Hmgb2 Acts As A Microglia Pro-inflammatory Mediator Via Ctss In Stroke

Background:Ischemic stroke or brain attack is caused due to the disruption of blood flow supplying to brain and is the the second leading cause of death worldwide.Despite of great advances in the understanding diverse mechanisms of stroke damages,thrombolysis with tissue plasminogen activator(t PA)is the only clinically effective therapy,but fewer than 5%stroke patients receive effective treatment due to the narrow therapeutic window.Currently,various studies aimed to the ischemic cascade indicated that suppression of inflammation offers potential therapeutic strategies.Stroke activates microglia that induces pro-and anti-inflammatory and immune responses.Under the physiological conditions,microglia produce anti-inflammatory response for neuronal survival and generate defensive reactions.However,after stroke onset,microglia are excessively activated,resulting in a series of pro-inflammatory responses that not only amplify the early tissue damages but also cause the secondary infarct expansion.Hence,intervention of microglia pro-inflammatory response while preserving anti-inflammatory functions is considered a promising strategy for the treatment of stroke.But,there are still no effective strategies to the excessive inflammatory reaction.Objective:To analyse the inflammatory response-related genes in the early phase of stroke and to explore the underlying mechanism of pro-inflammatory respone in microglia during stroke.Methods:Ischemic brain tissues isolated from 4 months old C57 mice at 1,3,7,14or 28 days after operation with sham or stroke were subjected to RNA sequencing(RNA-seq)and analyzed the differently expressed genes(DEGs).Brain damages were analyzed by using magnetic resonance image(MRI),2,3,5-triphenyltetrazolium chloride(TTC)and Fluoro-Jade(FJ)staining.DEGs associated with imflammatory response in the early phase of stroke were screened through time series analysis and GO enrichment analysis.Fluorescence-activated cell sorting(FACS)was used to acquire microglia in the cortex of transgenic mice.Western blot,immunofluorescence and ELISA analysis were practiced to analyze the expression of pro-inflammatory factors and the change of morphology and number of microglia after stroke.AAV-DIO-Virus were injected to Cx3cr1-cre mice to regulate the expression of high mobility group box 2(Hmgb2)in microglia specifically,then the inflammatory factors and neurological functions were analyzed to explore the function of Hmgb2 in stroke.Chromatin immunoprecipitation(CHIP)-q PCR and luciferase reporter assay were used to verified the relationship between Hmgb2 and Ctss.An irreversible Ctss inhibitor LHVS was used to research the potential strategy of stroke.Evans blue(EB)dye was used to assess the permeability of blood-brain barrier(BBB).Neurological performance was scored daily by using modified 7-point neurological scales.Results:Using the next-generation of RNA sequencing we found a total of 778genes increasingly expressed in the brain of stroke mice.These genes were classified into three different clusters in terms of the expression differences across the time course after stroke.Combined with the gene ontology(GO)enrichment analysis,we analyzed the expression of Hmgb2 in microglia after stroke.Then,we identified Hmgb2 acts as a microglia pro-inflammatory mediator and up-regulated at the first day after stroke onset.Inhibition of Hmgb2 in microglia specifically by using AAV protected against microglia pro-inflammatory response and stroke damage.We verified that the area of infarction and the number of M1 microglia and FJ~+cells were decreased by the inhibiton of Hmgb2 in stroke mice.Besides,the expression of pro-imflammatory factors such as IL-1,TNF-?were decreased in stroke mice while the anti-imflammatory factors such as IL-10unchanged.We next detected the expression of Ctss after stroke and comfirmed that Hmgb2 can regulate Ctss expression by binding the promotor sequence of Ctss gene.Inhibition of the protease activity of Ctss by using LHVS restored the blood-brain barrier function and stroke damages.Conclusions:Increased expression of Hmgb2 in microglia specifically is involved in microglia pro-inflammatory response after stroke by promoting the transcription of Ctss.The inhibition of Hmgb2 or Ctss blocks microglia pro-inflammatory response and protects against brain damages.