The Role And Mechanisms Of High-mobility Group Box 1(HMGB1) In Early Brain Injury After Experimental Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH), especially aneurysm subarachnoid hemorrhage, is a life-threatening disease of central nervous system (CNS). Although its relatively low incidence, its early age of onset and poor outcome results in a lot of life years lost. Clinical studies have shown that increased levels of pro-inflammatory factors in cerebrospinal fluid (CSF) and serum of SAH patients are associated with poor outcome. Elevated inflammatory factors contribute to the breakage of blood brain barrier (BBB), brain edema, neuroglia cells apoptosis and death. Administration of the antagonists of the pro-inflammatory factors confers neuroprotective effect in experimental studies. However, how the inflammatory response is initiated and the upstream of the inflammatory response is still poorly investigated.High-mobility group box 1(HMGB1), as a nonhistone protein binding with DNA, is widely expressed in the nucleus of nearly all eukaryotic cells, including the brain cells, and stabilizes nuclesome formation, facilitates gene transcription. Growing evidence shows that HMGB1 is passively released from necrotic cells or actively secreted from immune cells or non-immune parenchymal cells under various pathological conditions. Extracellular HMGB1 serves as alarmin or damage-associate molecular pattern (DAMP) that mediates cross-talk between damaged cells and relative healthy cells and triggers inflammatory response after interaction with toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4) and receptors for advanced glycation end-products (RAGE). Extracellular HMGB1 was regarded as a late inflammatory mediator in sepsis while as an early mediator in ischemia inducible models. However, little is known about the role of HMGB1 in the early brain injury after SAH. We supposed that the HMGB1 might translocate early from nucleus to cytoplasm after SAH and promote inflammation after being released into extracellular parts.The most urgent aim was to identify whether HMGB1 translocation occurred early after SAH and also to detect the expression level of HMGB1 in the early stage following SAH. Western blot, real-time PCR, immunohistochemistry and immunofluorescence were employed to observe the expression and location of HMGB1. Our research found that HMGB1 translocation occurred as early as 2 hours after experimental SAH with mRNA and protein levels up-regulated. Immunohistochemistry and immunofluorescence results indicated cytosolic HMGB1 was mainly from neurons while translocated HMGB1 could also be found in some microglia.Then we have detected the early translocation of HMGB1 from neurons. However, we are not sure whether HMGB1 released from neurons could trigger inflammatory response in surrounding glial cells. Thus hemoglobin(Hb) incubated neurons injury model and mixed glial cells cultured were employed. Supernatants from neurons treated by Hb incubation were collected and added to the medium of mixed glial cells. The results demonstrated that neurons released HMGB1 fast and early after Hb incubation. Then Hb were removed from supernatants. These supernatants could upregulated the inflammatory response in mixed glial cells. This effect could be inhibited by treatment of glycyrrhizic acid (GA), a specific HMGB1 inhibitor. These results indicated that HMGB1 from neurons could initiate the inflammatory response of glial cells. Further, to determine whether extracellular HMGB1 played an important role in the early brain injury, we designed experiments injecting recombinant HMGB1 (rHMGB) to the subarachnoid space in the rats and observed the inflammation and brain injury in brain tissue. P65 subunit is the primary pro-inflammatory subunit of NF-κB which located in the center of inflammatory response. Many researches used nuclear P65 protein level to evaluated the NF-κB activity. Thus in this study P65 protein level was used to evaluated the activity of NF-κB. Our results demonstrated that rHMGB 1 induced increased level of TLR4, IL-1β and nuclear P65 subunit. Thus, it was demonstrated rHMGB 1 up-regulated the inflammation in brain tissue. Further, compared with saline control group, the number of cells positive for cleaved caspase 3 and NeuN was increased in rHMGB 1 treatment group which suggested that rHMGB 1 might be a harmful molecule for brain cells. To identify through which signal pathway HMGB1 took effects, we used the TLR4 antagonist (TAK242) by intraventricular injection and observed the changes of NF-κB subunit P65 and inflammatory factors. Our results showed that NF-κB (P65) and downstream inflammatory factor was down-regulated after treatment of TLR4 antagonist, TAK242. This result suggested that TLR4 signal pathway took a great part in the inflammatory response induced by HMGB1.To find a treatment which targeted HMGB1, we used the HMGB1 inhibitor, glycyrrhizic acid (GA) which was reported to have benefical effect in brain ischemia models. GA were injected by vein. High dose (10mg/kg) and low dose of GA (5mg/kg) were both tested. Our result indicated that GA treatment could suppress the release of HMGB1, inhibit the cytokine activity of HMGB1, and down-regulate inflammatory response induced by SAH. Further, high dose GA treatment could reduce the brain edema significantly. Thus, numbers of TUNEL-positive or Fluoro-Jade C staining positive cells were reduced after GA treatment. These results suggested that GA can attenuate the early brain injury following SAH in rats.In conclusion, HMGB1 was released mainly from neurons early after SAH onset. Released HMGB1 could influence glial cells nearby and trigger inflammatory response through TLR4 and inflammation triggered by HMGB1 played an important role in the early brain injury after SAH. HMGB1 could be an important initiator of the inflammation after SAH. GA attenuated early brain injury through inhibition of HMGB1 activity and translocation of HMGB1.