缺失ABRO1抑制kupffer细胞TLR4信号通路保护LPS/D-Gal诱导的急性肝衰竭的机制研究

ABRO1 plays a pivotal role in the regulation of ubiquitination, but its function in liver injury is still unclear.Previous studies in our lab showed that: 1) ABRO1 -/- mice were resistance to LPS/D-Gal induced acute hepatic failure, showed higher survival rate, less severe liver injury, lower level of serum cytokines and fewer inflammatory cells invaded in liver. 2) That ABRO1 deficiency protects LPS/D-Gal induced acute hepatic failure was dependent on the hematopoietic cells. 3) Significant Less TNFα was detected in ABRO1-/- kupffer cells treated with LPS. 4) TNFα-NFκB signaling pathway was suppressed in ABRO1-/- MEF cells. Accordingly, we presumed that ABRO1 deficiency protects LPS/D-Gal induced acute hepatic failure in mice via suppressed TLR4 signaling pathway in kupffer cells. Therefore, We intend to focus on the following aspects: 1) The regulation of ABRO1 in the inflammatory activation of kupffer cells. 2) The molecular targets of ABRO1 in LPS/TLR4-NKkB pathay. 3) The mechanisms of ABRO1 in the regulation of it’s targets. 4) Preliminary study to confirm that ABRO1/BRCC3 can perform as a target for the treatment of hepatitis.

ABRO1是重要的泛素调节蛋白，其在肝损伤中的作用未见报道。我们前期的研究发现：1）ABRO1-/-小鼠抵抗LPS/D-Gal诱导的急性肝衰竭，表现为提高存活率、减轻肝损伤、降低细胞因子水平、减少炎症细胞侵入等；2）ABRO1-/-小鼠的抵抗能力依赖血液来源细胞，与肝实质细胞关系不大；3）LPS体外刺激ABRO1-/- kupffer细胞产生TNF-α减少；4）ABRO1-/-成纤维细胞TNFα-NFκB信号通路活化受阻。因此，我们推测，敲除ABRO1可能通过抑制kupffer细胞TLR4-NFκB信号通路活化来保护LPS/D-Gal诱导的急性肝衰竭。本项目拟重点开展以下研究：1）ABRO1调控kupffer细胞炎性活化；2）鉴定ABRO1调控LPS/TLR4-NFκB通路的靶分子；3）ABRO1调控靶分子的机制；4）ABRO1/BRCC3作为抗肝脏炎症靶点的初步验证。

TLRs通路介导的Kupffer细胞活化在维持肝脏免疫稳态中起关键作用，其异常活化会驱动多种肝脏炎症性疾病，但是Kupffer细胞TLRs通路活性调控机制并未完全揭示。BRISC复合体是细胞内重要的K63位去泛素化酶，可以通过调节NLRP3和IFNAR1活化参与炎症反应过程，但是在TLRs通路介导的Kupffer细胞炎性活化中的功能并不清楚。本项目重点研究了BRISC复合体调控Kupffer细胞TLRs通路活化的作用与机制，以及与肝病发生的关系。我们发现，BRISC缺失可以选择性抑制Kupffer细胞中TLR3、TLR4和TLR9配体诱导的促炎性细胞因子分泌，而不影响骨髓来源巨噬细胞、腹腔原位巨噬细胞及中性粒细胞中TLRs通路的活化。全身性、髓系特异性和Kupffer细胞特异性缺失BRISC，而非肝实质细胞特异性缺失BRISC，都可以提高小鼠抵抗D-GalN/TLRs配体诱导的急性肝损伤。机制上，BRISC可以调节线性泛素链组装复合体LUBAC的酶活性亚基HOIP的去泛素化修饰，激活LUBAC介导的NF-κB通路活化。我们证明HOIP蛋白在K1003和K1056位点发生K63/线性杂链泛素修饰，BRISC通过其亚基ABRO1结合HOIP，剪切HOIP蛋白K63位泛素链从而去除其线性泛素化修饰，解除HOIP的自抑制状态，激活LUBAC。更重要的是，BRISC酶活性抑制剂硫藤黄菌素可以显著抑制Kupffer细胞炎性活化，有效减轻D-GalN/LPS诱导的急性肝损伤。综上，本项目揭示了BRISC通过调节HOIP去泛素化修饰，选择性促进Kupffer细胞TLRs-NF-κB通路活化的新功能及新机制，发现靶向抑制BRISC可以减轻D-GalN/TLRs配体诱导的急性肝损伤，为肝脏炎症性疾病的治疗提供了候选靶点和先导化合物。