Role of Intestinal Proteostasis mediator HSP90 in alcoholic liver disease.

摘要 长期酒精改变肠道微生物组（生态失调），诱导炎症，损害肠道屏障功能 导致肠道“渗漏”、微生物产物进入门静脉循环并最终导致肝脏炎症。 目前靶向微生物组或宿主途径以恢复屏障功能的疗法已经利用抗生素， 益生菌，合生元，粪便微生物组移植（FMT），噬菌体治疗，FXR激动剂和营养素- 基于治疗。该提案的主要目标是研究蛋白质稳态介导剂的致病作用， HSP 90在肠道中通过肠道靶向在体内使用特异性药理学抑制剂来评价其对 酒精介导的宿主肠道炎症、屏障功能和生态失调。两种主要形式的 哺乳动物应激诱导的HSP 90旁系同源物是细胞质（CYT）HSP 90 AA 1和HSP 90 B1/GP 96， 内质网（ER）。HSP 90和GP 96的伴侣功能在免疫细胞的微调中至关重要 激活和炎症反应。蛋白稳态伴侣如HSP 90在慢性炎症中的确切作用 酒精介导的肠功能障碍尚未研究。我们报道了使用特异性的靶向HSP 90， 药理学抑制剂17-DMAG减少ALD中肠源性循环内毒素。髓系特异性 HSP 90 B1/GP 96缺陷型小鼠在ALD中也表现出减少的循环内毒素。进一步我们的初步数据 显示慢性酒精暴露小鼠肠中HSP 90 AA 1和HSP 90 B1/GP 96 mRNA诱导。在 此外，17-DMAG在肠道中是生物可利用的，并抑制空肠中的炎性细胞因子TNFα， 回肠我们假设慢性酒精诱导宿主肠道中的HSP 90，导致炎症， 损害肠屏障功能。此外，由于肠道炎症的治疗靶向可以恢复 保护性微生物群，以及考虑到17-DMAG的抗生素和抗真菌特性，很可能 17-DMAG治疗有利于保护肠道微生物群，通过减少宿主肠道炎症来促进。 总的来说，我们将探讨热休克蛋白90在酒精介导的肠道炎症和屏障功能中的作用。 并评估宿主HSP 90的药理学靶向是否影响肠道微生物群。具体目标 主要内容如下：1）通过检测HSP 90在酒精诱导的肠道炎症反应中的表达， HSP 90同种型，使用17-DMAG包封的纳米颗粒研究HSP 90的肠靶向， 小肠中的炎症反应和固有层中的免疫细胞，并评估是否有髓样细胞 特异性HSP 90 B1/GP 96缺陷影响肠道中的炎症反应。2)评估功能 通过评价通透性探讨HSP 90与酒精介导的肠屏障完整性和生态失调的相关性 和髓系GP 96抑制过程中的上皮紧密连接蛋白。B）评估 肠靶向纳米DMAG和髓样GP 96缺陷对微生物群的影响。成功完成 拟议的研究将揭示蛋白质稳态伴侣在肠道中的作用，并指导未来的研究，以确定 酒精介导的肠道损伤中的蛋白稳态途径。

ABSTRACT Chronic alcohol alters the enteric microbiome (dysbiosis), induces inflammation, impairs the gut barrier function resulting in a “leaky” gut, passage of microbial products into portal circulation and eventually liver inflammation. Current therapies targeting the microbiome or host pathways to restore barrier function have utilized antibiotics, probiotics, synbiotics, fecal microbiome transplant (FMT), bacteriophage therapy, FXR agonists and nutrient- based treatments. The major goal of this proposal is to investigate the pathogenic role of proteostasis mediator, HSP90 in the gut by intestinal targeting in vivo using specific pharmacological inhibitors to evaluate its effect on alcohol mediated host intestinal inflammation, barrier function and dysbiosis. Two predominant forms of mammalian stress inducible HSP90 paralogs are cytoplasmic (CYT) HSP90AA1 and HSP90B1/GP96 in the endoplasmic reticulum (ER). The chaperone function of HSP90 and GP96 is crucial in fine tuning immune cell activation and inflammatory responses. The precise role of proteostasis chaperones such as HSP90 in chronic alcohol mediated intestinal dysfunction is not yet investigated. We reported that targeting HSP90 using specific pharmacological inhibitor 17-DMAG, reduced gut derived circulating endotoxin in ALD. Myeloid specific HSP90B1/GP96 deficient mice also exhibit reduced circulating endotoxin in ALD. Further our preliminary data show induction of HSP90AA1 and HSP90B1/GP96 mRNA in intestine of chronic alcohol exposed mice. In addition, 17-DMAG is bioavailable in the intestine and inhibits inflammatory cytokine, TNFα in the jejunum and ileum. We hypothesize that chronic alcohol induces HSP90 in host intestine contributing to inflammation and compromising gut barrier function in ALD. In addition, since therapeutic targeting of gut inflammation can restore protective microbiota, as well as considering the antibiotic and antimycotic properties of 17-DMAG, it is likely that 17-DMAG treatment favors protective gut microbiota, facilitated by reduced host intestinal inflammation. Collectively, here we will explore the role of HSP90 in alcohol mediated gut inflammation and barrier function and also assess whether pharmacological targeting of host HSP90 impacts gut microbiota. The Specific Aims are: 1) To delineate the role of HSP90 isoforms on alcohol induced gut inflammation by- Evaluating expression of HSP90 isoforms, intestinal targeting of HSP90 using 17-DMAG encapsulated nanoparticles to study inflammatory responses in small intestine and immune cells in lamina propria and assessing whether myeloid specific HSP90B1/GP96 deficiency affects inflammatory responses in the gut. 2) To assess the functional relevance of HSP90 on alcohol mediated intestinal barrier integrity and dysbiosis by - Evaluating permeability and epithelial tight junction proteins during HSP90 and myeloid GP96 inhibition. B) Assessing the effect of intestine targeted nano-DMAG and myeloid GP96 deficiency on microbiota. Successful completion of the proposed studies will uncover the role of proteostasis chaperones in the gut and guide future studies to identify proteostasis pathways in alcohol mediated gut injury.