc RNA protects neurovascular unit recovery by modulating microglia activity through the SMAD7 signaling pathway.Section Ⅳ: Revealing that SMAD7/IκB-α signaling pathway inhibits neuroinflammatory response to protect the neurological function through negatively regulating NF-κB p50/p65 activation after TBI Objective: To illustrate the negative regulating effect of SMAD7/IκB-α on NF-κB p50/p65 activation which protect post TBI neurological function loss.Methods: The expression of microglial NF-κB p50/p65,IκB-α/p-IκB-α,and SMAD7 after SMAD7 overexpression was detected by western blot assay;CHX was used to determine the protein degradation of IκB-α;IP and IB were used to detect SMAD7 and IκB-α ubiquitination expression;Flow cytometry was used to detect the phenotypic transformation of microglia after ATS treatment in TBI mice model;IF was used to assess the recovery of BBB;The neurological and motor function of TBI mice were assessed by MWM and Rotarod test.Results: Upon LPS administration,expression of NF-κB p50/p65 expression was remarkable increased,while SMAD7 overexpression can attenuate NF-κB p50/p65 activation by increasing IκB-α expression;IκB-α ubiquitination was inhibited by SMAD7 overexpression;IκB-α can be degraded by CHX treatment,but overexpression of SMAD7 can reverse it;The expression of SMAD7/IκB-α were upregulated after asiaticoside treatment;SMAD7 overexpression attenuate microglia activation and rescue BBB as well as neurological after TBI.Conclusions: The SMAD7/IκB-α signaling pathway attenuate post TBI neuroinflammation by negatively modulating NF-κB p50/p65.