| Background:Ischemic stroke is a serious cerebrovascular disease with high morbidity,disability and mortality due to the interruption or significant reduction of blood flow to parts of the brain and metabolic disorders in brain tissue,followed by irreversible brain cell damage or death.At present,there are two main therapeutic measures,one is thrombolytic therapy,but its only 4.5h time window and its possible serious side effects limit its wide application;The other is a neuroprotective treatment.The existence of penumbra provides theoretical support for this treatment,but there is no specific drug.Therefore,enhancing endogenous cell protection or reducing the activation of metabolic pathways that lead to cell damage has become a new target for the treatment of ischemic stroke.Blood brain barrier(BBB)damage occurs after ischemic stroke,and a large number of studies have proved that BBB damage will aggravate brain injury and inflammation level after ischemic stroke.BBB injury plays an important role in ischemic stroke.The key to BBB formation is endothelial cells,which express IL1R1 receptor.Studies have shown that the activation of this receptor mediates the decrease of tight junction proteins ZO-1,Occludin and Claudin-5 in BBB,resulting in BBB damage,and increases the expression of adhesion molecules VCAM-1 and ICAM-1,resulting in increased infiltration of peripheral immune cells into the brain.GSDMD(Gasdermin D,GSDMD)mediating pyroptosis releases the interleukin-1β(IL-1β),the primary ligand for the IL1R1 receptor,which acts on endothelial cells to produce these changes.Microglia,as resident innate immune cells in the brain,are the first cells to respond to cerebral ischemia injury and play an important role in the occurrence of central nervous system inflammation.It has been proved that microglia can release large amounts of IL-1β through pyroptosis.Therefore,we speculated that microglia may act on endothelial cell IL1R1 receptor through IL-lβ released by pyroptosis after ischemic stroke,thus causing BBB injury.Objective:1.To clarify the role of GSDMD protein mediated microglial pyroptosis in blood brain barrier injury after ischemic stroke;2.To explore the mechanism of endothelial IL-1β/IL1R1 signaling pathway on blood brain barrier after ischemic stroke.Methods:At the in vivo study level,the middle cerebral artery occlusion(MCAO)model was established in mice,and the structure of the model was detected by laser speckle flow imaging system.The infarct volume was detected by magnetic resonance and Neurological deficits were mNSS score of mice.Immunofluorescence staining was used to detect the expression of GSDMD in microglia,astrocytes,oligodendrocytes and neurons.Western Blot,EB staining,RT-qPCR and immunofluorescence were used to detect the expression of BBB tight junction protein.At the in vitro study level,microglia were co-cultured with endothelial cells in Oxygenose deprivation/reoxygenation(Oxygenose deprivation/reoxygenation,OGD/R)condition and exogenously added Disulfiram(DSF)to inhibit microglia pyroptosis and IL1R1 receptor antagonist IL1RA,respectively,and LDH,WB,RT-qPCR techniques and immunofluorescence staining were applied to detect the effect of microglia on endothelial cells.Results:1.MCAO model construction and grouping of mice:Laser speckle imaging technology and magnetic resonance were used to evaluate the success of modeling,and finally only MCAO mice that met the standard were included in the experimental group.The mice were divided into four groups,including Sham-WT group,Sham-KO group,MCAO-WT group and MCAO-KO group,with 6 mice in each group.2.1n vivo experiment to investigate the effect of GSDMD protein on blood brain barrier in MCAO mice:(1)MRI of mice showed that compared with MCAO-WT group,cerebral infarction volume in MCAO-KO group decreased significantly(P<0.05).(2)Behavioral results showed that the neurological function of MCAO mice was impaired,and mNSS score was significantly higher than that of Sham group.mNSS score in MCAO-KO group was lower than that in MCAO-WT group,and the difference was statistically significant on the 2nd postoperative day(P<0.0001),indicating that GSDMD knockout has a protective effect on MCAO mice.(3)The Evans blue permeability test showed that there was obvious infiltration of Evans blue in the brain after MCAO,but no obvious infiltration was found in the brain of the sham operation group,indicating that MCAO damaged the blood-brain barrier.Compared with the MCAO-WT group,the MCAO-KO group significantly decreased the infiltration of Evans blue into the brain(P<0.01),suggesting that GSDMD knockout can reduce MCAO induced BBB damage.(4)Compared with Sham group,brain water content in MCAO-WT group was significantly increased,and that in MCAO-KO group was lower than that in MCAO-WT group(P<0.0001),suggesting that knockout OF GSDMD can alleviate cerebral edema induced by MCAO.(5)To investigate the occurrence of coke death in the brain of mice after MCAO.Western blot and fluorescence quantitative PCR were used to detect the expression of GSDMD and IL-1β.The expression of GSDMD and IL-1β in the MCAO group was significantly higher than that in the Sham group,indicating that pyroptosis occurred in the brain after MCAO.(6)To explore the effect of GSDMD on tight junction proteins and adhesion molecules,Western Blot was used to detect the expression of tight junction proteins ZO-1,Occludin,Claudin-5 and adhesion molecules ICAM-1 and VCAM-1,and immunofluorescence staining was used to detect the expression of ZO-1 and Claudin-5.The results showed that compared with Sham group,the expression of tight junction protein in MCAO group was significantly decreased,and the expression of adhesive molecules was significantly increased.Compared with the MCAO-WT group,the expression of tight junction protein in the MCAO-KO group increased and the expression of adhesive molecules decreased,indicating that GSDMD deletion could reduce the decrease of tight junction protein and the expression of adhesive molecules after MCAO.(7)To explore the main mechanism of GSDMD in the brain,immunofluorescence staining was used to detect the expression of GSDMD in astrocytes,oligodendrocytes,neurons and microglia in the brain,and the results showed that microglia expressed GSDMD protein most significantly.The expression of IL1R1 receptor in brain endothelial cells was detected by immunofluorescence staining.The results showed that THE expression of IL1R1 receptor was abundant in endothelial cells.3.In vitro experiment to explore the mechanism of GSDMD protein BBB effect(1)In vitro cultured BV2 cells,immunofluorescence staining,fluorescence quantitative PCR and LDH were used to detect the pyroptosis of microglia after OGD/R.The results showed that the expression of GSDMD protein and LDH release of microglia were significantly increased after OGD/R.The expression of GSDMD,IL-1β and LDH release were significantly decreased,indicating that microglia could undergo coke death after oxygen and glucose deprivation.(2)Microglia were co-cultured with microvascular endothelial cells in vitro,and the effect of microglia on endothelial cells was detected by Western Blot and immunofluorescence staining.The results showed that microglia after OGD/R reduced the expression of tight junction protein in endothelial cells.Compared with OGD/R group,the addition of GSDMD inhibitor DSF to microglia reduced the expression of tight junction protein in endothelial cells.Compared with OGD/R group,the addition of IL-1β inhibitor IL1RA also reduced the expression of tight junction protein in endothelial cells,suggesting that microglia pyroptosis maybe reduce the expression of tight junction protein in endothelial cells through the effect of IL1R1 receptor on the endothelial cell surface,affecting its function.Conclusion:1.Inhibition of GSDMD-mediated microglia pyroptosis has a protective effect on the BBB after ischemic stroke.2.GSDMD-mediated microglia pyroptosis may cause damage on the BBB after ischemic stroke by releasing IL-1β and acting on IL1R1 receptors in endothelial cells. |
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