The present study demonstrated that in both animal and cellular model, TBI triggered the elevation of S100B in the brain tissue and serum. The release of S100B might work with RAGE through paracrine and systemic response, resulting in further synthesis and secretion of S100B from astrocytes. The activation of S100B/RAGE signal after TBI could mediate endothelial glycocalyx shedding by enhancing the protein expression and enzyme activity of ADAM17 in endothelial cells. We further confirmed that the activation of S100B/RAGE could also induce the Golgi translocation of ADAM17 and its localization in W-P bodies of RAECs, subsequently promoting endothelial glycocalyx damage, and aggravating brain tissue injury. Systemically, the activation of S100B/RAGE-ADMA17 pathway could cause the damage of pulmonary microvascular endothelial glycocalyx, increased vascular permeability, and consequently leading to secondary lung injury.
TBI triggers the release of S100B
S100B has been considered as a biomarker of brain injury and the increase of S100B in serum can reflect the early changes of BBB function and nerve cell damage [26, 27]. Consistent with many clinical observations, in present study, the results from TBI models in vivo by experimental lateral fluid percussion injury in rats and in vitro by stretch injury in primary astrocytes demonstrated significant increases in S100B level both in brain and serum, but also and in astrocytes and medium early after injury. Several studies have shown that the overproduction of S100B by activated astrocytes after brain injury further enhances microglial and astrocyte activation, leading to neuroinflammation [7, 28]. Our study also found that inhibition of S100B could significantly alleviate the pathological damage of brain and lung tissues, indicating that S100B plays an important role in mediating the secondary injury of TBI by working as one of the DAMPs [29].
S100B and RAGE mutually regulate their expression and activation
S100B could exert its effects by binding with RAGE [30] In our study, the level of RAGE expression in brain tissue and astrocytes, and the level of sRAGE in serum and medium were all significantly up-regulated by TBI or stretch injury. The inhibition of S100B attenuated those up-regulation and the inhibition of RAGE reversed the enhancement of S100B either, indicating the mutual regulation of S100B and RAGE. The inhibition of RAGE also attenuated TBI-induced brain and lung damage, and improved astrocyte viability after stretch injury, suggesting that the activation of the ligand/RAGE signaling pathway may be an important factor in mediating the secondary damage of TBI.
The activation of S100B/RAGE results in endothelial glycocalyx shedding
Current studies have shown that the integrity of endothelial glycocalyx is damaged to varying degrees in TBI, especially in secondary injury [31, 32]. Indicating by the changes of syndeca-1 content and location in brain tissue and serum, as well as in endothelial and cultured medium, the results in present study demonstrated that astrocyte-derived S100B and the subsequent RAGE activation on endothelial cells after TBI and stretch injury can induce endothelial glycocalyx damage in brain and lung as well. These data suggested the S100B/RAGE-induced glycocalyx shedding from endothelial cells might be one of the critical steps in initiating secondary damage such as BBB dysfunction, brain edema or remote organ injury after TBI.
S100B/RAGE evokes EG damage by inducing ADAM17 expression and translocation
For the mechanism of S100B/RAGE-induced glycocalyx shedding, ADAM17, one of the major sheddases, emerged in our study. ADAM17 exists as immature proform (pADAM17) and as mature protease (mADAM17) in cells [33, 34]. In this study, we first demonstrated that the changes of syndecan-1 after TBI was accompanied with significant increased expression of both pADAM17 and mADAM17, implying the involvement of ADAM17 expression in endothelial glycocalyx shedding during the development of secondary TBI. A variety of inflammatory mediators are responsible in stimulating ADAM17 expression [33]. For the first time, present study found that the application of exogenous S100B enhanced the expression of pADAM17 and mADAM17, and the inhibition of S100B/RAGE signaling abolished TBI-induced ADAM17 expression, suggesting the activation of S100B/RAGE pathway triggered the expression of ADAM17 after TBI.
The sheddase activity of ADAM17 is modulated by the translocation of ADAM17 from ER to Golgi apparatus, where the maturation takes place [35]. This study found that the inhibition of S100B/RAGE signaling abolished the increase location of ADAM17 in Golgi apparatus in endothelial cells after TBI, confirming that TBI-induced activation of S100B/RAGE pathway also regulates the translocation and maturation of ADAM17. Most of the mature ADAM17 seems to be intracellularly located whilst only a small amount is actually at the cell surface, where shedding can take place [33, 36]. The S100B/RAGE-enhanced localization of mADAM17 with vWF in RAEC W-P bodies indicated that ADAM17 could be secreted through the release of W-P bodies from injured endothelial cells, then reach to distant organs and tissue [37]. Our findings imply that the detection of plasma ADAM17 level and activity might help to monitor disease progression in TBI with endothelial involvement [38].
The inhibition of ADAM17 activity is capable of attenuating EG damage
The activation of mADAM17 relied on trafficking to the cell surface, where shedding can take place [34]. In this study, ADAM17 inhibitor, TAPI-1, a specific hydroxamate inhibitor of metalloprotease disintegrins, reversed the elevation of syndecan-1, without changing the content of pADAM17 and mADAM17, confirming the activation of ADAM17 after maturation in inducing glycocalyx shedding. The impaired glycocalyx barrier is more conducive to inflammatory adhesion to endothelial cells, promoting thrombosis and cell damage, thus leading to a vicious circle [10]. In this study, the inhibition of ADAM17 activity with TAPI-1 could significantly reverse the shedding of endothelial glycocalyx both in endothelial cells and brain tissue, implying the effect of antagonizing ADAM activity in protecting endothelial barrier structure.
The S100B/RAGE-ADMA17-induced EG damage is involved in primary and secondary TBI
Astrocytes and endothelial cells are the key cells to maintain the normal function of BBB [39]. In this study, the protective effect of inhibiting S100B/RAGE signaling and ADAM17 activation on pathological injury and BBB dysfunction in TBI rat model confirms the involvement of this S100B/RAGE-induced astrocyte activation and ADAM17-evoked endothelial glycocalyx shedding in the development of primary and secondary TBI in vivo.
TBI-induced acute lung injury (TBI-induced ALI) is regarded as the most common complication of severe TBI that is an independent predictor of poor outcomes in TBI patients and strongly increases the mortality [40]. Concurred with those seen in other studies [41, 42], our previous study has shown that TBI-induced activation of S00B could mediated the development of neutrophil extracellular traps (NETs) in the lung, leading to subsequent ALI [43]. Present study provides more evidence to reveal that S100B/RAGE and ADAM17 activation triggered the shedding of pulmonary endothelial glycocalyx and this change could be rescued with inhibitors targeting this pathway, suggesting that S100B/RAGE/ADAM17 is not only a key signal of astrocyte and endothelial cell damage, but also an important participant in local or systemic secondary injury after TBI. Therefore, we should actively remedy the complications like lung injury while treating the primary injury, and prevent the secondary injury from further aggravating the pathogenetic condition of TBI patients.
There are some limitations to consider when drawing conclusions from this study. First, the activation of ADAM17 is a multi-step, gradually-progressed and strictly-regulated process, the critical regulatory mechanism of S100B/RAGE signal on this complex process remains to be further studied. Second, while the secondary brain and lung injuries cause the major organ dysfunctions after TBI, the effect of S100B/RAGE/ADAM17 pathway on other organ functions, such as coagulation and renal function, remains to be investigated. Third, pharmacological interventions used in study still have some unavoidable shortcomings, further RNA interference and gene knocking out should be considered in future research.