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，脓毒症仍然是导致 仅在美国，每年就有225,000人死于重症监护病房。黄斑狼疮的发病机制 脓毒症仍不清楚，但部分可归因于“早期”传播的动态调节炎症。 细胞因子(例如，肿瘤坏死因子和IL-1β)，但由“中间”(例如，SA)和“晚期”(例如，HMGB1)介体维持。 我们最近发现，LPS和SAA上调了连接蛋白43(Cx43)和/或PAnnexin的表达 1(Panx1)半通道促进ATP依赖的PKR激活和HMGB1释放，但不知道 脂多糖和沙门冬氨酸是否也诱导L原蛋白分泌介导致死性细菌感染 (LBI)。我们的初步数据表明，LPS和SAA诱导了PCTS-1的显著表达和分泌。 L在小鼠巨噬细胞和人单核细胞培养中的表达。因此，PCTS-L在 健康动物或人类受试者的循环，但在败血症动物和 病人。高纯度重组PCTS-L刺激原代人单核细胞释放多种 趋化因子，以及促炎细胞因子(如肿瘤坏死因子和白介素1β)和抗炎细胞因子(如白介素10)，以及 体内加重内毒素血症致死率。相比之下，PCTS-L中和抗体显著挽救 小鼠的致死性脓毒症，提示PCTS-L是另一种低密度脂蛋白I的晚期调节因子。与此同时，半高 800种天然产物和1360种美国药物的生产能力筛选 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)和黄体酮 (Pro)具有显著的结构相似性和相似的PCTS-L抑制活性。虽然局域网是一种 在一些药用植物中含有丰富的次生代谢产物，包括枸杞和阿维洛兹，它也是一种 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 建议调查PCTS-L在杠杆作用中的新角色，以及天然 产品兰及其衍生物PRO对PCTS-L诱导的炎症反应有抑制作用。中概述的实验 目的1验证PCTS-L在脓毒症患者循环中系统性蓄积的假说。 与脓毒症的其他替代标志物相关。在目标2中，我们将测试以下假设 PCTS-L水平(通过补充PCTS-L或基因敲除)或活动(通过使用中和IgG或 天然抑制剂LAN或PRO)不同程度地影响LBI的结果。目标3中概述的实验 将检验LAN和/或PRO通过损害PCTS-L诱导的炎症而抑制PCTS-CHO诱导的炎症的假说 TLR4/RAGE依赖的半通道和PKR的体外激活，并部分通过以下方式提供对LBI的保护 减轻全身炎症和体内相关的凝血失调。总体而言，这个项目 将加深我们对PCTS-L在杠杆作用中的作用的理解，并揭示其复杂的机制 潜在的天然产品局域网和亲介保护，以防止致命的细菌感染。

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.