Mechanisms of Novel Herbal Therapies for Sepsis.

项目摘要 尽管最近在抗生素治疗和重症监护方面取得了进展，脓毒症仍然是最常见的原因， 在重症监护室死亡，每年仅在美国就有超过225，000名受害者。的发病机制 脓毒症仍不清楚，但部分归因于“早期”炎症传播的失调。 细胞因子（例如，TNF和IL-1β），但通过“中间体”（例如，SAA）和“晚”（例如，HMGB 1）介质。 我们最近发现，LPS和SAA上调连接蛋白43（Cx43）和/或泛连接蛋白的表达， 1（Panx 1）半通道，以促进ATP依赖性PKR激活和HMGB 1释放，但不知道 LPS和SAA是否也诱导前组织蛋白酶L（pCTS-L）分泌介导致死性细菌感染 （LBI）。我们的初步数据表明，LPS和SAA诱导pCTS的显着表达和分泌， L在鼠巨噬细胞和人单核细胞培养物中。因此，pCTS-L在小鼠中未检测到。 健康动物或人类受试者的血液循环，但在脓毒症动物的血液中显著升高， 患者高度纯化的重组pCTS-L刺激原代人单核细胞释放各种 趋化因子，以及前-（例如，TNF和IL-1β）和抗炎细胞因子（例如，IL-10），和 加重了体内内毒素血症致死性。相比之下，pCTS-L-中和抗体显著挽救了 pCTS-L是LBI的另一种晚期介质。与此同时， 对800种天然产物的NatProduct Collection和1360种US Drug Collection进行通量筛选 生物活性化合物导致了一些先导化合物的发现[包括羊毛甾醇（LAN）和孕酮 （PRO）]具有显著的结构相似性和相似的pCTS-L抑制活性。虽然LAN是一个 枸杞子、牛油果等药用植物中含有丰富的次生代谢产物， 在动物和人类中合成PRO的底物。这些令人兴奋的发现促使目前 研究pCTS-L在LBI中的新作用的建议，以及天然LBI的复杂机制， 产物LAN及其衍生物PRO抑制pCTS-L诱导的炎症。中概述的实验 目的1将检验pCTS-L在脓毒症患者的循环中全身累积的假设， 与败血症的其他替代标志物相关。在目标2中，我们将检验以下假设： pCTS-L水平（通过补充pCTS-L或基因敲除）或活性（通过使用中和IgG或 天然抑制剂LAN或PRO）对LBI的结果有不同的影响。目标3中概述的实验 将检验LAN和/或PRO通过损害细胞的免疫功能来抑制pCTS-L诱导的炎症的假设。 TLR 4/RAGE依赖性半通道和PKR激活，并通过以下方式部分保护LBI： 减轻全身性炎症和相关的体内凝血失调。总的来说，这个项目 这将有助于我们更好地理解pCTS-L在LBI中的作用，并阐明其复杂的机制。 潜在的天然产物LAN和PRO介导的针对致命细菌感染的保护。

Project Abstract Despite recent advances in antibiotic therapy and intensive care, sepsis remains the most common cause of death in the intensive care unit, annually claiming >225,000 victims in the U.S. alone. The pathogenesis of sepsis remains obscure, but is partly attributable to dys-regulated inflammation propagated by “early” cytokines (e.g., TNF and IL-1β), but sustained by “intermediate” (e.g., SAA) and “late” (e.g., HMGB1) mediators. We recently discovered that LPS and SAA upregulated the expression of connexin 43 (Cx43) and/or pannexin 1 (Panx1) hemichannels to facilitate ATP-dependent PKR activation and HMGB1 release, but did not know whether LPS and SAA also induced procathepsin L (pCTS-L) secretion to mediate lethal bacterial infections (LBI). Our preliminary data indicated that LPS and SAA induced a marked expression and secretion of pCTS- L in both murine macrophage and human monocyte cultures. Consequently, pCTS-L was not detectable in the circulation of healthy animals or human subjects, but significantly elevated in the blood of septic animals and patients. Highly purified recombinant pCTS-L stimulated primary human monocytes to release various chemokines, as well as pro- (e.g., TNF and IL-1β) and anti-inflammatory cytokines (e.g., IL-10) in vitro, and exacerbated endotoxemic lethality in vivo. In contrast, pCTS-L-neutralizing antibodies significantly rescued mice from lethal sepsis, suggesting pCTS-L as another late mediator of LBI. Meanwhile, a semi-high throughput screening of a NatProduct Collection of 800 natural products and a US Drug Collection of 1360 bioactive compounds led to the finding of a few lead compounds [including lanosterol (LAN) and progesterone (PRO)] with striking structural resemblance and similar pCTS-L-inhibiting activities. Although LAN is an abundant secondary metabolite in some medicinal plants including wolfberry and aveloz, it also serves as a substrate for the synthesis of PRO in animals and humans. These exciting findings prompted the current proposal to investigate a novel role of pCTS-L in LBI, as well as intricate mechanisms by which a natural product, LAN, and its derivative, PRO, inhibit pCTS-L-induced inflammation. The experiments outlined in Aim 1 will test the hypothesis that pCTS-L systemically accumulates in the circulation of septic patients and correlates with other surrogate markers of sepsis. In Aim 2, we will test the hypothesis that alterations of pCTS-L levels (by supplementation of pCTS-L or genetic knockout) or activities (by use of neutralizing IgGs or natural inhibitor LAN or PRO) divergently influence the outcomes of LBI. The experiments outlined in Aim 3 will test the hypothesis that LAN and/or PRO inhibit the pCTS-L-induced inflammation through impairing the TLR4/ RAGE-dependent hemichannel and PKR activation in vitro, and confer protection against LBI partly by attenuating systemic inflammation and associated dysregulated coagulation in vivo. Collectively, this project will improve our understanding of the role of pCTS-L in LBI, and shed light on the intricate mechanism underlying natural product LAN and PRO-mediated protection against lethal bacterial infections.