HMGB1 inhibitor glycyrrhizin attenuates intracerebral hemorrhage-induced injury in rats
Highlights
► Glycyrrhizin attenuates thrombin-induced injury in rat slice cultures. ► The protective effects of glycyrrhizin are involved in HMGB1. ► Glycyrrhizin reduces brain edema in rat intracerebral hemorrhage model. ► Glycyrrhizin improves neurological deficit by intracerebral hemorrhage. ► Glycyrrhizin shows neuroprotection against hemorrhagic brain injury.
Introduction
Breakdown of cerebral blood vessels induces leakage of blood intrinsic factors, including a serine protease thrombin, into the brain parenchyma. So far, our studies have revealed that thrombin-induced activation of mitogen-activated protein kinases (MAPKs) and mobilization of microglia are deeply involved in induction of tissue injury of the striatum, a brain region liable to intracerebral hemorrhage (ICH), in organotypic slice cultures (Fujimoto et al., 2006, Katsuki, 2010). In addition, intrastriatal injection of thrombin in vivo induced acute necrosis and delayed apoptosis of neurons (Fujimoto et al., 2007). Corresponding mechanisms are also involved in pathogenic events in ICH model in vivo, where MAPKs support survival of cytotoxic microglia (Ohnishi et al., 2007). Accordingly, suppression of these deleterious factors can be new therapies for hemorrhagic brain injury. However, we have also shown that MAPKs play divergent roles in ICH insults. For example, the effects of inhibitors of each MAPK family member on the number of activated microglia did not always correlate with their neuroprotective effects. Moreover, activation of MAPKs was observed not only in microglia but also in neurons. For these reasons, direct inhibition of MAPKs is unlikely to be a realistic target for ICH therapy.
Meanwhile, a nucleoprotein high-mobility group box 1 (HMGB1) is expressed in various cells including neurons and microglia. HMGB1 is normally bound to DNA, and is released from cells undergoing necrosis but not from those undergoing apoptosis (Müller et al., 2001, Scaffidi et al., 2002). Although Hayakawa et al. suggested that HMGB1-expressing astrocytes contribute to neurovascular remodeling upon the chronic stage of focal cerebral ischemia in mice (Hayakawa et al., 2010), released HMGB1 behaves like an inflammatory mediator by acting on receptor for advanced glycation end products (RAGE) or Toll-like receptors (TLRs) 2 and 4 (Kokkola et al., 2005, Park et al., 2004). These receptors are expressed not only in peripheral macrophages, but also in microglia and neurons in the central nervous system (Hori et al., 1995, Tang et al., 2007). Emerging lines of evidence have suggested that neurons injured by thrombin may signal for activation of microglia (Choi et al., 2005). Released HMGB1 accelerates production of proinflammatory mediators through activation of MAPK family members (Kokkola et al., 2005, Tang et al., 2007). Moreover, anti-HMGB1 neutralizing antibody has been shown to ameliorate ischemic brain injury (Liu et al., 2007). Accordingly, HMGB1 may play an important role as the trigger of microglial activation to expand tissue injury, during acute stage of ICH.
Glycyrrhizin, an ingredient of the licorice root, has long been known to exhibit glucocorticiod-like anti-inflammatory actions by inhibiting 11β-hydroxysteroid dehydrogenase (Monder et al., 1989). More recently, glycyrrhizin has been also shown to bind to, and inhibit cytokine-like activity of, HMGB1 (Mollica et al., 2007). Here we examined the effect of glycyrrhizin on ICH-related pathogenic events in vitro and in vivo.
Section snippets
Drugs and chemicals
Drugs and chemicals were obtained from Nacalai Tesque (Kyoto, Japan), unless otherwise indicated. Thrombin from bovine plasma, collagenase type IV, glycyrrhizin, hydrocortisone, RU486 (mifepristone) and N-methyl-d-aspartic acid (NMDA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). HMGB1 was obtained from Chondrex (Redmond, WA, USA). Not less than 90% of the purified HMGB1 has been confirmed by SDS-PAGE and its cytokine-like activity has been assured by tumor necrosis factor (TNF)-α
Glycyrrhizin attenuated thrombin-induced cortical cell death by antagonizing HMGB1, but not through glucocorticoid system
As reported previously by us (Fujimoto et al., 2006, Fujimoto et al., 2008, Ohnishi et al., 2009, Ohnishi et al., 2010a, Ohnishi et al., 2010b), application of 100 U/ml thrombin to cortico-striatal slice cultures for 72 h led to necrotic cell death in the cortical region as indicated by increased intensity of PI fluorescence, and also to prominent shrinkage of the striatal region. Co-application of glycyrrhizin (10–100 μM) with thrombin significantly attenuated thrombin-induced cortical cell
Discussion
The present study addressed the effect of glycyrrhizin on pathogenic events related to ICH. We demonstrated that glycyrrhizin produced cytoprotective effect against thrombin toxicity in cortico-striatal slice cultures. This in vitro model well reflects thrombin toxicity as part of ICH-associated damage. Glycyrrhizin is known to possess glucocorticoid-like anti-inflammatory properties, due to its inhibitory activity on 11β-hydroxysteroid dehydrogenase (Monder et al., 1989). In the present study,
Acknowledgments
This work was supported by Strategic Support Project of Research Infrastructure Formation for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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