ADAR1通过抑制RIG-I受体介导的信号途径及IFN的产生保护肝缺血再灌注损伤

The innate immune response and excessive activation of inflammatory pathways are important mechanisms in the development of liver ischemia and reperfusion (IRI), in which, Type I interferons (IFNs) play central role. Damage-associated molecular patterns (DAMPs) and pattern recognition receptors(PRR) play crucial roles in the initiation of the innate immune response. RIG-I like RNA receptors(RLRs) are important cytosolic PRRs to sense and recognize double stranded RNA to further activate cytosolic RNA sensing pathway, which has been known to induce antiviral innate immune responses. Adenosine deaminase acting on RNA1(ADAR1) is an RNA-editing protein that changes RNA sequences by converting adenosine to inosine,ADAR1 is also a strong RNA-binding protein; ADAR1 may affect cellular RNA transportation, localization, and functions through its RNA-binding activity. Recent studies indicated that ADAR1 limited cytosolic RNA sensing pathway. we demonstrated that Liver specific ADAR1 knockout mice showed dramatic growth retardation and high mortality at young ages due to severe liver structural and functional damage; Overwhelming production of RIG-I, IRF as well as type I IFN were observed in liver tissue and hepatocytes after induced deletion of the ADAR1 gene; In addition ,our results indicated that ADAR1 decreased while liver injury aggravated during IRI. So we infer that ADAR1 plays a protective role in the process of IRI by limiting RIG-I RNA sensing pathway and suppresses IFN production to protect against IRI. We subjected isolated hepatocytes to hypoxia-reoxygenation or mice to IRI to explore the role of ADAR1 on signaling pathways induced by these insults. ADAR1 expresssion was suppressed by shRNA treatment, or conversely, enhanced by cDNA targeting in hepatocytes in vitro or in mouse livers in vivo. Indexes for the cell viability or liver injury were measured. To explore the role of RIG-I RNA sensing, we knocked-down the IPS-1 or/and RIG-I genes or blocking TBK1/IKKε in ADAR1 knockdown or overexpression liver and hepatocytes. Indexes for the cell viability or liver injury were measured as well. In addition, interaction between ADAR1 and RIG-I was assessed by coimmunoprecipitation and pull-down assays. This study is to uncover the mechanisms of hepatic IRI and provide new clues to the prevention and treatment of hepatic IRI, as well as new medicine target to deal with such conditions.

固有免疫过度活化引起的瀑链式炎症反应是肝脏缺血再灌注损伤(IRI)重要机制。胞内RNA受体RIG-Ⅰ及其信号途径介导Ⅰ型IFN诱发的固有免疫反应，且可被双链RNA特异性腺苷酸脱氨酶（ADAR1）抑制，而在应激时伴有RNA表达水平显著增加。我们结果显示ADAR1维持肝结构、生长及功能；其缺失将诱导肝组织或细胞高表达RIG-Ⅰ/IFN信号途径相关分子，且ADAR1的表达与IRI负相关。故推测ADAR1通过干扰RIG-Ⅰ信号通路与RNA之间作用，减少Ⅰ型IFN表达，进而减轻肝脏IRI。我们拟降低或增加肝脏或肝细胞ADAR1表达，分析肝损伤及RIG-Ⅰ通路在IRI及应激肝细胞中的变化，进而阻断该通路，验证ADAR1抑制IFN依赖RIG-Ⅰ信号途径，进而研究ADAR1干扰RIG-Ⅰ信号通路的分子机制。从RNA受体的角度探讨抑制IRI固有免疫的途径及机制，为临床防治IRI提供新的药物靶点。

固有免疫异常激活在肝缺血再灌注损伤的过度炎症反应中发挥至关重要的作用。病原体相关分子模式分子（PAMP)引发的感染与危险相关分子模式分子（DAMP)诱导的炎症反应激活共同信号通路。胞浆维甲酸诱导基因Ⅰ（RIG-Ⅰ)样RNA受体（RLR)感知RNA病毒并介导I型干扰素产生，而双链RNA特异性腺苷酸脱氨酶（ADAR1）调控RLR对病毒RNA的识别。我们通过小鼠缺血再灌注模型，证实RIG-Ⅰ样受体也可被IRI内源性配体激活，且ADAR1也可调控IRI激活的RIG-Ⅰ/IFN信号通路。抑制ADAR1明显增强IRI诱导的炎症反应和肝损伤，并伴随I型干扰素明显升高。与体内实验结果一致，敲降肝细胞ADAR1加剧双链RNA或内毒素导致的炎症反应及I型干扰素过度表达，而抑制RIG-Ⅰ可消除ADAR1增强的上述反应。由此，我们证实肝细胞以ADAR1依赖方式保护自己，减轻响应急性肝IR诱发的过度胞浆RLR-RNA-感知信号通路激活。ADAR1抑制肝非感染性炎症过度激活的病毒RNA-感知通路。