FOXO3A抑制Kupffer细胞中STAT1/c-FLIP轴介导的坏死性凋亡途径调控机体内毒素耐受

Endotoxin tolerance induced by Kupffer cells (KCs) is an important protective mechanism for limiting the endotoxin shock. The accepted main mechanism to induce the endotoxin tolerance of KCs is to inhibit the activation of NF-κB. It is reported that inhibition of cellular necroptosis can relieve the inflammatory response mediated by nuclear factor kappa B (NF-κB). However, it has not been studied whether inhibition of cellular necroptosis can induce the endotoxin tolerance of KCs, and its mechanism is still a mystery. Recent findings assert that cellular FLICE-inhibitory protein (c-FLIP) is a positive regulatory factor which promotes necroptosis induced by LPS in macrophages. In addition, FOXO3A down-regulates the expression of c-FLIP by inhibiting the DNA location of signal transducer and activator of transcription 1 (STAT1), which is the main transfer factor of c-FLIP. Thus, it is reasonable to assume FOXO3A can induce endotoxin tolerance of KCs through negatively regulating necroptosis via STAT1/c-FLIP axis. In this study, we will focus on the KCs and take FOXO3A-STAT1/c-FLIP axis as the leading clue, to explore the effects of necroptosis on endotoxin tolerance. We hope these works could provide a novel idea for the treatment of inflammation-related diseases.

Kupffer细胞（KCs）内毒素耐受是机体限制内毒素休克的重要保护机制。目前认为，诱导KCs内毒素耐受的主要机制是抑制NF-κB的活化，现有报道称抑制细胞坏死性凋亡途径后可抑制NF-κB介导的炎症反应，但并无文献报道此途径是否参与诱导KCs内毒素耐受，机制也尚不可知。而据国内外研究发现，细胞凋亡抑制蛋白（c-FLIP）是促进LPS介导巨噬细胞坏死性凋亡的正向调控因子，FOXO3A则可通过抑制c-FLIP的转录因子STAT1的DNA定位，下调c-FLIP的表达。因此，我们推测FOXO3A通过抑制STAT1/c-FLIP信号轴，负性调控坏死性凋亡途径，诱导KCs内毒素耐受。本项目选择KCs为靶细胞，以FOXO3A-STAT1/c-FLIP为研究轴线，探讨坏死性凋亡途径是否参与调控KCs内毒素耐受，以及其确切机制，为治疗炎症相关性疾病提供新的思路。

Kupffer细胞（KCs）内毒素耐受是机体限制内毒素休克的重要保护机制。有报道称抑制细胞坏死性凋亡途径后可抑制NF-κB介导的炎症反应，但并无文献报道此途径是否参与诱导KCs内毒素耐受，机制也尚不可知。在本研究中，我们在体外KCs内毒素耐受发现KCs的坏死性凋亡激活情况与KCs内毒素耐受能力密切相关。抑制坏死性凋亡途径后，可抑制KCs非内毒素耐受模型中NF-κB通路的活性，提高KCs对内毒素的耐受能力。接着，我们观察改变c-FLIP的蛋白表达对KCs内毒素耐受的影响。结果发现，沉默c-FLIP表达可抑制KCs非内毒素耐受模型中NF-κB通路的活性，提高KCs对内毒素的耐受能力。之后，我们观察抑制STAT1表达及活性后对KCs内毒素耐受的影响。结果发现，沉默STAT1表达或抑制STAT1活性后可抑制KCs非内毒素耐受模型中NF-κB通路的活性，提高KCs对内毒素的耐受能力。且c-FLIP基因和蛋白的表达均显著下降；而抑制STAT1表达或活性后，均能显著降低内毒素非耐受模型KCs中 RIPK1的蛋白表达和RIPK3磷酸化水平。而在沉默c-FLIP后的KCs内毒素非耐受模型中，Fludarabinum对KCs死亡率、NF-κB磷酸化水平、TNF-α和IL-10的分泌以及RIPK1的蛋白表达和RIPK3磷酸化水平影响并不显著。最后，我们观察改变FOXO3A表达后，STAT1/c-FLIP对坏死性凋亡调控作用以及对KCs内毒素耐受的影响。结果发现，FOXO3A可显著抑制LPS诱导的STAT1/c-FLIP激活，从而抑制坏死性凋亡途径介导的KCs死亡。过表达c-FLIP可抵消FOXO3A降低LPS介导的炎症反应的效应。由此，我们研究证实了FOXO3A可通过负性调控STAT1/c-FLIP信号轴，从而抑制坏死性凋亡途径，进而提高KCs及小鼠对内毒素的耐受能力。这是我们首次观察到坏死性凋亡途径参与调控KCs内毒素耐受的现象，这可能对进一步阐明内毒素休克内源性保护机制有重要意义。