NOD1/TLR4/ NF-ΚB/MAPK和miR-10b-5p在肝脏缺血再灌注损伤中的作用机制

Hepatic ischemia-reperfusion injury (IRI) is an important cause of postoperative complications and survival rate that often occurs in liver resection, liver transplantation process, but its pathogenesis still remains unclear. The pre-experiment results showed that toll like receptor and nod like receptors in IRI expressed higher by using RNA-seq analysis of high-throughput technologies , the expression of NF-ΚB also increased, NOD1 expresses in various types of cells, but the study of nod like receptor signaling pathway in IRI is not much, the interrelationship of TLR4 and NOD1 and its specific mechanism is not clear. The sequencing results also found that miR-10b-5p is involved in liver ischemia-reperfusion injury, and the predict results shows it may targets NOD1, but whether it has therapeutic effects in IRI treatment still need to be confirmed. The aim of this project is to study mechanisms of TLR4/NOD1/NF-κB/MAPK and to explore the role of miR-10b-5p by establishing liver ischemia-reperfusion models both in vitro and in vivo and using TLR4-/- and NOD1-/- mice.

肝脏缺血再灌注损伤（IRI）常发生于肝切除、肝移植等过程中，是影响患者术后并发症及生存率的重要原因，但其发生机制目前尚不清楚。本课题的预实验中通过mRNA和miRNA的高通量测序结果发现Toll样受体和NOD样受体在缺血再灌注过程中高表达，它们都在先天免疫应激中起重要作用，此外，MAPK、NF-ΚB的表达量也增多，NOD1在各种类型的细胞中表达，但NOD样受体信号通路在肝脏缺血再灌注中的作用尚未有人研究，TLR4和NOD1在IRI的具体作用机制及其相互之间的作用尚不十分清楚。另外测序结果也发现miR-10b-5p参与肝脏肝脏缺血再灌注损伤过程，并且预测出其与NOD1可能有靶向关系，但其是否在肝脏IRI中有治疗作用尚需印证。本项目通过在体内、体外建立肝脏缺血再灌注模型并使用TLR4及NOD1基因敲除小鼠来研究TLR4/NOD1/NF-ΚB/MAPK作用机制并探讨miR-10b-5p的作用。

肝脏缺血再灌注损伤（ischemia-reperfusion injury，IRI）是肝脏缺血缺氧在恢复供血和供氧后损伤加重的病生理过程，是外科实践中一种常见的组织器官损伤。因此，阐明肝脏IRI的机制是亟待解决的临床问题。在前期的miRNA的测序结果也表明miR-10b-5p参与肝缺血再灌注过程，通过targetscan对NOD1进行miRNA的靶向预测，发现miR-10b-5p对NOD1有靶向关系，我们推测miR-10b-5p可能靶向结合NOD1而激活NF-κB和MAPK通路以及影响TLR4通路来抑制免疫炎症反应，减轻肝脏缺血再灌注损伤。实验结果显示，与Sham组相比，再灌注6h后，NOD1、TLR4、NF-κBmRNA表达水平逐渐升高（p<0.05），而miRNA-10-5p从2h至6小时表达水平逐渐降低。此外，与Sham组相比，免疫组化分析显示肝脏组织中NOD1蛋白的表达有所增加，同时，BNIP3、LC3II、P62蛋白的表达水平随着再灌注后时间的变化而增加。且焦亡指标Caspase1和GSDMD的表达也随再灌注时间延长而增多，用NOD1 siRNA转染的AML12细胞中的Atg5和LC3 II表达显著下降。综上所述，miR-10b-5p通过调控NOD1介导细胞自噬参与肝脏IRI，同时，TLR4与IRF-1调控的免疫炎症与细胞焦亡亦伴随其中。本项目研究结果为减轻肝缺血再灌注损伤、保护肝细胞功能的治疗寻求可能的理论依据。