Early release of high-mobility group box 1 (HMGB1) from neurons in experimental subarachnoid hemorrhage in vivo and in vitro

Abstract

BackgroundTranslocation of high-mobility group box 1 (HMGB1) from nucleus could trigger inflammation. Extracellular HMGB1 up-regulates inflammatory response in sepsis as a late mediator. However, little was known about its role in subarachnoid hemorrhage-inducible inflammation, especially in the early stage. This study aims to identify whether HMGB1 translocation occurred early after SAH and also to clarify the potential role of HMGB1 in brain injury following SAH.MethodsSprague-Dawley (SD) rats were randomly divided into sham group and SAH groups at 2 h, 12 h and on day 1, day 2. SAH groups suffered experimental subarachnoid hemorrhage by injection of 0.3 ml autoblood into the pre-chiasmatic cistern. Rats injected by recombinant HMGB1(rHMGB1) solution were divided into four groups according to different time points. Cultured neurons were assigned into control group and four hemoglobin (Hb) incubated groups. Mixed glial cells were cultured and stimulated in medium from neurons incubated by Hb. HMGB1 expression is measured by western blot analysis, real-time polymerase chain reaction (PCR), immunohistochemistry and immunofluorescence. Downstream nuclear factor kappa B (NF-κB) subunit P65 and inflammatory factor Interleukin 1β (IL-1β) were measured by western blot and real-time PCR, respectively. Brain injury was evaluated by cleaved caspase-3 staining.ResultsOur results demonstrated HMGB1 translocation occurred as early as 2 h after experimental SAH with mRNA and protein level increased. Immunohistochemistry and immunofluorescence results indicated cytosolic HMGB1 was mainly located in neurons while translocated HMGB1 could also be found in some microglia. After subarachnoid injection of rHMGB1, NF-κB, downstream inflammatory response and cleaved caspase-3 were up-regulated in the cortex compared to the saline control group. In-vitro, after Hb incubation, HMGB1 was also rapidly released from neurons to medium. Incubation with medium from neurons up-regulated IL-1β in mixed glial cells. This effect could be inhibited by HMGB1 specific inhibitor glycyrrhizic acid (GA) treatment.ConclusionHMGB1 was released from neurons early after SAH onset and might trigger inflammation as an upstream inflammatory mediator. Extracellular HMGB1 contributed to the brain injury after SAH. These results might have important implications during the administration of specific HMGB1 antagonists early in order to prevent or reduce inflammatory response following SAH.

RETRACTED: Regulatory T cells ameliorate intracerebral hemorrhage-induced inflammatory injury by modulating microglia/macrophage polarization through the IL-10/GSK3β/PTEN axis

“…On the following day, the microglia was stimulated using 10 mM hemoglobin (Sigma-Aldrich) for 6 h and washed with PBS twice. 31,33 CD4 þ CD25 þ regulatory T cells isolation kits (Miltenyi) and AutoMACS pro separators (Miltenyi) were used for Tregs sorting according to the instructions of the manufacturer. The purity of the CD45…”

HMGB1 Level in Cerebrospinal Fluid as a Marker of Treatment Outcome in Patients with Acute Hydrocephalus Following Aneurysmal Subarachnoid Hemorrhage

“…22 The 3 target receptors for HMGB1 (TLR2, TLR4, and RAGE) are normally expressed on the brain cells. 22,23 Their mutual downstream mediator, nuclear factor kappa B, not only triggers expression of proinflammatory mediators (iNOS, COX-2, ICAM-1, VCAM1, E-selectin, IL-1b, II-6, and TNF alpha) but also upregulates upstream receptors (TLR2, TLR4, and RAGE.). 7,23-27 As a result, overexpression of the above mediators and receptors activates both damaging and repair processes following SAH.…”

The Role of HMGB1 in Pial Arteriole Dilating Reactivity following Subarachnoid Hemorrhage in Rats

“…In one study, the plasma HMGB1 in patients with SAH upon admission was significantly higher than that in healthy controls and the levels of HMGB1 were associated with long-term poor neurological outcomes [10]. In experimental SAH, cytosolic HMGB1 has been significantly increased in neurons and microglia in rats, contributing to the induction of neuroinflammation and brain injury following SAH [11,12]. An increase in the expression of HMGB1 and its receptor TLR4 in the basilar artery after SAH has been recently demonstrated [13,14].…”