The Role Of S100B/RAGE Induced ADAM17 Expression And Activity In Traumatic Brain Injury

Research background:Traumatic brain injury(TBI)is still one of the main causes of disability and mortality in the world.More than 10 million people are hospitalized for TBI every year.Although primary mechanical injury cannot be avoided and intervened,how to prevent secondary injury from causing further damage to TBI patients is a critical and difficult problem in the management of TBI today.S100B is not only a biomarker of brain injury with its serum level closely related to the severity and prognosis of TBI patients,but also a damage associated molecular pattern,which can combine with RAGE to mediate a series of pathophysiological reactions.Glycocalyx is the first line of defense against endothelial injury.The damage of glycocalyx can lead to thrombosis,vascular hyper-permeability,inflammation and other pathological reactions.Various molecules released into the blood after cell injury may affect the stability of endothelial glycocalyx.Among them,the sheddase ADAM 17 may play an important role in endothelial glycocalyx shedding after TBI.At present,the mechanism of TBI secondary injury is not yet clear.Whether S100B/RAGE signal affects the structure and function of endothelial glycocalyx by regulating the expression and activity of ADAM 17,thereby mediating secondary injury after TBI has not yet been studied.Objectives:(1)To clarify whether TBI induces S100B/RAGE signal expression and activation;(2)To clarify whether S100B/RAGE mediates endothelial glycocalyx damage after TBI;(3)To clarify whether S100B/RAGE signaling mediates endothelial glycocalyx damage by up-regulating the expression and activity of ADAM 17 after TBI,thereby aggravating secondary damage.Methods:In this study,we established animal TBI model in rats by fluid percussion injury,cell model of stretch-injuried in astrocytes,and endothelial injury model in rat aortic endothelial cells(RAECs)stimulated by conditioned medium.The expressions of S100B,RAGE,syndecan-1 and AD AM17 were detected by western blot and immunofluorescence.The TBI secondary damage in animals and cells was evaluated by HE staining,Evans blue,and tissue water content,CCK-8 assay.Then,the exogenous S100B,S100B inhibitor ONO-2506,RAGE inhibitor FPS-ZM1,and ADAM 17 inhibitor TAPI-1 were used to intervene the activity of the S100B/RAGE/ADAM17 signaling pathway,respectively,for the confirmation of its effect on endothelial glycocalyx shedding and secondary injury in TBI.Results:(1)The results of animal experiments showed that the protein expressions of S100B and RAGE in brain tissue of TBI rats were increased in a time-dependent manner,and the glycocalyx in brain vascular endothelium was shed and its content was decreased,all the changes were most obvious at 6 h after TBI.The levels of serum S100B,sRAGE and syndecan-1 levels were increased as well.(2)The stimulation of RAECs with exogenous S100B could lead to the enhancement of RAGE expression and damage of endothelial glycocalyx.The application of S100B inhibitor ONO-2506 or RAGE inhibitor FPS-ZM1 could attenuate the endothelial glycocalyx shedding in RAECs stimulated with conditioned medium,as well as in brain tissue of TBI rats,while the levels of syndecan-1 in medium and serum were reduced.(3)The expression and activity of ADAM17 protein was increased in RAECs stimulated with conditioned medium and in brain tissue of TBI rats.The application of ADAM 17 inhibitor TAPI-1 could prevent glycocalyx shedding in RAECs and in brain tissue without affecting the expression and activity of ADAM 17 protein.At the same time,the levels of sRAGE,syndecan-1,and levels in the medium and serum were down-regulated.(4)The stimulation of RAECs with exogenous S100B could enhance the protein expression,Golgi translocation,enzyme activity of ADAM17,and the release of microparticles containing AD AM 17,the levels of mADAM17 in the medium and serum were up-regulated.The application of S100B inhibitor and RAGE inhibitor could attenuate the protein expression,Golgi translocation,enzyme activity of ADAM17,and the release of microparticles containing AD AM 17 in RAECs stimulated with conditioned medium,and in brain tissue of TBI rats,the levels of mADAM17 in the medium and serum were down-regulated.(5)The stimulation with exogenous S100B could inhibit the cell viability of astrocytes.The application of S100B inhibitor ONO-2506,RAGE inhibitor FPS-ZM1,or ADAM 17 inhibitor TAPI-1,respectively,could increase the cell viability of stretch-injuried astrocytes.The pretreatment of those inhitors could also attenuate the damage of brain tissue with lower blood brain barrier permeability and less brain water content.While TBI induced obvious secondary lung injury,the application of those inhitors could alleviate lung damage with reduced pulmonary microvascular endothelial glycocalyx damage,lower lung permeability and less lung water content in TBI rats.Conclusions:This project confirms that S100B/RAGE mediates endothelial glycocalyx injury by upregulating ADAM 17 protein expression and enzyme activity,which promotes blood-brain barrier dysfunction,accelerates the formation of brain edema,and participates in the process of secondary injury after TBI.