The Mechanism Of HSPD1 Mediated Apoptosis Of Brain Microvascular Endothelial Cells Induced By Streptococcus Suis 2 Enolase

Streptococcus suis Type 2(SS2)is an important pathogen which can induce bacterial meningitis.Enolase(ENO)is an important virulence molecule in the process of meningitis induced by SS2 infection.In the early research,the expression of heat shock protein 60(HSPD1)was found to increase significantly during the apoptosis of brain microvascular endothelial cells(BMEC)induced by Streptococcus suis type 2 enolase(SS2-ENO),but its specific role is not clear.Therefore,this study focused on the mechanism by which HSPD1 mediates SS2-ENO induced apoptosis in brain microvascular endothelial cells.To investigate the role of HSPD1 in the process of ENO induced apoptosis of BMEC,the expression level of HSPD1 was analyzed by Western blot,and the results showed that ENO can promote the intracellular and extracellular HSPD1 expression and secretion.Further studies found that intracellular overexpression of HSPD1 and extracellular addition of HSPD1 promoted ENO-induced apoptosis.Conversely,intracellular knockdown of HSPD1 and extracellular antibodies blocked HSPD1 could inhibite it.This result indicated that high expression of HSPD1 could promote ENO induced apoptosis.In order to study the change of HSPD1 during ENO-induced apoptosis,this study was performed by some mothods,such as fluorescent labeling.The results showed that the expression of HSPD1 in mitochondria decreased gradually,and the expression of HSPD1 in cytoplasm increased gradually during ENO-induced apoptosis.In this process,HSPD1 was transferred from mitochondria to cytoplasm.At the same time,the mitochondrial permeability function was also changed.These results indicated that the permeability of mitochondrial membrane changed during ENO-induced apoptosis,which caused HSPD1 to migrate from mitochondria to cytoplasm and then to extracellular.Further studies were aimed at the molecular mechanism of intracellular and extracellular HSPD1 mediated apoptosis.Co-IP,MS and immunofluorescence staining verfied the interaction protein of intracellular HSPD1,and the involved signal pathway was analyzed.It was found that the interaction protein of intracellular HSPD1 was actin,and the cell apoptosis induced by HSPD1 and actin was through the Smac-XIAP-Caspase 3 signaling pathway.Extracellular HSPD1 mediates the development of apoptosis via TLR4.Lastly,in order to verify the effect of HSPD1 on the permeability of blood-brain barrier(BBB)in vivo and in vitro,the in vitro BBB co-culture model and healthy mice were used for experiments.The results showed that the addition of HSPD1 in the vitro model led to a low TEER value,and HSPD1 injected in the tail vein of the mice showed high concentration of the evans blue dye in the brain tissue,confirming that HSPD1 can increase the permeability of BBB.The results of immunohistochemistry also confirmed that the expression of HSPD1 in mouse brain tissue was increased under the treatment of SS2 or ENO.This study confirmed that in the case of ENO-induced apoptosis of brain microvascular endothelial cells,the expression of intracellular and extracellular HSPD1 was increased,and the HSPD1 was transferred from mitochondria to the cytoplasm and extracellular,resulting in increased permeability of the blood-brain barrier.This study elucidated the effect of HSPD1 on apoptosis and its effect on BBB permeability,enriched the understanding of HSPD1 function,and provided an important theoretical basis for the study of the pathogenesis of SS2 meningitis.