Mechanisms of Novel Herbal Therapies for Sepsis.

 描述(申请人提供)：尽管最近抗生素治疗和重症监护方面取得了进展，脓毒症仍然是重症监护病房中最常见的死亡原因，仅在美国每年就有225,000名患者死亡。脓毒症的发病机制尚不清楚，但部分原因是由晚期促炎介质(如HMGB1、CIRP和NO)维持的动态调节的炎症反应。我们的开创性发现HMGB1是致死性全身炎症(LSI)的晚期介质(Science，285：248-51,1999)，这一发现促使我们研究了传统草药对HMGB1分泌的复杂分子机制。甘草(甘草)传统上用于治疗消化性溃疡、肝损伤和肝炎，但其保护机制仍不清楚。我们的初步数据表明，甘草主要成分甘草酸的衍生物甘草酮(CBX)可剂量依赖地抑制脂多糖诱导的PKR(双链RNA激活蛋白激酶R)磷酸化和HMGB1分泌，并使小鼠免于盲肠结扎和穿孔(CLP)所致的致死性脓毒症(即使是延迟给药)。与此同时，有令人信服的证据支持：i)STAT1信号在细胞质HMGB1易位中的关键作用(PNAS，111：3068-73,2014)；以及ii)PKR在炎症体激活和HMGB1分泌中的关键作用(自然，488：670-4,2012)。鉴于Cx43和Panx1半通道蛋白在ATP外流中的可能作用，以及超纯脂多糖诱导的HMGB1分泌对ATP的需求，有必要检验一个新的假说，即粗制的内毒素或其他关键的HMGB1分泌刺激物(如SAA和CIRP)持续刺激可能导致Panx1和/或Cx43半通道蛋白上调，从而促进依赖于ATP的PKR和STAT1的磷酸化，从而促进HMGB1的分泌和致死性全身炎症(LSI)。因此，我们建议检验：1)新的假设，粗制的内毒素和其他重要的炎症刺激(例如，SAA和CIRP)上调Panx1和Cx43半通道的表达，并触发PKR磷酸化和HMGB1的分泌(目标1)；2)Panx1和Cx43半通道在致死性全身炎症(LSI)发病机制中的新作用(目标2)；以及3)Panx1或Cx43半通道抑制物(如模拟肽拮抗剂、中和抗体以及草药抑制剂如GZA和CBX)影响LSI结果的新机制(目标3)。总之，这些拟议的研究将提高我们对草药有效抑制外源性PAMP或内源性促炎介质诱导的HMGB1分泌的机制的理解，并有助于开发新的半通道靶向治疗LSI。

 DESCRIPTION (provided by applicant): Despite recent advances in antibiotic therapy and intensive care, sepsis remains the most common cause of death in the intensive care units, claiming > 225,000 victims annually in the U.S. alone. The pathogenesis of sepsis remains obscure, but is partly attributable to dys-regulated inflammatory responses sustained by late pro-inflammatory mediators (e.g., HMGB1, CIRP, and NO). Our seminal discovery of HMGB1 as a late mediator of lethal systemic inflammation (LSI) (Science, 285: 248-51, 1999) has prompted an investigation of the intricate molecular mechanisms underlying the pharmacological modulation of HMGB1 secretion by traditional herbal medicine. Gancao (Radix glycyrrhizae, or licorice) has been traditionally used in the treatment of peptic ulcer, hepatic injury, and hepatitis, but its protective mechanisms remain elusive. Our preliminary data indicated that carbenoxolone (CBX), a derivative of the major Gancao component, glycyrrhizin (glycyrrhizic acid, GZA), dose-dependently abrogated LPS-induced PKR (double-stranded RNA-activated protein kinase R) phosphorylation and HMGB1 secretion, and rescued mice from lethal sepsis (induced by cecal ligation and puncture, CLP) even if given in a delayed fashion. Meanwhile, compelling evidence has emerged to support the critical roles of: i) STAT1 signaling in cytoplasmic HMGB1 translocation (PNAS, 111: 3068-73, 2014); and ii) PKR in inflammasome activation and HMGB1 secretion (Nature, 488: 670-4, 2012). In light of the possible role of Cx43 and Panx1 hemichannels in ATP efflux, as well as the requirement of ATP for ultrapure LPS-induced HMGB1 secretion, it is important to test a novel hypothesis that prolonged stimulation with crude LPS or other key HMGB1 secretion stimuli (such as SAA and CIRP) may lead to upregulation of Panx1 and/or Cx43 hemichannel proteins, which facilitate ATP-dependent PKR and STAT1 phosphorylation, thereby contributing to HMGB1 secretion and lethal systemic inflammation (LSI). Accordingly, we propose to test: 1) the novel hypothesis that crude LPS and other important inflammatory stimuli (e.g., SAA and CIRP) up-regulate Panx1 and Cx43 hemichannel expression, and trigger PKR phosphorylation and HMGB1 secretion (Aim 1); 2) the novel roles of Panx1 and Cx43 hemichannels in the pathogenesis of lethal systemic inflammation (LSI) (Aim 2); and 3) the novel mechanisms by which Panx1 or Cx43 hemichannel inhibitors (such as mimetic peptides antagonists, neutralizing antibodies, and herbal inhibitors such as GZA and CBX) affect the outcomes of LSI (Aim 3). Collectively, these proposed studies will improve our understanding of the mechanisms by which herbal medicine effectively inhibits exogenous PAMPs or endogenous proinflammatory mediators-induced HMGB1 secretion, and shed light on the development of novel hemichannel-targeting therapies for LSI.