TLR4介导NLRP3炎症小体活化对肠外营养肝损害影响机制研究

The pathogenesis of parenteral nutrition-associated liver damage ( PNALD ) remains unclear. Our previous studies found that intestinal bacteria translocation and its generated endotoxemia, as well as oxidative stress, were related to the development of PNALD. NLRP3 inflammasome has been shown to be involved in several diseases, through induction of inflammatory responses after activation by a wide spectrum of pathogenic or danger signals. Studies found that the Toll like receptor 4 (TLR4) was engaged in priming NLRP3 inflammasome, while oxidative products might work as a stimuli of NLRP3 inflammasome in "activation step". Hence, we made the following hypothesis: during parenteral nutrition, increased gut derived endotoxin leads to NLRP3 inflammasome activation through TLR4 signal pathway, with the results of inflammation and liver injury, which might be an important mechanism of PNALD. In order to demonstrate this, we will carry out the following experiments:.1. To find the relationship between NLRP3 inflammasome activation and parenteral nutrition induced liver injury: we will observe the mRNA and protein expression of NLRP3 inflammasome components and the levels of downstream inflammatory markers in mice on parenteral nutrition of different duration..2. To observe the influence of NLRP3 overexpression and suppression on liver damage and associated signal pathway ,using NLRP3 expression and shRNA Adeno-associated virus vectors..3. To observe the impact of TLR4 signal change on NLRP3 inflammasome activation, using TLR4 knockout mice and a TLR4 upexpression model through intraperitoneal LPS injection..Our study will help to elucidate the molecular mechanism of PNALD,and identify key molecular targets for further prevention and therapy.

肠外营养相关肝损害（PNALD）至今机制未明。我们前期实验发现氧化应激和肠源性内毒素与PNALD密切相关。NLRP3 炎症小体可由多种病原体和危险信号激活，诱发炎症反应，是否参与PNALD发生尚无文献报道。我们提出以下假设：肠源性内毒素可激活Toll 样受体4（TLR4）导致NLRP3炎症小体活化引发炎症造成肝损害。本研究拟通过PNALD小鼠模型，检测不同时间节点NLRP3炎症小体活化标志物的mRNA和蛋白表达水平，明确NLRP3炎症小体活化与肝脏病变的相关性；构建NLRP3过表达和shRNA腺病毒载体，观察NLRP3过表达与抑制对肝损害及相关信号通路的影响,应用TLR4基因敲除小鼠，观察TLR4信号通路改变对NLRP3炎症小体活化的影响，从多个层面探讨TLR4介导NLRP3 炎症小体活化在PNALD中的可能作用机制，找出关键分子靶点，为防治研究提供依据。

肠外营养相关肝损害（PNALD）至今机制未明，本课题组前期研究显示内毒素血症与氧化应激与PNALD密切相关。NLRP3 炎症小体可由多种病原体和危险信号激活，诱发炎症反应，但是否参与PNALD发生尚无文献报道。本研究发现原代肝细胞经LPS刺激后可通过激活Myd88非依赖的TLR4/TRIF通路，活化NLRP3炎症小体释放成熟的IL-1β以及IL-18，导致肝细胞发生焦亡。通过PNALD小鼠模型发现使用shNLRP3抑制NLRP3表达后可减轻肝损害并下调相关信号通路蛋白及mRNA表达，而使用TLR4拮抗剂后可以显著抑制NLRP3信号通路。本研究首次验证肠外营养时可通过TLR4介导NLRP3炎症小体活化引发炎症进而导致肝损害，对于进一步阐明PNALD的发病机制及防治研究有重要意义。

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